Environmental Mass Spectrometry: Emerging Contaminants and

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Environmental Mass Spectrometry: Emerging Contaminants and Current Issues Susan D. Richardson National Exposure Research Laboratory, U.S. Environmental Protection Agency, Athens, Georgia 30605, United States

’ CONTENTS Introduction Major Analysis Trends Sampling and Extraction Trends Chromatography Trends Use of Nanomaterials in Analytical Methods Other Trends with Emerging Contaminants General Reviews Sucralose and Other Artificial Sweeteners Antimony Nanomaterials General Reviews Nanosilver and Nanogold Fullerenes and Other Carbon-Based Nanomaterials Nanomaterials in Foods, Plants, and Biota PFOA, PFOS, and Other Perfluorinated Compounds Measurements in Biota Paper Coatings for Food Packaging Drinking Water Landfill Leachates Seawater and Sediments Air and Soils around a Fluorochemical Manufacturing Plant Sewage Sludge New PFC Substitutes Fate and Sources New Methods Pharmaceuticals and Hormones Environmental Impacts of Pharmaceuticals General Reviews New Methods Illicit Drug Methods Marine Sample Methods Biota Methods Direct Analysis and Novel Approaches Occurrence Studies Fate of Pharmaceuticals: Wastewater Treatment, Drinking Water Treatment, and Photolysis Hormones Drinking Water and Swimming Pool Disinfection Byproducts This article not subject to U.S. Copyright. Published 2011 by the American Chemical Society

Drinking Water DBPs Combining Chemistry with Toxicology Discovery of New DBPs N-DBPs Nitrosamines Alternative Disinfection Technologies Using Iodine, UV, and Other Treatments Other Formation/Fate Studies DBPs of Pollutants New Swimming Pool Research Sunscreens/UV Filters Brominated Flame Retardants Benzotriazoles Dioxane Siloxanes Naphthenic Acids Musks Pesticide Transformation Products Perchlorate Algal Toxins Microorganisms Biography Acknowledgment References

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’ INTRODUCTION This biennial Review covers developments in environmental mass spectrometry for emerging environmental contaminants over the period of 2010
2011. Analytical Chemistry’s policy is to limit reviews to a maximum of 250 significant references and to mainly focus on new trends. Even with a narrow focus, only a small fraction of the quality research publications could be discussed. As a result, as with the previous review on Environmental Mass Spectrometry in 2010,1 this Review will not be comprehensive but will highlight emerging contaminant groups and discuss representative papers. I write a similar review article on Water Analysis, which also focuses on emerging contaminants.2 That review article is somewhat different from this one, in that it focuses on only water contaminants and

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Table 1. List of Acronyms APCI

atmospheric pressure chemical ionization

APPI

atmospheric pressure photoionization

BP-3

benzophenone-3

CCL

Contaminant Candidate List

DBPs E1

disinfection byproducts estrone

E2

17β-estradiol

E3

estriol

EE2

17α-ethinylestradiol

EDCs

endocrine disrupting compounds

EPA

Environmental Protection Agency

ESI

electrospray ionization

FT FTOHs

Fourier-transform fluorinated telomer alcohols

GC

gas chromatography

HAAs

haloacetic acids

IC

ion chromatography

ICP

inductively coupled plasma

IR

infrared

LC

liquid chromatography

MALDI 4-MBC

matrix-assisted laser desorption ionization 4-methylbenzylidene camphor

MCL

maximum contaminant level

MRM

multiple reaction monitoring

MS

mass spectrometry

NCI

negative chemical ionization

NDMA

N-nitrosodimethylamine

NMR

nuclear magnetic resonance

NOM N-EtFOSAA

natural organic matter N-ethyl perfluorooctane sulfonamide acetate

OC

octocrylene

ODPABA

octyl-dimethyl-p-aminobenzoic acid

PCBs

polychlorinated biphenyls

PBDEs

polybrominated diphenyl ethers

PFCs

perfluorinated compounds

PFCAs

perfluorocarboxylic acids

PFHxA PFNA

perfluorohexanoic acid perfluorononanoic acid

PFOA

perfluorooctanoic acid

PFOS

perfluorooctane sulfonate

PFOSA

perfluorooctane sulfonamide

REACH

Registration, Evaluation, and Authorization of Chemicals

SPE

solid phase extraction

SPME

solid phase microextraction

SRM THMs

selected reaction monitoring trihalomethanes

TOF

time-of-flight

UCMR

Unregulated Contaminant Monitoring Rule

UPLC

ultraperformance liquid chromatography

Numerous abstracts were consulted before choosing the best representative ones to present here. Abstract searches were carried out using Web of Science, and in many cases, full articles were obtained. A table of acronyms is provided (Table 1) as a quick reference to the acronyms of analytical techniques and other terms discussed in this Review. Major Analysis Trends. One of the hottest trends continues to be the use of high resolution mass spectrometry (MS) with liquid chromatography (LC) to identify unknown contaminants, typically environmental transformation products. In this regard, time-of-flight (TOF) and quadrupole (Q)-TOF mass spectrometers, as well as Orbitrap mass spectrometers, are increasing in use. In addition, there is increased use of nuclear magnetic resonance spectroscopy (NMR) with LC/MS/MS to confirm tentative structures proposed by LC/MS/MS. Because NMR is not as sensitive as MS, preparative LC is often used to collect enough material in fractions to enable the analysis of unknowns in complex environmental mixtures. Examples in this Review include the identification of pharmaceutical and pesticide transformation products. Atmospheric pressure photoionization (APPI) is also increasingly being used with LC/MS because it provides improved ionization for more nonpolar compounds, such as polybrominated diphenyl ethers (PBDEs) and musks discussed in this Review. Sampling and Extraction Trends. Solid phase extraction (SPE) remains the most popular means of extraction and concentration, and a new SPE device called Bag extraction was reported during the last 2 years. This bag-SPE consists of polystyrenedivinylbenzene enclosed in a woven polyester fabric, which can be immersed in water samples for solid phase extraction. Measured concentrations of pharmaceuticals have been shown to be comparable for bag-SPE vs Oasis HLB extraction. Benefits include the ease of handling, unattended water extraction, and that no filtration is needed. In addition, new SPE sorbents are available, including Oasis MCX and hypercrosslinked polymer resin (HXLPP), that are being used to capture a broader range of analytes within a single extraction. Solventless extraction techniques, such as solid phase microextraction (SPME), also continue to be used in many applications. Chromatography Trends. Ultraperformance liquid chromatography (UPLC) continues to increase in use. UPLC uses small diameter particles (typically 1.7 μm) in the stationary phase and short columns, which allow higher pressures and, ultimately, narrower LC peaks (5
10 s wide). In addition to providing narrow peaks and improved chromatographic separations, UPLC dramatically shortens analysis times, often to 10 min or less. Another significant chromatography trend is the use of twodimensional GC (GCxGC). GCxGC enables enhanced separations of complex mixtures through greater chromatographic peak capacity and allows homologous series of compounds to be easily identified. It also enables the detection of trace contaminants that would not have been identified through traditional GC. TOF-MS is often used as the detector for GCxGC because of its rapid acquisition capability. An example of the use of GCxGC in this Review includes the identification of naphthenic acids. Use of Nanomaterials in Analytical Methods. In addition to nanomaterials being a class of emerging contaminant, they are also being applied in creative ways to aid in the measurement of other emerging contaminants. For example, as described later in this Review, gold nanoparticle labeling was used with inductively coupled plasma mass spectrometry (ICPMS) to measure E. coli O157:H7 in water. This method took advantage of the signal amplification property of gold nanoparticles, monoclonal antibody

includes additional analytical methods beyond mass spectrometry. This Review on environmental mass spectrometry focuses on methods and occurrence/fate studies utilizing mass spectrometry but also includes the study of air, soil/sediment, and biological samples, in addition to water. I welcome any comments you have on this review ([email protected]). 748

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recognition, and the high sensitivity of ICPMS. Silica-supported Fe3O4 magnetic nanoparticles were also used to extract and preconcentrate pharmaceuticals from environmental waters. Other Trends with Emerging Contaminants. The analysis of environmental transformation products has become a major trend in environmental chemistry, and increasingly, researchers are taking this a step further in proposing complex transformation pathways, with detailed mechanisms deduced by LC/MS/ MS and sometimes confirmed by NMR. In addition, more researchers are combining toxicology with chemistry, in particular with the testing of transformation products and disinfection byproducts for toxicity, but also in effect-directed research to identify unknowns responsible for adverse environmental effects. There are also significant advances the last 2 years for nanomaterials, such that uptake in plants and animals (even humans) is now being demonstrated. Finally, a major trend in the area of perfluorinated chemicals is in investigating potential sources and their fate in the environment. New types of PFCs are also being investigated, including perfluorinated iodides.

’ GENERAL REVIEWS This section includes general reviews relating to environmental mass spectrometry and emerging contaminants. Reviews that relate to specific areas (e.g., PFCs, pharmaceuticals, DBPs) can be found in those specific sections. Many reviews have been published over the last 2 years that relate to environmental mass spectrometry, and some focus specifically on emerging contaminants. Because of the large number of reviews, only a few could be cited here. My other biennial review on Water Analysis published in 2011 (together with Thomas Ternes) discussed advances in mass spectrometry research for the same emerging contaminants discussed in this current Review, along with ionic liquids.2 Another biennial review published in Analytical Chemistry by Ballesteros-Gomez and Rubio covers new developments in environmental analysis and provides excellent discussions of sampling, sample preparation, separation, and detection techniques (including mass spectrometry).3 This review includes methods for a much broader range of environmental contaminants, such as volatile organic compounds (VOCs), pesticides, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), nitroaromatics, phthalates, organotins, and heavy metals, in addition to some emerging contaminants. Emerging contaminants were the focus of several reviews the past 2 years. For example, Alvarez and Jones-Lepp published a new review on sampling and analysis of emerging contaminants in surface water, groundwater, and soil and sediment pore water.4 Personal care products were the focus of two reviews. One by Brausch and Rand included discussions of environmental concentrations and toxicity,5 and the other by Pedrouzo discussed analytical methods for measuring them in environmental waters.6 Emerging food contaminants were the focus of another review by Kantiani et al., which included PFCs, PBDEs, nanomaterials, pharmaceuticals, and marine biotoxins.7 Clarke and Smith published a review of emerging contaminants in biosolids and ranked the chemicals based on their environmental persistence, human toxicity, bioaccumulation, ecotoxicity, and the number and quality of international studies.8 Several reviews were related to the use of different mass spectrometry techniques for emerging contaminants. Mass spectrometry analysis of phenolic endocrine disruptors and related compounds was the focus of a review by Gallart-Ayala et al.,

which included practical aspects of the use of GC/MS and LC/ MS with different ionization and monitoring modes.9 Petrovic et al. reviewed LC/MS methods used for pharmaceuticals, drugs of abuse, polar pesticides, PFCs, and nanomaterials.10 While not reviews themselves, other papers are worthy of note for broad applicability in the analysis of emerging contaminants. For example, Hernandez et al. discussed the use of GC with high resolution-TOF-MS for wide-scope target screening and unknown identification in environmental and food samples.11 Diaz et al. presented optimal experimental conditions for the creation of an empirical TOF-MS library to aid in the identification of environmental contaminants by UPLC or LC/MS/MS.12 Exact mass data for protonated or deprotonated molecular ions and up to 5 product ions, along with UPLC retention times, are presented for approximately 230 chemicals, including pharmaceuticals, hormones, pesticides, and transformation products. Palma et al. reported recent developments and applications of the use of electron ionization (EI) with LC/MS.13 In particular, two modern approaches, supersonic molecular beam and direct-EI LC/MS, can offer several advantages to classic atmospheric pressure ionization techniques (including electrospray ionization [ESI], atmospheric pressure chemical ionization [APCI], and atmospheric pressure photoionization [APPI]). These direct EI approaches allow automated library identification, easier identification of unknown compounds (through extensive fragmentation information), and lack of matrix interferences that cause problems with traditional LC/MS approaches. Effect-directed analysis is increasing in use. Examples include the identification of 8 androgen-disrupting compounds14 and a neurotoxic brominated ether in river sediments.15 In the first study by Weiss et al., androgenic and antiandrogenic assays were coupled with LC/accurate mass-MS, which enabled the identification of the chemicals responsible for the androgen-disrupting effects. In the second study by Qu et al., a neurotoxicity assaydirected analysis was coupled to LC/APCI-MS/MS, which enabled the identification of tetrabromobisphenol A diallyl ether as the causative toxicant in sediment samples collected from a river near a brominated flame retardant plant in China.

’ SUCRALOSE AND OTHER ARTIFICIAL SWEETENERS Sucralose (also known as Splenda or SucraPlus) is a relatively new artificial sweetener that is now widely used in North American and Europe. It may seem like an odd compound to include as an emerging contaminant, but it is now being found in environmental waters and is extremely persistent (half-life up to several years).2 It is made by chlorinating sucrose, where three hydroxyl groups are replaced by chlorine atoms. Sucralose is heat stable, which is why it has replaced other artificial sweeteners (such as aspartame) for baking and is now widely used in soft drinks because of its long shelf life. Several research groups have reported measurements of sucralose in the environment (including river water, groundwater, and coastal waters), and research has expanded to include other artificial sweeteners, such as acesulfame, saccharin, cyclamate, and aspartame. Due to its stability in the environment, sucralose has received a lot of attention as a potential tracer of anthropogenic inputs into environmental waters. A very interesting paper by Soh et al. follows the fate of sucralose through various environmental processes (microbial degradation, hydrolysis, sorption), water treatment processes (chlorination, ozonation, sorption to activated carbon, and UV irradiation), and impact on plants.16 Results showed some degradation due 749

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Analytical Chemistry to hydrolysis, ozonation, and microbial processes, but it was slow, suggesting that sucralose will be persistent in the environment and can be used as a tracer for anthropogenic activity. Sucralose was not found to inhibit plant uptake of sucrose in a variety of plant cotyledons investigated, nor did sucralose exhibit any toxicity on aquatic plant species studied. The authors brought up an interesting point that sucralose is one of very few contaminants that are highly persistent but do not bioaccumulate and have little or no reported toxicity at environmentally relevant concentrations. Then, an intriguing question is posed: “Is persistence reason enough for concern or regulation?”, but at the same time, it is recognized that the risks of chronic low-dose exposure to sucralose are unknown. Torres et al. investigated the fate of sucralose in wastewater treatment from 7 wastewater treatment plants in Arizona.17 Sucralose did not degrade in aerobic or anaerobic biological reactors, either metabolically or cometabolically after 42
62 days. Prolonged exposure to UV radiation did not oxidize sucralose significantly, and chlorine and ozone only showed a slow oxidation, such that, under typical wastewater treatment conditions, no significant sucralose degradation would be expected. Average treatment plant effluent levels were 2.8 μg/L. Two artificial sweeteners, sucralose and acesulfame, were included with 4 other contaminants (carbamazepine, diatroic acid, 1Hbenzotriazole, and tolyltriazole) in a study tracing their fate from wastewater treatment plants to receiving waters to riverbank filtration wells.18 Data were collected over 7 months at 7 sampling locations on the Rhine and Main Rivers in Germany. Sucralose, acesulfame, and carbamazepine showed pronounced stability during activated sludge wastewater treatment, and the concentrations for acesulfame and carbamazepine were well correlated, such that ratios could be used to identify a carbamazepine point source. In addition, soil aquifer treatment was tested at a site in Israel, where sucralose was found to degrade somewhat, possibly due to biodegradation or slow hydrolysis in the upper layer of the recharge basins, rather than by sorption. However, remaining levels were relatively high (2.1
3.5 μg/L), such that sucralose could still be used as an anthropogenic marker. Ferrer and Thurman developed a SPE-LC/TOF-MS method to measure sucralose, aspartame, and saccharin in wastewater, surface water, groundwater, and soft drinks.19 The presence of the artificial sweeteners could be confirmed by accurate mass measurements. Analysis of several wastewater, surface water, and groundwater samples revealed relatively high levels of sucralose, up to 2.4 μg/L. Sucralose was frequently detected, whereas saccharin was only detected in one wastewater sample and aspartame was not detected in any samples. It is likely that aspartame and saccharin are easily biodegraded, due to reactive chemical moieties in these molecules. Zygler et al. created a method to analyze for 9 artificial sweeteners in various food products (e.g., beverages, dairy, and fish products).20 Acesulfame, sucralose, aspartame, alitame, cyclamate, dulcin, neohesperidin dihydrochalcone, neotame, and saccharin were measured using LC/ESI-MS/MS. Buerge et al. used LC/MS/MS to analyze sucralose, acesulfame, saccharin, and cyclamate in soils to estimate inputs from agriculture and households, degradation, and leaching to groundwater.21 It is interesting to note that saccharin is registered as an additive in piglet feed, and it is largely excreted and can be found in manure up to 12 mg/L, with stability during 2 months of storage. As a result, saccharin has a high probability of winding up in significant quantities from the application of manure to agricultural land. Sweeteners can also get into soils from the

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irrigation of wastewater-contaminated surface water or through leaky sewers. In this study, half-lives ranging from 0.4 to 124 days were reported for the sweeteners. Data suggested that the detection of saccharin in the groundwater was most likely due to the application of manure but that the high levels of acesulfame were primarily due to infiltration of wastewater-contaminated surface water through stream beds. Van Stempvoort et al. investigated the presence of artificial sweeteners in groundwater from 8 urban sites in Canada.22 Acesulfame was detected at all 8 urban sites, and data suggested that it might be a good tracer for “young” wastewater (100 mg/kg of antimony. Recent studies have shown that antimony can leach from these plastic bottles over prolonged storage and especially at warm temperatures.2 This is a concern because of the growing popularity of bottled water. Compared to PET bottles, low density polyethylene bottles contain much lower levels (∼1%) of antimony.24 Human exposure to antimony was reviewed by Belzile et al., who discussed sources and intake of antimony through air particulate matter, drinking water, bottled water, and food.24 Exposure from bottled water (with PET bottles) was highlighted as a major source of antimony, especially after prolonged storage. High levels have also been observed in fish and in food grown near contaminated sites. Reimann used ICPMS to investigate the type of bottle on the leaching of antimony (and other metals/elements) into bottled water.25 Glass bottles, hard PET bottles, and soft PET bottles of different colors were investigated by purchasing bottled waters in supermarkets across Europe, rinsing the bottles and refilling with high purity (deionized) water at pH 6.5, and also at pH 3.5 to investigate the effect of pH. Antimony was found to have a 21 higher concentration when sold in PET bottles, but glass could also leach antimony in acidified waters, up to 0.45 μg/L after 150 days in a dark green glass bottle. For plastic bottles, the soft PET bottles and dark blue hard PET bottles leached the most antimony at near-neutral pH (6.5). Finally, Cheng et al. assessed antimony and other metal leaching into water from plastic bottles that had been previously recycled.26 They investigated factors that could affect leaching, including cooling with frozen water, heating with boiling water, microwaving, having low pH, exposure to 750

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outdoor sunlight irradiation, and exposure to in-car storage. Heating and microwaving led to the highest antimony leaching relative to controls, whereas low-pH, outdoor sunlight irradiation, and in-car storage had no significant effect. Results also revealed partial antimony leaching from PET bottles comes from the surface of plastic during the manufacturing process, while major antimony leaching comes from conditional changes.

larger size ZnO formulations. Levels in the blood were low, but it is clear that the zinc can penetrate the skin, which was not believed possible before. This study highlights the possibility for internal exposures to NPs (as well as slightly higher sized particles). General Reviews. As mentioned earlier, there were numerous reviews published for nanomaterials, even in the environmental arena. As a result, only a very few reviews could be cited here, such that I could also highlight new studies. In 2011, two special issues of Trends in Analytical Chemistry (TrAC), edited by Barcelo and Farr
e, focused on characterization, analysis, and risks of nanomaterials in environmental and food samples. In one of these special issues, Farr
e et al. led off with a review of the analysis and assessment of the occurrence, fate, and behavior of nanomaterials in the environment.28 Qualitative and quantitative methods were detailed, along with recent studies. In addition, the authors discuss a number of research gaps and issues regarding the assessment of engineered NPs, including the lack of analytical methods capable of quantifying real environmental samples (and therefore, lack of real concentration data), the need for standards and reference materials, the need for transparency of data with full documentation of experimental procedures, sample preparation, and analysis, and the need for analytical procedures to distinguish the origins of NPs. In 2010, a special series of nano papers was published in Journal of Environmental Quality. Top experts in the field led off this special issue with a review of the environmental occurrence, behavior, fate, and ecological effects of NPs.29 Within this review article, there are discussions of risks and release of engineered nanomaterials, key research areas and needs, and sustainable development of engineered nanomaterials. Important questions raised include: How much will be released? In which environmental compartments will they reside? What are the environmentally relevant forms? How do environmental conditions determine the form of nanomaterials? Peralta-Videa et al. also wrote an excellent review on nanomaterials in the environment and summarized work on risk assessment/toxicity, characterization and stability, toxicity, fate, and transport of nanomaterials in terrestrial ecosystems, as well as new engineered nanomaterials.30 Gottschalk and Nowack published a review, which included discussion of life cycle considerations of nanomaterials (from production, use in products, and recycling/disposal), release scenarios, models to predict their release into the environment, and experimental measurements of nanomaterial release in the environment.31 Nanosilver and Nanogold. Fabrega et al. reviewed the behavior and effects of silver NPs in the aquatic environment.32 Included is a discussion of the synthesis and characterization of silver NPs, their release in the environment, and uptake and effects in aquatic organisms, including fish. Farkas et al. measured silver NPs released in effluents from a new commercially available silver nanowashing machine.33 This washing machine released silver at an average of 11 μg/L, as measured by ICPMS. The presence of silver NPs was confirmed by single particle ICPMS, as well as ion selective electrode measurements and filtration techniques. The average size measured was 60
100 nm, and the effluent was shown to have an adverse effect on a natural bacterial community. Consumer aerosol sprays were the subject of another study by Lorenz et al., who used ICPMS to measure inorganic NPs in an antiperspirant, shoe impregnation sprays, and a plant strengthening agent.34 Nanosized aerosols were observed in products that contained propellant gas. Highest levels were found in the antiperspirant containing nanosilver.

’ NANOMATERIALS Nanomaterial research continues to rise exponentially, with companies and universities expanding their efforts. University departments have been developed around the study of nanomaterials, and government investment in nanotechnology has dramatically increased in the last 10 years. In my searching on Web of Science this year, nearly 5000 citations appeared in the literature for just the last 2 year period that this Review covers. This included >500 review articles on nanomaterials. There is even a monthly journal called ACS Nano (created in 2008). Special issues of journals highlighted nanomaterials, as well as numerous symposia held at scientific meetings the last 2 years. Most nanomaterial research is centered on developing new uses for nanomaterials and new products with unique properties, but on the other side, there is also significant concern regarding nanomaterials as environmental contaminants. As such, nanomaterials are the focus of a large initiative at the U.S. EPA, under which research on nanomaterial fate, transport, and health effects is being conducted. Nanomaterials are 1 to 100 nm in size and can have unique properties, including high strength, thermal stability, low permeability, and high conductivity. In the near future, nanomaterials are projected to be used in areas such as chemotherapy, drug delivery, and labeling of food pathogens (“nanobarcodes”). The chemical structures of nanomaterials are highly varied, including fullerenes, nanotubes, quantum dots, metal oxanes, TiO2 nanoparticles (NPs), nanosilver, nanogold, and zerovalent iron NPs. Most environmental concerns center on the potential human and ecological effects, and most methods use techniques other than mass spectrometry, such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), quartz crystal microbalance, energy dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy, static light scattering (SLS), particle electrophoresis, LC/UV, Raman spectroscopy, and NMR spectroscopy. In addition, most studies are carried out in “clean” systems and not in real environmental systems. Mass spectrometry techniques used for measuring nanomaterials include ICPMS and single particleICPMS (for metal-containing nanomaterials) and ESI- and APPI-MS/MS for fullerenes. Previous studies have shown the release of nanosilver from socks and other clothing treated with nanosilver, as well as from other consumer products including toothpaste, shampoo, and detergent.2 One particularly important finding this year was the discovery that zinc NPs in sunscreens can penetrate the skin and get transported into the bloodstream.27 In this fascinating study, Gulson et al. used human volunteers to test sunscreen formulations containing radiolabeled zinc (68Zn) in outdoor settings on a beach. One formulation contained ZnO NPs (which is commonly used in sunscreens that go on clear, rather than white and pasty), and the other contained larger ZnO particles (>100 nm). After 2 days of sunscreen application, 68Zn was observed in all the volunteers’ blood and urine, not only from the nano-ZnO but also from the 751

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Analytical Chemistry In a new method for silver NPs, Poda et al. used flow-field flow fractionation and ICPMS to measure silver NPs in aqueous suspensions and in biological tissues from aquatic organisms.35 Flow-field flow fractionation produced comparable results to other established sizing methods, and ICPMS provided increased sensitivity and selectivity relative to other detection techniques. Laborda used single particle detection with ICPMS in a new method to measure nanosilver in aqueous samples.36 Size limits for the detection of pure silver NPs were 18 nm, and detection limits were 1  104 particles/L. In another method, Chao et al. used cloud point extraction and ICPMS to measure silver NPs in antibacterial products and environmental waters.37 Limits of quantification for antibacterial products were 0.4 μg/kg and 0.2 μg/kg for silver NPs and total silver, respectively. This method was then used to measure nanosilver in 6 different antibacterial products. Interestingly, results revealed NPs in only 3 of the 6 tested products, even though all 6 were labeled as containing NPs. The first evidence of trophic transfer of NPs from a terrestrial primary producer to a primary consumer was demonstrated in a new study, along with the first evidence of biomagnification of a nanoparticle within a terrestrial food web. In this interesting fateuptake study, Judy et al. used tobacco plants and tobacco horn worms to investigate plant uptake and the potential for trophic transfer of 5, 10, and 15 nm diameter gold NPs.38 ICPMS, laser ablation-ICPMS, and X-ray fluorescence were used for measurement of the gold NPs. Results revealed trophic transfer and biomagnification of the nanogold from the plant to the animal by factors of 6.2, 11.6, and 9.6 for the 5, 10, and 15 nm-sized particles. Due to these findings, the authors highlighted the importance of considering dietary uptake as a pathway for nanoparticle exposure and also potential issues with long-term land application of biosolids containing NPs. Fullerenes and Other Carbon-Based Nanomaterials. Pycke reviewed strategies for quantifying C60 fullerenes in environmental and biological samples, along with implications for studies in environmental health and ecotoxicology.39 The authors stressed that our ability to quantify fullerenes in biological samples largely depends on our ability to extract these compounds from the complex matrixes, and so far, liquid
liquid extraction is considered the most robust method for fullerene extraction. However, there is a need for automated SPE extractions to improve detection limits for low-level environmental determinations and reduce solvent consumption. In another review, Scida et al. highlighted recent novel applications of carbon-based nanomaterials, including their use in sample preparation, separation, and detection.40 This included such applications as the extraction of flavonoids, proteins, peptides, and phosphopeptides using SPE with C60 fullerenes bound to silica particles; separation of chiral compounds through the use of carbon nanotubes coated with β-cyclodextrins; and the application of carbon nanotubes to the electrochemical detection of organic compounds. Cosmetics were the focus of another investigation by Benn et al., who measured C60 and C70 fullerenes using LC/APCI-MS/MS.41 C60 was detected in four commercial cosmetics (face serums/ creams), ranging from 0.04 to 1.1 μg/g, and C70 was qualitatively detected in two samples. A single use (0.5 g) can contain up to 0.6 μg of C60. Hydroxylated fullerenes were the subject of a new LC/MS/ MS method developed by Chao et al.42 A hydrophilic interaction liquid chromatography (HILIC) column was used to achieve

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good separation of the commercial fullerols, and method detection limits of 0.19 ng/L were achieved. This method was then tested in the presence of Suwannee River fulvic acid (as a model for natural organic matter commonly present in environmental waters), which significantly interfered with LC/UV measurements but only minimally impacted the LC/MS/MS measurement. Finally, van Wezel et al. developed an LC/linear ion trap (LTQ)-Orbitrap-MS method for measuring C60 fullerene and its transformation products in environmental waters.43 This method enabled measurements as low as 5 ng/L and revealed that C60 transformation products can exceed levels of their parent compounds in freshly prepared aqueous standards and also in standards stored at room temperature in light for 1
2.5 h. Transformation products resulted from oxidation and could be determined with accurate mass measurements. However, no C60 or its transformation products were detected in a wide array of surface waters collected in The Netherlands. This was attributed to the possibility of low emissions or losses in the aqueous solution phase by sedimentation, sorption, or transformation. Nanomaterials in Foods, Plants, and Biota. Determination of nanomaterials in food was the subject of a review by Blasco and Pico.44 This review highlighted current applications of nanomaterials in foods, food additives, and food-contact materials and discussed analytical methods for measuring them. Food matrixes included bread, biscuits, fish, and food packaging. In a groundbreaking study, Rico et al. demonstrated the first evidence for differential biotransformation and genotoxicity of ZnO and CeO2 NPs in terrestrial plants.45 In this study, ZnO and CeO2 NPs affected the growth of soybean plants. CeO2 NPs were found in the roots, and ZnO NPs biotransformed. Both were genotoxic to the soybean plants. In a separate review by Rico et al., the interaction of NPs with edible plants and their possible implications in the food chain were addressed.46 Several studies have shown effects on seed germination, but few have shown biotransformation of NPs in food crops. Possible transmission of NPs and potential biomagnification from edible plants is not known. Finally, Cassee et al. reviewed the exposure, health, and ecological effects of cerium and CeO2 NPs associated with its use as a fuel additive.47 CeO2 NPs have been used as an additive in diesel fuels since 1999 to increase mileage by performing as a combustion catalyst, increasing fuel combustion efficiency, and decreasing diesel soot emissions. However, engine tests have shown that small amounts of nano-CeO2 can be released in particulate matter in the exhaust. Ongoing exposure is occurring in large populations, but the impacts to environmental and public health are not known.

’ PFOA, PFOS, AND OTHER PERFLUORINATED COMPOUNDS Perfluorinated compounds (PFCs) (also referred to as fluorotelomer-acids, alcohols, and sulfonates) have been manufactured for more than 50 years and have been used to make stain repellents (such as polytetrafluoroethylene and Teflon) that are widely applied to fabrics and carpets. They are also used in the manufacture of paints, adhesives, waxes, polishes, metals, electronics, fire-fighting foams, and caulks, as well as grease-proof coatings for food packaging (e.g., microwave popcorn bags, French fry boxes, hamburger wrappers, etc.). PFCs are unusual chemically, in that they are both hydrophobic (repel water) and lipophobic (repel lipids/grease), and they contain one of the strongest chemical bonds (C
F) known. Because of these properties, they are highly 752

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stable in the environment (and in biological samples) and have unique profiles of distribution in the body. During 2000
2002, an estimated 5 million kg/yr was produced worldwide, with 40% of this in North America. Two of these PFCs, perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), have received the most attention. PFOS was once used to make the popular Scotchgard fabric and carpet protector, and since 2002, it is no longer manufactured in the U.S., due to concerns about widespread global distribution in the blood of the general population and in wildlife, including remote locations in the Arctic and North Pacific Oceans. Like PFOS, PFOA is ubiquitous at low levels in humans, even in those living far from any obvious sources.1 In January 2005, the U.S. EPA issued a draft risk assessment of the potential human health effects associated with exposure to PFOA (www.epa.gov/oppt/pfoa/pubs/pfoarisk.html), and in January 2006, the U.S. EPA invited PFC manufacturers to participate in a global stewardship program on PFOA and related chemicals (www.epa.gov/oppt/pfoa/pubs/stewardship). Participating companies agreed to commit to reducing PFOA from emissions and product content by 95% by 2010 and to work toward eliminating PFOA in emissions and products by 2015. The U.S. EPA has now listed PFOA and PFOS on the new Contaminant Candidate List (CCL-3), a priority list for consideration for future regulation in drinking water (http://water.epa. gov/scitech/drinkingwater/dws/ccl/ccl3.cfm). In Europe, the European Food Safety Authority has established tolerable daily intakes for PFOA and PFOS (www.efsa.europa.eu), and there are new restrictions on the use of PFOS as part of the European Union’s REACH program (http://ec.europa.eu/enterprise/ sectors/chemicals/files/reach/restr_inventory_list_pfos_en.pdf). Potential health concerns include developmental toxicity, cancer, and bioaccumulation. Research questions include understanding the sources of PFOA and other PFCs, their environmental fate and transport, pathways for human exposure and uptake, and potential health effects. It is hypothesized that the widespread occurrence of PFOA and other fluoro-acids is partly due to the atmospheric or oceanic transport of the more volatile fluorinated telomer alcohols (FTOHs) and subsequent transformation into PFOA and other fluoro-acids via metabolism and biodegradation. Recent studies support this hypothesis. In addition, there is evidence that PFOA itself is volatile (can sublime in its solid form) and that it can also partition from water to air.48 Even the ammonium salt form of PFOA can sublime into air. In addition to providing another unexpected mechanism for atmospheric transport for PFOA, these results also suggest that extra care should be taken in manufacturing facilities that make or use PFOA in formulations in order to minimize workplace exposure from inhalation. PFOS, PFOA, and other PFCs are included in the National Health and Nutrition Examination Survey (NHANES) conducted by the Centers for Disease Control and Prevention (CDC) to provide a better assessment of the distribution of these chemicals in adults and children in the United States (www. cdc.gov/nchs/nhanes.htm). This survey is carried out on a continual basis, with blood and urine collected from thousands of participants in the United States. The most recent report that includes population serum levels of PFCs can be found at www.cdc.gov/exposurereport/pdf/FourthReport.pdf. The National Toxicology Program is also studying PFOA and several other perfluorocarboxylic acids (PFCAs) and perfluorosulfonates (PFSAs) to better understand their toxicity and persistence in

human blood (http://ntp.niehs.nih.gov). Unlike other contaminants that accumulate in humans (e.g., dioxins, polychlorinated biphenyls), PFCs do not accumulate in fatty tissues but bind to serum proteins and accumulate instead in blood. As such, they have unique profiles of distribution in the body, owing to their unique chemical properties. While PFOS and PFOA were the first fluorinated surfactants to receive considerable attention, research has rapidly expanded beyond these two contaminants to other long-chain perfluorinated acids and various precursors. In addition, there is increased focus on shorter-chain forms, e.g., perfluorobutanoic acid (PFBA) and perfluorobutane sulfonate (PFBS), as manufacturers are beginning to shift to lower molecular weight PFCs that are not bioaccumulative. Butt et al. published a nice review detailing the levels and trends for PFCs in the Arctic environment.49 This review details measurements in snow from various regions of the Arctic, lake water and sediments, seawater and marine sediments, marine ecosystems, freshwater ecosystems, and terrestrial ecosystems. Transport pathways are also discussed. The authors point out that the detection of PFCAs and PFSAs in snow deposition is consistent with the volatile precursor hypothesis. Gaps in our knowledge include lack of knowledge regarding PFCs in the Russian Arctic, lack of measurements in abiotic systems, and limited measurements in seawater. Pico et al. published a review on the global perspective of PFCs in food.50 Food contamination levels and dietary intake risks posed by PFCs are outlined, as well as specific methods for their determination. Measurements in Biota. Studies continue to investigate PFCs in biota. Thomsen et al. examined changes in PFC levels in breast milk during 12 months of lactation for 9 mothers living in Oslo, Norway.51 This study was the first to measure depuration rates of PFOS and PFOA from human milk. Significant decreases in breast milk concentrations per month were observed, with depuration rates of 3.8 and 7.8% per month for PFOS and PFOA, respectively. After one year of breast feeding, concentrations were reduced by 37 and 94%, respectively, such that lactation was an important route of excretion for mothers. However, infants receive approximately 112 and 61 ng/day, such that lactation is a significant source of exposure for them. Human milk from China was the focus of another study by Liu et al., who measured 10 PFCs in 24 pooled samples from 1237 individual human milk samples collected from 12 provinces in China.52 PFOS and PFOA were the dominant PFCs found in all samples, with a mean of 46 pg/mL for each. High levels included 814 pg/ mL for mothers living in rural areas and 616 pg/mL for those living in urban settings (e.g., Shanghai). The estimated mean and maximum dietary intake were 17.8 ng/kg/d and 129.1 ng/kg/d, respectively. In another study by Harada et al., blood serum samples were collected from women in Japan, Korea, and Vietnam to assess PFOS and PFOA levels since the voluntary phase-out by 3 M Corporation of PFOS in 2002 in the region.53 Serum PFOS levels ranged from 4.86 ng/mL in Japan to 9.36 ng/mL in Korea (2007
2008). Serum PFOA levels ranged from 0.575 ng/mL in Vietnam to 14.2 ng/mL in Japan. PFOA levels increased in Korea from 1994 to 2008, but PFOS levels did not change. Serum PFOS levels from Japan in 2008 showed significant decreases (by 22
67%) compared to 2003/2004 levels. PFOA trends were mixed, as one highly exposed area of Japan showed a clear decline from 2003 to 2008, but low-exposure areas showed no change. 753

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Freberg et al. reported a fascinating human exposure study involving professional ski waxers from two national winter sports teams participating in the World Cup.54 Perfluorinated alkanes are used with petroleum-derived straight-chain aliphatic hydrocarbons in ski waxing products, and ski waxes are widely used by ski teams to increase performance. The product is applied either through the use of solid blocks, which are melted and dripped onto the ski using a heated iron, or through the use of powders, both of which are heated with an iron to create a smooth surface. During this process, significant aerosols can be generated, creating a risk for inhalation of PFCs. This human exposure study involved the measurement of 11 PFCAs and 8 PFSAs in the serum of 13 professional ski wax technicians. Highest median levels were for PFOA (50 ng/mL), which were 25 higher than background levels. Perfluorotetradecanoic acid was also reported for the first time in human serum. Positive, statistically significant associations were observed for years exposed as a ski waxer and 7 PFCAs in human serum. During an 8-month interval where no exposures were occurring, C8
C11 PFCAs decreased by 5
20%. This study was the first to link PFCAs in ski waxers’ serum to exposure from work room aerosols. In a related study from Sweden, Nilsson et al. investigated inhalation exposure to PFCs in air collected from the breathing zone of ski wax technicians during work.55 Air samples were collected with cartridges connected to portable air pumps. PFCAs were measured using LC/ MS/MS, and FTOHs (6:2, 8:2, and 10:2) were measuring using GC/MS/MS. Daily inhalation exposures of 8:2 FTOH were 800 higher than for PFOA. Overall levels ranged from 830 to 255 000 ng/m3. Together with other results showing increased levels of PFOA in blood serum, these results suggest the biotransformation of 8:2 FTOH to PFOA. PFCs in bird eggs were the focus of two recent studies. In the first, Holmstrom et al. investigated the temporal trends of PFCs in Swedish Peregrine falcon eggs from 1974 to 2007.56 This was the first study to establish temporal trends for PFCs in terrestrial biota. PFCAs, PFSAs, and perfluorooctane sulfonamide (PFOSA) were measured. PFOS was predominant (83 ng/g wet weight in samples from 2006), followed by perfluorotridecanoic acid (PFTrA) (7.2 ng/g wet weight) and perfluoroundecanoic acid (PFUnA) (4.2 ng/g wet weight). PFCA concentrations increased exponentially over time, whereas PFOS and PFHxS leveled off after the mid-1980s. In the second study, Gebbink et al. measured linear and branched PFOS isomer patterns in herring gull eggs from bird colonies in different regions of the Great Lakes (U.S.).57 Eggs were collected in 2007 from 15 bird colonies, and linear PFOS was consistently the dominant isomer in all eggs, comprising 95
98% of the total PFOS isomers. Linear PFOS was enriched in the gull eggs relative to technical PFOS, and higher proportions were observed in eggs from the lower lakes (Lake Erie and Lake Ontario). Fernandez-Sanjuan et al. used a solid
liquid extraction-LC/ MS/MS method to evaluate the occurrence of PFOS, PFOA, perfluorononanoic acid (PFNA), PFHxS, and PFBS in aquatic organisms.58 Freshwater organisms were studied along with those from marine ecosystems and included insect larvae, oysters, zebra mussels, sardines, and crabs. Among the organisms studied, none of the bivalves accumulated PFCs, but instead, insect larvae contained the highest levels, followed by fish and crabs, ranging from 0.23 to 144 ng/g wet weight for PFOS and 0.14 to 4.3 ng/g wet weight for PFOA, with only traces of PFNA and PFHxS. PFBS was not detected in any of the organisms. Fish were the focus of another occurrence study published by Schuetze et al.59 Wild fish

(including eels) were caught from different rivers in Northern Germany, as well as from the North Sea and Baltic Sea. PFOA was not found in any of the fish filet samples above the quantification limit of 0.27 μg/kg (fresh weight), whereas PFOS was detected up to 225 μg/kg (fresh weight), particularly from densely populated areas. Marine samples and samples from remote locations showed fewer positive detections, with a maximum to 50.8 μg/kg. On the basis of a provisional tolerable daily intake proposed by the European Food Safety Authority, 33 of the 112 fish samples might be classified as a potential risk for consumers with high fish consumption. Paper Coatings for Food Packaging. Trier et al. explored the identity of PFCs used for food contact materials used to impart oil and water repellency.60 Information was combined from patents, chemical suppliers, and analyses of industrial blends (using UPLC/MS/MS). More than 115 compounds were found in industrial blends from the European Union, U.S., and China. PFCs identified included polyfluoroalkyl-mono- and diester phosphates (monoPAPS, diPAPS, and S-diPAPS), -ethoxylates, -acrylates, -amino acids, -sulfonamide phosphates, and -thio acids, together with residuals and synthesis byproducts. In addition, a large number of starting materials, such as perfluorooctane sulfonamide N-alkyl esters, were analyzed. Fourteen different products were then tested for PFC migration, including microwave popcorn (from 6 manufacturers), a hamburger box, chocolate cake mix, bread mix, prepackaged frozen dinners, noodles, and coffee cups. Standardized migration testing used for plastics (60 °C, 2 h test, 1 dm2) was used to evaluate potential migration from the coated paper/cardboard packaging. Of the 14 samples, diPAPS and S-diPAPS were detected in 5 and 4 food migrate samples, respectively, with highest levels from microwave popcorn bags, up to 0.7 mg/kg. Besides microwave popcorn, the only other product that showed migration was a hamburger box, but levels were much lower. While PFOS and its derivatives are now prohibited for manufacture in the U.S. and in Europe, these derivatives were still present in the food packaging for these products sold in the U.S. and Europe. Because diPAPS are known to metabolize to PFCAs, the authors suggest that more types of PFCs should be monitored in order to map the sources in humans and the environment. Drinking Water. Thompson et al. conducted an extensive drinking water occurrence study from 34 locations across Australia, including capital cities and regional centers.61 SPE with LC/MS/MS was used for measurement, and a wide range of PFCs were measured. PFOS and PFOA were the most commonly detected, present in 49 and 44% of the samples, respectively. PFOS was found at the highest level, up to 16 ng/L, with PFHxS and PFOA up to 13 and 9.7 ng/L, respectively. The contribution from drinking water for PFOS and PFOA was 2
3%, with a maximum of 22 and 24%, respectively. Landfill Leachates. Because many PFC-containing products (paper packaging, nonstick coated pans, etc.) are disposed of in municipal landfills, there is a concern regarding PFCs leaching into groundwater and surface waters. Landfill leachates were the focus of two new studies. Busch et al. measured 43 PFCs in 22 landfill sites in Germany.62 Total PFC concentrations ranged from 31 to 12 819 ng/L in untreated leachate and from 4 to 8060 ng/L in treated leachate. Dominant compounds were PFBA and PFBS. PFC discharges varied according to the type of treatment used. Membrane treatment (reverse osmosis and nanofiltration) and activated carbon released the lowest concentrations into the environment than those using wet air oxidation or only biological 754

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treatment. Mass flows of total PFCs ranged from 0.08 to 956 mg/ day. Another landfill leachate study by Huset et al. used SPE with dispersive carbon sorbent cleanup and LC/MS/MS to measure 24 PFCs in landfill leachates from 4 locations in the U.S.63 In addition, leachate generated in a laboratory bioreactor containing residential refuse was investigated. All 7 leachates showed higher levels of short-chain PFCAs or PFSAs (C4
C7) as compared to longer-chain homologues (gC8). PFCAs were the most abundant, followed by the PFSAs, with PFBS levels up to 2300 ng/L. Sulfonamide derivatives were also observed, at ∼8% of the total PFCs measured. Seawater and Sediments. The distribution of PFCs in seawater from the North Sea and the Baltic Sea was the focus of a study by Theobald et al.64 Observed gradients could be explained by oceanographic mixing processes and currents, with large rivers as major sources. For example, 9 ng/L PFOA and 8 ng/L PFOS were found at the mouth of the river Elbe, but concentrations decreased to 3.8 and 1.8 ng/L, respectively, along the coast, and dropped to 0.13 and 0.09 ng/L, respectively, toward the open sea. Along the Dutch coast, high PFBS levels (3.9 ng/L) were observed. In the Baltic Sea, even distributions of PFCs were observed, with PFOA and PFOS predominant. In another study, Zushi et al. investigated time trends of PFCs from sediment cores taken from Tokyo Bay, Japan from the 1950s until 2004.65 The authors demonstrated that sediment cores can serve as a tool for reconstructing past PFC contamination and provided evidence on their environmental dynamics and time trends. Concentrations of 24 PFCs were measured. PFOS decreased gradually from the early 1990s, and its precursors, N-ethyl perfluorooctane sulfonamide acetate (N-EtFOSAA) and N-methyl perfluorooctane sulfonamide acetate (N-MeFOSAA), decreased rapidly in the late 1990s, whereas PFOA increased rapidly. Observed trends revealed a shift from perfluorooctyl sulfonyl fluoride (PFOSF)based product to telomer-based products after the phase-out of PFOSF-based products in 2001. Air and Soils around a Fluorochemical Manufacturing Plant. An interesting study by Ruan et al. investigated the presence and partitioning behavior of polyfluorinated iodides (PFIs) in environmental matrixes around a fluorochemical manufacturing plant in China.66 This study confirmed the presence of 4 perfluorinated iodine alkanes (FIAs) and 3 polyfluorinated telomer iodides (FTIs) in the environment. Thermal desorption-GC/high resolution-MS was used for air measurements, and a SPME-GC/low resolution-MS method was used for soil. Ambient air collected around the plant showed a wide concentration range, from 1.4 to 3.08  104 pg/L for FIAs and 1.39 to 1.32  103 pg/L for FTIs. In surrounding soils, most of the PFIs were not detected, with only a few higher carbon-chain analytes sporadically detected, up to 499 pg/g. Measurements suggest unintentional release of PFIs during the telomer-based manufacturing process. Because a majority of the PFIs partition to the gas phase, it is likely that these PFCs are transported atmospherically. In addition, PFIs have the potential to be converted into PFCAs. Because perfluorooctane iodide was also detected in fluorotelomer raw materials at high ppm levels, these other PFC sources should be investigated further. Sewage Sludge. Sun et al. developed a method based on solvent extraction and LC/MS/MS to measure long-chain PFCs in digested sewage sludge from Switzerland.67 Total PFCAs ranged from 14 to 50 μg/kg dry matter, and PFOS ranged from 15 to 600 μg/kg. In three wastewater treatment plants, PFOS levels were 6
9 higher than those from other plants. Because

the elevated PFOS levels did not correlate with higher levels of PFCAs, different local sources were suggested. New PFC Substitutes. As mentioned earlier, due to the phase-out of PFOS and PFOA, there has been a transition in manufacturing toward shorter-chain PFCs that are not bioaccumulative or are more biodegradable. Quinete et al. recently investigated the degradability of 5 new perfluorinated surfactants: PFBS, fluorosurfactant Zonyl (a fluorinated alkyl ether), two fluoroaliphatic esters (NOVEC FC-4430 and NOVEC FC4432), and 10-(trifluoromethoxy)decane-1-sulfonate using advanced oxidation based on UV, hydrogen peroxide, or combinations, followed by more conventional biodegradability tests, including a fixed-bed bioreactor, biological oxygen demand, and the closed-bottle test with microorganisms from the Rhine River.68 Most of these fluorinated surfactants are well established in the marketplace and have been used in applications as alternatives to PFOS- and PFOA-based surfactants. LC/MS/ MS was used to measure the degradation of the parent PFCs. Results showed that PFBS is not biodegradable. However, microorganisms could degrade 10-(trifluoromethoxy)decane-1-sulfonate in 6 days. The fluoroaliphatic ether and esters degraded slowly and did not meet the criterion for ready biodegradation. PFBS degradation by UV and oxidation was not significant within 120 min; other PFCs degraded upon exposure to UV, especially when coupled with hydrogen peroxide. In another study, Arakaki et al. investigated the microbial degradation of a new fluorotelomer alcohol, 1H,1H,2H,2H,8H,8H-perfluorododecanol.69 A mixed bacterial culture obtained from activated sludge was used to test for biological degradation, and LC/MS was used to analyze metabolites. PFBA, perfluoropentanoic acid, and perfluoropentanedioic acid were found as degradation products. The parent perfluorinated surfactant was cleaved into two short-chain fluorinated carboxylic acids that have less bioaccumulative potential and lower toxicity that the biodegradation products of 8:2 FTOH. Fate and Sources. Lee et al. investigated the biodegradation of polyfluoroalkylphosphates (PAPs) as a source of PFCAs to the environment.70 Headspace sampling revealed production of the fluorotelomer alcohols (FTOHs) from the hydrolysis of the PAP phosphate ester linkages. Analysis of the aqueous phase revealed microbial transformation to the final PFCA products. Most products were consistent with beta-oxidation-like mechanisms, but the detection of odd-chain PFCAs suggested that other pathways are likely. Results suggest that PAP-containing commercial products may be a significant contributor to the increased PFCA mass flows observed in wastewater treatment effluents. Fr€omel and Knepper examined the biotransformation of fluorotelomer ethoxylates (FTEOs) as sources of PFCs in the environment.71 The fluorotelomer ethoxylates are high production chemicals but have not been seriously considered as potential sources of PFCs in the environment. Experiments showed that the FTEOs are aerobically degraded, with a half-life of 1 d. Structural elucidation of the transformation products was made using LC/ESI-MS/MS, and a pathway was proposed. In addition to short-chain FTEO carboxylates, perfluorohexanoic acid (PFHxA) and PFOA were detected but were likely formed by degradation of residual fluorotelomer alcohols present in the commercial product. Sea spray was investigated in another study by Webster and Ellis as a means of generation of PFCAs into the gas phase.72 Using observed concentrations of perfluorooctanoate in oceans of the Northern Hemisphere and estimated spray generation rates, this mechanism was shown to have potential for contributing large 755

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amounts of PFOA to the atmosphere and may contribute to the concentrations observed in remote locations. Thompson et al. examined the fate of PFSAs and PFCAs in two water reclamation plants in Australia.73 One plant uses adsorption and filtration with ozonation, and the second uses membrane processes and advanced oxidation to produce purified recycled water from treated wastewater. Concentrations of PFOS and PFOA were reduced somewhat after treatment with ozone, and all PFCs were removed in the finished water by reverse osmosis. New Methods. New methods continue to be developed for PFCs. Gosetti et al. developed an automated online SPE-UPLC/ MS/MS method for measuring 9 PFCs in biological, environmental, and food samples.74 Limits of detection ranged from 3 to 15 ng/L, and the method was used to measure PFCs in river water, blood serum, blood plasma, and fish. Chromatographic separations could be made in 7 min. Lacina created a new UPLC/ MS/MS method for measuring 23 PFCs in milk and fish.75 Limits of quantification were 1
6 ng/L and 2
13 ng/L for milk and fish, respectively. This method was then used to measure PFCs in milk and canned fish samples, which revealed a wide spectrum of PFCAs, PFOS, and perfluoro-1-octanesulfonamide (PFOSA) in canned fish. Umbilical cord blood was the focus of a new UPLC/MS/MS method by Lien et al.76 This method allowed limits of quantification of 0.15 to 3.1 ng/mL for 12 PFCs. PFOA, PFOS, PFUnA, and PFNA were then detected in 68% of umbilical cord plasma in samples from the Taiwan Birth Panel Study. Beser developed a microwave-assisted extraction-LC/MS/MS method to measure 12 per- and polyfluorinated compounds in airborne particulate matter.77 Recoveries ranged from 83 to 120%, and quantification limits of 1.4 pg/m3 were achieved. This method was then used to measure PFCs in air samples collected in Valencia, Spain. Eight of the 12 PFCs were quantified in at least one sample, with levels ranging from 1.4 to 34.3 pg/m3. Llorca et al. used pressurized solvent extraction with LC/MS/ MS in a method to measure 13 PFCAs, 4 PFSAs, and perfluorooctane sulfonamide in sewage sludge.78 Method detection limits of 15 to 79 ng/kg were achieved, and the method was subsequently used to measure PFCs in 5 different sludge samples. Results showed all 18 PFCs present in all sludge samples, but many were below the limit of quantification. PFOS was present at the highest level, up to 121 μg/kg. Matrix-assisted laser desorption ionization (MALDI)-TOFMS was used in a new method by Cao et al. to measure PFCs in environmental waters.79 In this method, 1,8-bis(tetramethylguanidino)-naphthalene was used as the matrix, and SPE was used for preconcentration of PFCs. Very low limits of detection of 0.015 ng/L for PFOS were obtained, which were lower than LC/MS/MS methods. Finally, Sun et al. used derivatization with benzylamine and LC/MS in a new method to measure perfluorooctane sulfonyl fluoride (PFOSF) in environmental samples.80 Detection limits of 2.5 pg were possible, and the derivatization was selective for PFOSF.

wildlife and humans. A major concern for pharmaceuticals also includes the development of bacterial resistance (creation of “Super Bugs”) from the release of antibiotics to the environment, and there are also new concerns that antibiotics will decrease biodegradation of leaf and other plant materials, which serves as the primary food source for aquatic life in rivers and streams. It is estimated that approximately 3200 different substances are used as pharmaceutical ingredients, including painkillers, antibiotics, antidiabetics, betablockers, contraceptives, lipid regulators, antidepressants, chemotherapy drugs, and impotence drugs. However, only a very small subset of these compounds has been investigated in environmental studies so far. Pharmaceuticals are introduced not only by humans but also through veterinary use for livestock, poultry, and fish farming. Various drugs are commonly given to farm animals to prevent illness and disease and to increase the size of the animals. One lingering question is whether the relative low environmental concentration levels of pharmaceuticals (generally ng/L range) would cause adverse effects in humans or wildlife. Pharmaceuticals and hormones are now included on the U.S. EPA’s final CCL-3 (http://water.epa. gov/scitech/drinkingwater/dws/ccl/ccl3.cfm). One antibiotic (erythromycin) and one explosive (nitroglycerin) that is also used as pharmaceutical and 8 natural and synthetic hormones (17α-ethinylestradiol [EE2], 17α-estradiol, 17β-estradiol [E2], equilenin, equilin, estriol [E3], estrone, mestranol, and norethindrone) are included as priority drinking water contaminants, on the basis of health effects and occurrence in environmental waters. For the revision of the priority substances list within the EU water framework directive (2000) describing the chemical status of European rivers, streams, and lakes, two pharmaceuticals (diclofenac and ibuprofen) and two hormones (EE2 and E2) are suggested. There are also increasing “sourceto-tap” studies considering the fate of pharmaceuticals from wastewaters to river waters, to source waters, and to finished drinking water, such that the complete cycle of pharmaceutical fate is being considered. Innovative analytical instrumentation, such as hybrid mass spectrometry, enables the identification and quantification of organic pollutants, including pharmaceuticals and hormones, down to the lower ng/L and ng/kg range in environmental matrixes and drinking water. While most organic contaminants enter wastewater without being metabolized, pharmaceuticals are frequently transformed in the body and a combination of nonaltered pharmaceuticals and their metabolites are excreted by humans. Microbial transformation products (TPs) of pharmaceuticals and hormones can be formed during biological wastewater treatment, from contact with sediment and soil, as well as during bank filtration. Furthermore, TPs can be formed by UV irradiation in surface waters and during oxidative treatment processes, such as ozonation and chlorine disinfection. Still, LC/MS/MS is the method of choice for the determination of all classes of pharmaceuticals in aqueous matrixes. ESI and APCI are the most commonly used LC interfaces. Major innovations have been made in modern hybrid mass spectrometry systems (e.g., linear ion trap/ FT-MS, Q-TOF-MS) coupled to liquid chromatography, providing accurate masses of the analytes and information for mass fragments, which can be used to identify the chemical structures. Further innovations have been made in rapid online extraction and bag extraction, as well as online derivatization techniques in combination with GC/MS(/MS) detection. Environmental Impacts of Pharmaceuticals. Research on pharmaceuticals continues to grow exponentially, and this is

’ PHARMACEUTICALS AND HORMONES Pharmaceuticals and hormones have become important emerging contaminants, due to their presence in environmental waters (following incomplete removal in wastewater treatment or point-source contaminations), threat to drinking water, and concern about possible estrogenic and other effects, both to 756

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again evidenced by the vast number of reviews published the last 2 years, as well as the number of special issues of journals covering them. While many pharmaceuticals can have an acute or chronic effect on aquatic or other organisms, most of the lowest observed effect concentrations (LOECs) are substantially above the environmental concentrations that have been observed (ng/L to low μg/L). However, there are a few notable exceptions, where chronic toxicity LOECs approach levels observed in wastewater effluents. For chronic toxicity, these include salicylic acid, diclofenac, propranolol, clofibric acid, carbamazepine, and fluoxetine. For example, for diclofenac, the LOEC for fish toxicity was in the range of wastewater concentrations, and the LOEC of propranolol and fluoxetine for zooplankton and benthic organisms was near the maximum measured in wastewater effluents. The antibiotic ciprofloxacin has also been shown to have effects on plankton and algae growth at environmentally relevant concentrations.1 Estrogenic effects on wildlife are quite possible with the contraceptive α-ethinylestradiol (EE2), as it can induce estrogenic effects in fish at extremely low concentrations (low and sub-ng/L). Effects include alteration of sex ratios and sexual characteristics and decreased egg fertilization in fish.1 An article in Nature (Oaks, J. L.; Gilbert, M.; Virani, M. Z.; Watson, R. T.; Meteyer, C. U.; Rideout, B. A.; Shivaprasad, H. L.; Ahmed, S.; Chaudhry, M. J. I.; Arshad, M.; Mahmood, S.; Ali, A.; Khan, A. A. Nature 2004, 427, 630
633) highlighted that residues of veterinary used diclofenac probably caused renal failure of vultures and hence lead to a dramatic decline (>95%) of the vulture population in India and Pakistan. Experts estimate the vulture loss at 40 million, and it is being called the “worst case of wildlife poisoning ever”, far eclipsing the numbers of birds affected by DDT a few decades ago. Reviews were published the last 2 years summarizing ecotoxicity to aquatic organisms. For example, Santos et al. presented a nice comprehensive listing and discussion of many pharmaceuticals from different classes, including known ecotoxicity data for the parent drugs as well as metabolites and environmental transformation products.81 Reported concentration ranges in the environment were also included. Corcoran et al. presented a review on the presence and reported biological effects of pharmaceuticals in fish.82 General Reviews. A thought-provoking critical review by Ort et al. raised the question: “Sampling for pharmaceuticals and personal care products (PPCPs) and illicit drugs in wastewater systems: are your conclusions valid?”83 Issues discussed included the importance of short-term pollutant variations and whether reported variations can be attributed to real variations or if they simply reflect sampling artifacts. In another review, Howard and Muir identified commercial pharmaceuticals that might be persistent and bioaccumulative and were not being considered in current wastewater and environmental measurement studies.84 Two models, KOWWIN and BIOWIN1/BIOWIN5 (from EPI Suite software) were used to predict their potential bioaccumulative ability and persistence. A database of 3193 pharmaceuticals was developed from two U.S. Food & Drug Administration databases and lists of top-selling drugs. Of the 3193 pharmaceuticals, 275 have been found in the environment, and 92 of these were rated as potentially bioaccumulative, 121 as potentially persistent, and 99 as high production volume (HPV) pharmaceuticals. Other than these 275 pharmaceuticals, 58 HPV compounds were identified that were both persistent and bioaccumulative, and 48 others were identified as persistent only. Of the non-HPV compounds, 364 were identified as persistent and bioaccumulative. As a result, the authors highlighted several pharmaceuticals

that should be considered for future environmental studies. A detailed listing of these compounds was provided. In a mass spectrometry-focused review, Niessen detailed the fragmentation of toxicologically relevant drugs in positive-ion LC/MS/MS.85 The MS/MS spectra for ∼570 compounds was interpreted, and fragmentation was discussed. Fatta-Kassinos et al. presented a review of pharmaceuticals in environmental waters and wastewater.86 Occurrence of many pharmaceuticals of different classes is discussed, with concentration ranges given, as well as the ability of different treatment technologies to remove them. The occurrence, transformation, and fate of antibiotics in municipal wastewater treatment plants was reviewed by Zhang and Li.87 Major removal pathways included adsorption, biodegradation, disinfection, and membrane separation, and the majority of antibiotics are only partially eliminated in wastewater treatment. Daughton published a review on pharmaceutical ingredients in drinking water and discussed their occurrence and significance for human exposure.88 The 6 pharmaceuticals with the highest consistently reported concentrations were: ibuprofen, triclosan, carbamazepine, phenazone, clofibric acid, and acetaminophen (paracetamol). Except for ibuprofen and its methyl ester metabolite, all of these pharmaceuticals were ciprofloxacin ∼ norfloxacin ∼ lomefloxacin . pipemidic acid. The piperazine ring was the primary reactive group toward chlorine dioxide. Reaction pathways were proposed, but antibacterial activity is likely not eliminated because there was little destruction of the quinolone ring. Chemical and toxicity evaluation of the reaction of ozone with the antibiotic sulfamethoxazole was investigated by GomezRamos et al.138 Two main transformation pathways were proposed, which involved the preferential attack of molecular ozone or OH radicals, leading to the formation of 6 intermediates that were identified using LC/Q-TOF-MS. Reactions involved hydroxylation of the benzene ring, oxidation of the amino group on the benzene ring, oxidation of the methyl group and the double bond in the isoxazole ring, and S
N bond cleavage. Reaction of antibiotics with potassium permanganate was the focus of another study by Hu et al.139 LC/MS/MS was used to identify several DBPs, including 12 from ciprofloxacin, which were consistent with oxidation of the tertiary aromatic and secondary aliphatic amine groups on the piperazine ring and the cyclopropyl group. Seven DBPs were identified for both lincomycin and trimethoprim. Detailed oxidation pathways were proposed. Subsequent bacterial growth inhibition assays showed that the products had lost most of their antibacterial potency, suggesting that permanganate could be effective for eliminating their pharmaceutical activity in drinking water. Dioxin photolysis products had been reported for triclosan recently, and Buth et al. followed up this work by investigating sediment cores for their presence in real environmental samples.140 In this study, two sediment cores from a wastewater-impacted depositional zone of the Mississippi River were analyzed for triclosan using UPLC/MS/MS and for a suite of polychlorinated dioxins and furans using GC/high resolutionMS. 2,8-Dichlorodibenzo-p-dioxin was detected at levels that 761

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correlated with the use of triclosan since the 1960s. Three other dioxin congeners, 2,3,7-trichlorodibenzodioxin, 1,2,8-trichlorodibenzodioxin, and 1,2,3,8-tetrachlorodibenzodioxin, which are known photoproducts of chlorinated derivatives of triclosan, were also detected with similar trend profiles. The profiles did not correlate with higher chlorinated dioxin homologues or any chlorinated furan homologues but were consistent with the photolysis of triclosan and its chlorinated derivatives that form during wastewater chlorine disinfection. These dioxin derivatives have increased over time, such that they now constitute up to 31% of the total dioxin pool. The photolysis of diclofenac was investigated by Schulze et al.141 Effect-directed analysis revealed that the photolysis product, 2-[2-(chlorophenyl)amino]benzaldehyde, was responsible for the enhanced toxicity to green algae observed following the reaction. LC was used to fractionate products in the reaction mixture, and GC/MS was used to identify this TP. Its EC50 was a factor of 10 lower than for diclofenac. Photolysis products of the drug Tamiflu (oseltamivir) and oseltamivir carboxylate were investigated by Goncalves et al., who used UPLC/Q-TOF-MS to propose their structures.142 Reactions involved hydration of the cyclohexane ring, ester hydrolysis, intramolecular cyclization, and cleavage of the ethylpropoxy side chain. Finally, Yuan et al. studied the photodegradation and toxicity changes of 3 antibiotics with UV and UV/H2O2 treatment.143 GC/MS was used to identify the photoproducts. Toxicity, as assessed using Vibrio fischeri, increased following UV photolysis, with the photoproducts preserving the characteristic structure of the parents. On the other hand, UV/ H2O2 treatment resulted in more extensive reactions and subsequent detoxification. Hormones. Wise et al. reviewed sources of estrogens in surface water, source waters, and drinking water and posed the question: “Are oral contraceptives a significant contributor to the estrogenicity of water?”144 The removal of estrogens through water disinfection process and formation of byproducts was the focus of another review by Pereira et al.145 Oxidative treatments are effective for removing estrogens, but DBPs are generated. Structures of 46 natural estrogen and synthetic estrogen (EE2) DBPs are presented, along with the oxidation/disinfection processes that give rise to them. Liu et al. created a new method for 28 steroids, including 4 estrogens, 14 androgens, 5 progestogens, and 5 glucocorticoids in surface water, wastewater, and sludge.146 LC/MS/MS was used, combined with SPE, ultrasonic extraction, and silica gel cleanup. Method detection limits ranged from 0.01 to 1.4 ng/L and 0.08
2.1 ng/g for environmental waters and sludge, respectively. Langdon et al. published an extensive, Australia-wide survey of personal care products and endocrine disruptors in biosolids, including estrone (E1), E2, E3, and EE2.147 Levels were higher in samples from anaerobic treatment than aerobic treatment. E1 was the only hormone detected, in 4 of the 14 samples, up to 0.28 mg/kg in the biosolids. GC/MS with isotope dilution was used for analysis.

particularly important discovery in this regard was the formation of high levels of N-nitrosodimethylamine (NDMA) in drinking water that resulted from the reaction of ozone with a fungicide (tolylfluanide) used in Europe.2 New areas in drinking water DBP research include the study of highly genotoxic or carcinogenic DBPs that have been recently identified, issues with increased formation of many of these with the use of alternative disinfectants (e.g., chloramines and ozone), and routes of exposure besides ingestion. In this regard, there have been several recent studies of DBPs in swimming pools. Other trends include the development of UPLC/MS/MS methods and the combination of analytical chemistry with toxicology to account for toxicological effects with DBPs measured. In addition, near real-time methods are being developed, which could give drinking water utilities a better understanding and control over DBP levels received by consumers and improve exposure characterizations for epidemiologic studies. Toxicologically important DBPs include brominated, iodinated, and nitrogen-containing DBPs (“N-DBPs”). Brominated DBPs are generally more carcinogenic than their chlorinated analogues, and new research is indicating that iodinated compounds are more toxic than their brominated analogues.1 Brominated and iodinated DBPs form due to the reaction of the disinfectant (such as chlorine) with natural bromide or iodide present in source waters. Coastal cities, where groundwaters and surface waters can be impacted by salt water intrusion, and some inland locations, whose surface waters can be impacted by natural salt deposits from ancient seas or oil-field brines, are examples of locations that can have high bromide and iodide levels. A significant proportion of the U.S. population and several other countries now live in coastal regions that are impacted by bromide and iodide; therefore, exposures to brominated and iodinated DBPs are of growing interest. This year, another source of iodine has been discovered, X-ray contrast media, which contributes to the formation of iodo-DBPs. This new discovery is detailed in the section on DBPs of Pollutants. Early evidence in epidemiologic studies indicates that brominated DBPs may be associated with reproductive and developmental effects, as well as cancer. Brominated and iodinated DBPs of interest include iodo-acids, bromonitromethanes, iodo-trihalomethanes (iodo-THMs), brominated forms of MX (3-chloro4-(dichloromethyl)-5-hydroxy-2(5H)-furanone), haloaldehydes, and haloamides. Iodinated DBPs are increased in formation with chloramination, and bromonitromethanes are increased with the use of preozonation. Besides haloamides, other N-DBPs of interest include NDMA and other nitrosamines, which can form with either chloramination or chlorination (if nitrogen-containing coagulants are used in treatment). Five nitrosamines (NDMA, N-nitrosodiethylamine, N-nitrosodipropylamine, N-nitrosodiphenylamine, and N-nitrosopyrrolidine), as well as formaldehyde (which is a DBP from treatment with ozone, chlorine dioxide, or chlorine), are currently listed on the U.S. EPA’s new Contaminant Candidate List (CCL-3) (http://water.epa.gov/scitech/ drinkingwater/dws/ccl/ccl3.cfm). Chloramination has become a popular alternative to chlorination for plants that have difficulty meeting the regulations with chlorine, and its use has increased with the new Stage 2 Disinfectants (D)/DBP Rule (www.epa.gov/ safewater/disinfection/stage2). Potential health risks from DBPs include cancer and reproductive/developmental effects, with bladder cancer showing the most consistency in human epidemiologic studies from several countries. While this Review does not typically cover toxicology

’ DRINKING WATER AND SWIMMING POOL DISINFECTION BYPRODUCTS Drinking Water DBPs. Drinking water DBPs are formed by the reaction of disinfectants (chlorine, chloramines, ozone, chlorine dioxide, etc.) with natural organic matter (NOM) and bromide or iodide in source waters. They can also form by the reaction of disinfectants with other organic contaminants, and there is an increasing amount of research in this area. One 762

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or epidemiology studies, an important epidemiologic study was just published that bears mentioning here. Cantor et al. conducted a new case-control bladder cancer study and found an enhanced risk for bladder cancer (odds ratio 5.9) for people with a particular genotype, which can be found in approximately 25% of the U.S. population.2 Their study also found that dermal/ inhalation exposure from showering, bathing, and swimming was a significant risk factor. The findings strengthen the hypothesis that DBPs cause bladder cancer and suggest possible mechanisms, as well as classes likely to be implicated. Several reviews have been published the last 2 years on DBPs. Charrois published a nice review on the analysis of emerging DBPs in drinking water, which included detailed discussions on different analytical techniques that can be used to measure them.148 In addition, Charrois presented a nice historical perspective on the beginnings of this research area, with mention of the Water Chlorination Conferences begun by Robert Jolley (also published in a series of books), on up to the establishment of a Gordon Research Conference on Drinking Water DBPs in 2006. Swimming pool DBPs were also discussed. Richardson published a new review on DBP formation and occurrence in drinking water.149 This review provides a comprehensive listing of >600 DBPs identified from different disinfectants and disinfectant combinations (updating a 1998 encyclopedia article containing these original comprehensive lists) and includes discussion of formation and occurrence, issues with alternative disinfectants, route of exposure, and formation of “pollutant” DBPs. Combining Chemistry with Toxicology. More studies are combining DBP identification/measurement efforts with toxicology to understand their potential health effects. For example, Pressman et al. reported the second phase of a large integrated multidisciplinary study (called the Four Lab Study) involving the collaboration of chemists, toxicologists, engineers, and risk assessors from the 4 National Research Laboratories of the U.S. EPA, as well as collaborators from academia and the water industry.150 This paper described a new procedure for producing chlorinated drinking water concentrates for animal toxicology experiments, the comprehensive identification of >100 DBPs, and quantification of 75 priority and regulated DBPs. Complex mixtures of DBPs were produced by concentrating natural source waters with reverse osmosis membranes, followed by addition of bromide and treatment with chlorine. By concentrating the NOM first and disinfecting with chlorine afterward, DBPs (including volatiles and semivolatiles) were formed and maintained in a water matrix suitable for animal studies. DBPs were relatively stable over the course of the animal studies (125 days) with multiple chlorination events (every 5
14 days), and a significant proportion of the total organic halogen was accounted for through a comprehensive identification approach. Many DBPs were reported for the first time, including previously undetected and unreported haloacids and haloamides. The new concentration procedure not only produced a concentrated drinking water suitable for animal experiments but also provided a greater TOC concentration factor (136), enhancing the detection of trace DBPs that are often below detection using conventional approaches. Discovery of New DBPs. Increasingly, ESI-MS/MS is being used to discover new, highly polar DBPs. For example, Zhao et al. identified three new haloquinone DBPs in drinking water using LC/ESI-MS/MS: 2,6-dichloro-3-methyl-1,4-benzoquinone, 2,3,6trichloro-1,4-benzoquinone, and 2,6-dibromo-1,4-benzoquinone.151 Following their discovery in chlorinated drinking water, they were

quantified, along with 2,6-dichloro-1,4-benzoquinone. Levels ranged from 0.5 to 165 ng/L. An unusual feature about these compounds is that, using negative ion-ESI, they form (M + H)
ions through a reduction step, rather than the classic (M
H)
ions that are typically observed. The authors used tandem-MS and accurate mass measurements to confirm the identity of these unusual ions. This effort was followed up by an interesting study to investigate interactions of these haloquinones with oligodeoxynucleotides to understand potential binding to DNA.152 As measured using ESI-MS, the binding affinity of 2,6-dibromo-1,4benzoquinone to oligodeoxynucleotides was similar to that of ethidium bromide, a carcinogen that is well-known for intercalating with DNA. Tandem MS confirmed the formation of 1:1 and 2:1 complexes of 2,6-dibromo-1,4-benzoquinione with oligodeoxynucleotides. The chlorobenzoquinones also formed 1:1 adducts, but their binding was much weaker. Binding affinities followed the order: 2,6-dibromo-1,4-benzoquinone . 2,6-dichloro-1,4-benzoquinone > 2,6-dichloro-3-methyl-1,4-benzoquinone ∼ 2,3,6-trichloro-1,4-benzoquinone. Zhai and Zhang used precursor ion scans with UPLC/MS/MS to identify new polar brominated DBPs formed in chlorinated water, including 2,4,6-tribromophenol, 3,5-dibromo-4-hydroxybenzoic acid, 2,6-dibromo-1,4-hydroquinone, and 3,3-dibromopropenoic acid.153 In this study, various polar brominated DBPs were found to reach maximum levels at different chlorine contact times, suggesting that high molecular weight brominated DBPs might undergo decomposition or further reactions to form lower molecular weight DBPs and finally to haloacetic acids and trihalomethanes. Reaction pathways were suggested for the formation of brominated DBPs from Suwannee River humic acid, bromide, and chlorine. A newly synthesized chloroformate derivatizing agent was used in another paper to aid in the identification of 13 unknown highly polar DBPs in ozonated fulvic and humic acid solutions and ozonated drinking water.154 This derivatizing agent was specifically designed to derivatize carboxyl, hydroxyl, and amine groups, forming multiply substituted nonpolar derivatives that can be easily extracted from water and determined by GC/ negative chemical ionization (NCI)-MS. N-DBPs. Bond et al. published a review on the occurrence and control of N-DBPs in drinking water.155 It was stressed that the impact of water treatment processes on N-DBP formation is complex and variable, such that coagulation and filtration can efficiently remove cyanogen halide precursors, but they are less efficient for removing other N-DBP precursors, such as amino acids and amines. Oxidation before final disinfection can increase halonitromethane formation but decrease NDMA formation. Chloramination can increase both NDMA and cyanogen halides relative to chlorination. Several new studies focused on the formation and source of N-DBPs. Yang et al. investigated the formation of cyanogen chloride, dichloroacetonitrile, and chloropicrin during chloramination of several different precursors, including alpha-amino acids, amines, dipeptides, purines, and pyrimidines.156 Reaction pathways were proposed. 15N-labeled monochloramine revealed that the nitrogen in N-DBPs can originate from both NH2Cl and organic-N compounds. All precursors formed cyanogen chloride, with highest levels from glycine; dichloroacetonitrile was formed from the chloramination of glutamic acid, cytosine, cysteine, and tryptophan, and most precursors generated chloropicrin. Aldehydes and nitriles were identified as intermediates using negative ion-ESI-MS. Chu et al. investigated precursors of dichloroacetamide, the most common haloamide formed in chlorinated and chloraminated 763

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drinking water.157 In a reservoir in China, dissolved organic matter was separated into 6 fractions by a series of resin elutions. The hydrophilic dissolved organic matter fraction formed the highest levels of dichloroacetamide. Fluorescence excitation
emission matrix spectra revealed protein-like substances in this fraction, made up of amino acids, which were likely the dichloroacetamide precursors. Subsequent reactions of 20 amino acids with chlorine revealed that 7 amino acids (aspartic acid, histidine, tyrosine, tryptophan, glutamine, asparagine, and phenylalanine) could form dichloroacetamide during chlorination, with yields of 0.231, 0.189, 0.153, 0.104, 0.078, 0.058, and 0.050 mmol/mol amino acid. Nitrosamines. Nitrosamines continue to be of interest, since they were discovered to be DBPs in 2002. NDMA is a probable human carcinogen, and there are toxicological concerns regarding other nitrosamines. NDMA was initially discovered in chlorinated drinking waters from Ontario, Canada, and has since been found in other locations. The detection of NDMA in drinking water is largely due to improved analytical techniques that have allowed its determination at low ng/L concentrations. NDMA is generally present at low ng/L in chloraminated/ chlorinated drinking water, but it can be formed at much higher levels in wastewater treated with chlorine. It was also recently shown to form when waters containing a microbial degradation product of the fungicide, tolylfluanide, were ozonated.2 NDMA is not currently regulated in the United States for drinking water, but the U.S. EPA has recently made a preliminary decision to recommend it for regulation in drinking water, along with a group of other nitrosamines, for which the U.S. EPA has national occurrence and toxicity data. NDMA is regulated in Ontario, Canada (at 9 ng/L), under Ontario’s Safe Drinking Water Act (http://www.e-laws.gov.on.ca/html/regs/english/elaws_regs_ 030169_e.htm), and a Canadian national drinking water guideline for NDMA is also under development (www.hc-sc.gc.ca/ ewh-semt/consult/_2010/ndma/index-eng.php). NDMA was included in the U.S. EPA’s second Unregulated Contaminant Monitoring Rule (UCMR-2), along with 5 other nitrosamines (N-nitrosodiethylamine, N-nitrosodibutylamine, N-nitrosopropylamine, N-nitrosomethylethylamine, and N-nitrosopyrrolidine), and national occurrence data are currently available (http://water. epa.gov/lawsregs/rulesregs/sdwa/ucmr/data.cfm#ucmr2010). In addition, NDMA and 4 other nitrosamines are also on the U.S. EPA’s final CCL-3 (http://water.epa.gov/scitech/drinkingwater/ dws/ccl/ccl3.cfm). Nawrocki and Andrzejewski published an excellent review on nitrosamines, which included methods for their analysis, occurrence in drinking water and wastewater, precursors of NDMA in drinking water and wastewater, removal of nitrosamine precursors in water treatment, mechanisms of formation for NDMA, and methods to remove NDMA.158 The authors conclude with a list of research needs, which includes the need to identify specific N-containing groups present in NOM, systematic research on the presence of secondary amines in surface water and their behavior in drinking water treatment, and the investigation of NDMA contamination in surface and groundwaters in Europe. A new GC/MS nitrosamine method, capable of measuring 9 nitrosamines, was created using GC/CI-ion trap-MS/MS.159 Because NDPhA decomposes at common GC injection port temperatures (and is why this nitrosamine is not included in the EPA Method that also uses GC/CI-MS/MS), the authors monitor its decomposition product, diphenylamine, to enable its determination. It also uses methanol as the CI reagent gas and does not require large volume injectors. Detection limits for

NMEA were 2 ng/L; all of the other nitrosamines could be detected down to 1 ng/L. The method was subsequently used to measure nitrosamines in drinking water and in swimming pool water, where NPYR was found in all samples at concentrations greater than 50 ng/L. Several studies examined the formation and fate of nitrosamines. Patterson et al. examined the fate of trace organic compounds, including NDMA and NMOR, in recycled water during managed aquifer recharge.130 NDMA and NMOR did not degrade under either aerobic or anaerobic conditions (half-life > 50 days), such that natural attenuation during aquifer passage alone may not allow extracted water to meet regulatory limits. Van Huy et al. investigated the formation potential for NDMA in groundwater and river water in Tokyo.160 Twenty-three groundwaters and 18 river waters were collected, and NDMA was analyzed using LC/MS/MS. NDMA precursors ranging from 4 to 84 ng-NDMA equivalents/L in groundwater and from 11 to 185 ng in river water. Molecular size fractionation of the river waters revealed that NDMA precursors were mostly in the BDE-206 did not mirror any known technical PBDE mixture and provided evidence for BDE-209 degradation. Oysters and mussels were the focus of a study by Ueno et al., who examined the spatial distribution of PBDEs, hexabromocyclododecanes (HBCDs), and other contaminants in bivalves from the coast of Japan.196 HBCDs were found up to 5200 ng/g (lipid weight), and PBDEs were found up to 86 ng/g (lipid weight). No differences were seen in bioaccumulation between oysters and mussels, indicating that oysters could be used as an alternative species to mussels for examining bioaccumulation. Estimated dietary exposure through consumption of seafood was 0.45
34 ng/kg body weight per day, which was ∼1000 lower than the lowest observed effect level. Honey was the focus of an extensive study by Wang et al., who measured 27 PBDEs in 50 honey samples originating from different parts of the world.197 Concentrations of BDE-209 ranged from nondetect to 9260 pg/g, while the other 26 PBDEs ranged from 300 to 10 550 pg/g. Honey samples from developed countries generally had higher levels than developing countries. BDE-209 was the dominant congener in all honey samples, accounting for 16% and 65% of the total PBDE concentration in honey from developed and developing countries, respectively. These findings were consistent with a long, historical use of PBDE-containing products in developed countries and a current, heavy use of BDE-209 in developing countries. Findings also indicate a source of human exposure through consumption of honey. Fish oil was examined by Ortiz et al. for HBCDs.198 Concentrations ranged from 0.09 to 27 ng/g, and specific enantiomers and diastereomers were also determined. HBCD levels were similar to other pollutants and correlated with dioxin and PCBs. Dietary intake was estimated at 0.08 to 5.38 ng per day. Air samples near a municipal solid waste incinerator were the focus of another study by Wang et al.199 GC/high resolution-MS with isotope dilution was used for measuring 30 PBDEs and other contaminants. Total PBDE levels ranged from 25.7 to 100 pg N/m3 in these samples from Taiwan, which were similar to levels reported in urban air from North America and Japan. New methods developed included those using LC/APPI/ MS/MS and GC/MS. Zhou et al. developed a new LC/APPIMS/MS method for measuring PBDEs and other halogenated flame retardants in fish.200 The analytes eluted within 14 min, with detection limits of 4.7 pg. Good agreement was found between results from this method and from GC/high resolutionMS. Gonzalez-Gago et al. reported a new GC/MS method using synthesized 81Br-labeled standards for measuring PBDEs in fish and other solid samples.201 Low limits of detection (0.02 to 0.9 ng/g) could be achieved. It is also possible to use this procedure with even more sensitive GC/NCI-MS analyses, which cannot be done with more common 13C-labeled standards. Finally, Lupton et al., created a new LC/APPI-MS/MS method for measuring hydroxylated PBDE metabolites.202 This method alleviates the 768

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Analytical Chemistry need for derivatization used with current GC/MS techniques and was demonstrated for the analysis of BDE-47 and -99 in human liver microsomes.

’ BENZOTRIAZOLES Benzotriazoles are complexing agents that are widely used as anticorrosives (e.g., in engine coolants, aircraft deicers, or antifreeze liquids) and for silver protection in dish washing liquids. The two common forms, benzotriazole (1H-benzotriazole) and tolyltriazole (a mixture of 4- and 5-methyl-1H-benzotriazole), are soluble in water, resistant to biodegradation, and only partially removed in wastewater treatment. There is also new evidence for estrogenic effects in vitro but, so far, not in vivo, in recent fish studies.1 There is also some evidence that benzotriazole may be a human carcinogen, and Australia now has a drinking water guideline limit of 7 ng/L for tolyltriazole.203 Because of their water solubility, LC/MS and LC/MS/MS methods have been recently developed for their measurement in environmental waters. While reports of benzotriazoles are fairly recent (∼last 8 years), studies indicate that they are likely ubiquitous environmental contaminants. Janna et al. reported an interesting study entitled, “From dishwasher to tap? Xenobiotic substances benzotriazole and tolyltriazole in the environment”.203 This study demonstrated their presence in UK wastewaters, rivers, and drinking water and suggested that their use as silver polishing agents in dishwasher tablets and powders may account for a significant proportion of inputs to wastewaters. Benzotriazole and tolyltriazole ranged from 840 to 3605 ng/L and 2685
5700 ng/L, respectively, in sewage effluents and from 0.6 to 79.4 ng/L and 50% of the groundwaters sampled, up to 1.03 and 0.52 μg/L, respectively.107 New methods include those developed for benzotriazole UV stabilizers in biota. Kim et al. created a new multiresidue method using UPLC/MS/MS to measure benzotriazole UV stabilizers and other contaminants in fish.204 Method detection limits ranged from 0.0002 to 0.009 ng/g for the 8 benzotriazoles measured. These were subsequently found in fish from the coast of the Philippines, up to 179 ng/g for 2-(2H-benzotriazol-2-yl)-4,6-di-tert-pentylphenol (UV-328). Nakata et al. developed a new GC/high resolutionMS method for measuring benzotriazole UV stabilizers in the blubber of porpoises.205 Mean concentrations were 38 and 19 ng/g for UV-328 and 2,4-di-tert-butyl-6-(5-chloro-2H-benzotriazol-2yl)phenol (UV-327) in porpoises from the coast of Japan. The bioconcentration factor was 33 300 for UV-327, which was an order of magnitude higher than for fish found in the same region. A new method for indoor dust was created by Carpinteiro et al, who used pressurized solvent extraction followed by GC/MS/ MS to measure benzotriazole UV stabilizers.206 Limits of quantification below 10 ng/g could be achieved, and this method allowed the first detection of 4 benzotriazole UV stabilizers in dust from indoor environments. Mean concentrations ranged from 71 to 780 ng/g. Headspace-SPME-GC/MS was used in another method by Carpinteiro et al. for measuring 5 benzotriazole UV stabilizers in environmental waters.207 Limits of quantification below 2 ng/L could be achieved, and this method was demonstrated on raw wastewaters.

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’ DIOXANE 1,4-Dioxane is a widespread industrial contaminant in environmental waters (often exceeding water quality criteria and guidelines), has also been found in drinking water, and is a probable human carcinogen. Dioxane is a high production chemical and is used as a solvent stabilizer in the manufacture and processing of paper, cotton, textile products, automotive coolants, cosmetics, and shampoos, as well as a stabilizer in 1,1,1-trichloroethane (TCA), a popular degreasing solvent. In 2002, more than 500 t of dioxane were produced or imported in the United States. The U.S. EPA has identified dioxane as a high priority contaminant, and it is currently listed on the new CCL-3 (http://water.epa.gov/ scitech/drinkingwater/dws/ccl/ccl3.cfm). There is also an EPA Method (522) for its measurement (www.epa.gov/microbes/ Method%20522_final%20for%20OGWDW%2009_22_08.pdf). Dioxane is problematic to extract and measure because it is miscible with water. It is also difficult to remove from water by air stripping or carbon adsorption. Environmental investigation and remediation of dioxane and other solvent stabilizers was the focus of a new book by Mohr.208 This book included a discussion of the chemistry, uses, and occurrence; environmental fate and transport; sampling and analysis; toxicology; regulation and risk assessment; remediation technologies; case studies of releases, treatment, and drinking water contamination; and forensic applications. Ramirez et al. developed a thermal desorption-GC/MS multiresidue method for measuring dioxane and 98 other contaminants in air emissions from an industrial wastewater plant.209 This method was rapid and did not require the use of organic solvents. Detection limits of 1.33 μg/m3 were possible, and dioxane was found up to 105 μg/m3 in air from one of the two sites sampled. ’ SILOXANES Siloxanes have become a relatively new area of research. They include cyclic siloxanes, octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), dodecamethylcyclohexasiloxane (D 6 ), and tetradecamethylcycloheptasiloxane (D 7 ), and linear siloxanes, which are used in a number of products, such as cosmetics, deodorants, soaps, hair conditioners, hair dyes, car waxes, baby pacifiers, cookware, cleaners, furniture polishes, and water-repellent windshield coatings. There is concern about potential toxicity and transport into the environment. They have been previously measured in wastewater, river water, and landfill biogases.1,2 In a new study, Sparham reported the first measurements of D5 in river and estuarine sediments in the UK.210 Two extraction methods were used with GC/MS for measurement. Accelerated solvent extraction (ASE) was useful for measuring the higher concentrations in river sediments (up to 1450 ng/g in sediments from the Great Ouse River), and liquid
solid extraction was useful for lower concentrations found in the estuarine sediments (up to 256 ng/g in the Humber estuary). Limits of quantification of 57
110 ng/g and 4 ng/g were possible using ASE and liquid
solid extraction, respectively. ’ NAPHTHENIC ACIDS Naphthenic acids (NAs) are a complex mixture of alkylsubstituted acyclic and cyclo-aliphatic carboxylic acids that dissolve in water at neutral or alkaline pH and have surfactant-like properties. They occur naturally in crude oil deposits across the world (up to 4% by weight) and have also been recently 769

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Analytical Chemistry discovered in coal, which could be a source of contamination for groundwater. NAs are toxic to aquatic organisms, including phytoplankton, daphnia, fish, and mammals and are also endocrine disrupting. With decreased conventional crude oil resources, it has become economically feasible to extract heavier oils from oil sand deposits. One of the world’s largest accumulation of oil sands occurs in North and South America. Venezuelan oil sand deposits contain the largest known petroleum deposits in the world, and the Athabasca oil sands in Alberta, Canada, are a close second.211 The Athabasca oil sands represents more than 25% of Canada’s annual oil production, and most research on NAs has been conducted in this region. Caustic hot water is used in the extraction of crude oil from oil-sands, which results in a residual tailing water (0.1 to 0.2 m3 of tailings per ton of oil-sands processed) that contains high levels of NAs (80 to 120 mg/L levels are common) and is very toxic. The total volume of tailing ponds is projected to exceed 109 m by the year 2020. Whitby published a very nice review on microbial napthenic acid degradation, which included a discussion of chemical properties, toxicity, sources, and environmental contamination.211 Biodegradation studies included those carried out on model compounds and commercial NAs, as well as actual environmental degradation. Bioremediation methods were also discussed. New methods continue to be developed, including one by Rowland et al., which used GCxGC/TOF-MS to identify individual tetra- and pentacyclic NAs in oil sands process water.212 Headley et al. evaluated the potential of negative ion-ESI-Fourier transform ion cyclotron resonance (FTICR)-MS for comparing polar organics (including NAs) from tailing ponds, interceptor wells, groundwater, rivers, and lake water.213 This work was done to expand investigations beyond NAs because aquatic toxicity and environmental chemistry are attributed to the total organics, not only to the NAs. The ratios of species containing oxygen and nitrogen were useful for differentiating organics derived from oil sands process water from those found in river and lake waters. New fate studies have also been conducted, including one by Headley et al. who investigated the degradation of NAs with UV/ TiO2 and microwave radiation.214 A higher oxygen content was observed in the treated samples using FTICR-MS, consistent with oxidation of the parent acids. Microbial degradation was the focus of another controlled laboratory study by Johnson et al., who found that degradation was affected by the degree of alkyl side-chain branching.215 Degradation products were generally less toxic than the parent compounds. In each case, biodegradation of the carboxyl side chain proceeded through beta-oxidation. Oxidation pathways were proposed. Rowland et al. reported the synthesis and characterization of 6 alkylcyclohexylethanoic NAs, which had been tentatively identified as biodegradation transformation products.216 GC/MS was used to characterize their trimethylsilyl derivatives, and toxicity results showed lower relative toxicity of these transformation products relative to the parent NAs. Finally, an interesting new study by Hersikorn and Smits investigated the effect of tailing ponds (containing NAs) on wood frogs.217 Time to metamorphosis, thyroid hormone status, and detoxification enzyme induction were measured in tadpoles raised in reclaimed oil-sands wetlands and were compared to tadpoles raised on control (nonimpacted) wetlands. Metamorphosis was delayed or never occurred in tadpoles raised in young tailings (e7 years old), but no effects were seen for tadpoles raised in older tailings. This suggests that tailings wetlands become less toxic with age.

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’ MUSKS Synthetic musk compounds are widely used as fragrance additives in many consumer products, including perfumes, lotions, sunscreens, deodorants, and laundry detergents. They can have nitroaromatic structures, as in the case of musk xylene (1-tert-butyl3,5-dimethyl-2,4,6-trinitrobenzene) or musk ketone (4-tert-butyl2,6-dimethyl-3,5-dinitroacetophenone), or polycyclic structures, as in the case of 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene (AHTN; trade name, tonalide) or 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-(g)-2-benzopyran (HHCB; trade name, galaxolide). Because they are widely present in environmental samples, including wildlife and humans, there is growing concern. Musks are highly lipophilic, so they tend to accumulate in sediments, sludges, and biota. Up to 190 ng/g lipid has been reported in humans.2 Several new methods have been recently developed for measuring musks. Lung and Liu created a new UPLC/APPIMS/MS method for measuring 6 musks.218 Chromatographic separation could be achieved in 7 min in the positive ion mode and 5.1 min in the negative ion mode. Limits of detection were below 6 pg. Hu and Zhou compared microwave-assisted extraction, simultaneous distillation-solvent extraction, Soxhlet extraction, and ultrasound extraction for extracting polycyclic musks from sediments and other environmental samples.219 Microwave-assisted extraction and ultrasound extraction were found to be the most effective. This method was subsequently used to measure the uptake of musks in wheat plants, which correlated significantly to concentrations found in the sediments. Sapozhnikova et al. used ASE with gel permeation chromatography/SPE cleanup and GC/MS to measure musks in sediments and shrimp.220 HHCB was detected in all shrimp measured, with levels of 48 to 683 ng/g lipid in farmed shrimp and 66 to 762 ng/g in wild shrimp, revealing very similar levels and widespread distribution. Sediment collected from 3 regions of the Chesapeake Bay showed HHCB up to 9.2 ng/g (dry weight). Finally, Reiner and Kannan measured polycyclic musks in river water, sediment, fish, and mussels from the Upper Hudson River, New York.221 HHCB and AHTN were found in river water (up to 25.8 ng/L), sediment (up to 544 ng/g dry weight), fish (up to 125 ng/g lipid weight), and zebra mussels (up to 65.9 ng/g lipid weight). Bioaccumulation factors of HHCB calculated for white perch, catfish, smallmouth bass, and largemouth bass ranged from 18 to 371 (on a wet weight basis) and 261 to 12 900 (on a lipid weight basis). ’ PESTICIDE TRANSFORMATION PRODUCTS Herbicides and pesticides continue to be the focus of much environmental research. Recent studies have focused more on their transformation products because their hydrolysis, oxidation, biodegradation, or photolysis transformation products can be present at greater levels in the environment than the parent pesticide and can be as toxic or more toxic. Several pesticide degradation products are on the U.S. EPA’s new CCL-3: alachlor ethanesulfonic acid (ESA), alachlor oxanilic acid (OA), acetochlor ESA, acetochlor OA, metolachlor ESA, metolachlor OA, 3-hydroxycarbofuran, and terbufos sulfone (http://water.epa. gov/scitech/drinkingwater/dws/ccl/ccl3.cfm), as well as on the UCMR-2 (alachlor ESA and OA, acetochlor ESA and OA, and metolachlor ESA and OA). LC/MS and LC/MS/MS are now common-place for measuring pesticide degradates, which are generally more polar than the 770

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Analytical Chemistry parent pesticides, making LC/MS ideal for their detection. In addition, researchers are increasingly using UPLC to enable simultaneous analysis of larger groups of pesticides and their degradation products, and TOF-MS and Q-TOF-MS are being used to identify new pesticide degradates. Botitsi et al. published a comprehensive review of MS strategies for analyzing pesticides and their metabolites in food and water matrixes.222 Sample preparation techniques included the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) approach, and various methods using UPLC and LC/MS/MS are nicely summarized. Methods include those applied to fruits, vegetables, milk, meat, eggs, honey, flour, rice, grains, baby foods, cereals, fruit juices, soft drinks, olive oil, wines, and other alcoholic beverages. Durand et al. used NMR, LC/NMR, and LC/MS as complementary techniques to investigate the biodegradation pathways of the herbicide mesotrione.223 The use of LC/NMR enabled the unambiguous identification of 6 metabolites, whereas only 4 metabolites were suggested by LC/MS. In addition, NMR was able to uncover a new metabolic pathway. Degradation mechanisms of the pesticide phoxim was the focus of another study by Lin et al., who used LC/MS/MS to identify the transformation products.224 Results showed a first-order degradation. UV irradiation and increased pH and temperature accelerated the degradation. Five intermediates were identified, and degradation pathways were proposed. Hydrolysis products of the pesticide dyfonate were the focus of another study by Wang et al., who used GC/MS to identify the transformation products, thiophenol and phenyl disulfide.225 Hydrolytic pathways were also proposed. Kern et al. compared model predictions to actual field data for 16 pesticides and 46 transformation products (TPs) measured in a small river draining an agricultural catchment in Switzerland.226 Twenty TPs were measured quantitatively using SPE-LC/MS/ MS, and the remaining 26 were detected qualitatively, due to the lack of reference standards. Comparison of predicted and measured exposure ratios for 20 pairs of TPs and parent pesticides showed agreement within a factor of 10, except for chloridazon desphenyl and chloridazon-methyl-desphenyl, which were found at elevated levels during baseflow conditions and in groundwaters across Switzerland. A model-based approach was proposed to identify TPs that exhibit a high aquatic exposure potential. Helbling et al. published a new high-throughput procedure for the elucidation of TPs for a broad and diverse group of pesticides.93 Samples coming from batch reactors seeded with activated sludge were separated by LC and analyzed by linear ion trap-Orbitrap-MS. TPs were tentatively identified using a postacquisition data processing method, which was based on target and nontarget screening of full-scan MS data, and structures were proposed by interpretation of MS/MS fragments. Using this procedure, new microbial TPs were reported. Results showed that the complementary use of target and nontarget screening allowed for more comprehensive identification of TPs. UPLCMS/MS was used by Benvenuto et al. for a new method to simultaneously determine triazine and its TPs in surface water and wastewaters.227 Confirmation of identity was achieved by acquiring 3 selected reaction monitoring (SRM) transitions and matching ion ratios, which was possible down to 0.025 μg/L. Finally, in the pan-European survey mentioned earlier by Loos et al., pesticide transformation products were included and were among the most frequently detected and highest concentration of the many analytes measured in European groundwaters.107

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For example, desethylatrazine and desethylterbutylazine were found in 55% and 49% of the samples, up to 487 and 266 ng/L, respectively.

’ PERCHLORATE Perchlorate became an important environmental contaminant following its discovery in a number of water supplies in the western United States. It has since been found in environmental waters across the United States and in other parts of the world at μg/L levels, as well as in fresh produce, foods, wines, and beverages from many countries, including those in Europe and the Far East. Perchlorate has also been found in biological samples, and it can be transported by pregnant mothers to their developing babies across the placental barrier. Perchlorate is increasingly being found in environmental waters following fireworks displays. As a result, it is now recognized as a worldwide environmental issue, rather than only being limited to the United States. Ammonium perchlorate has been used in solid propellants used for rockets, missiles, and fireworks, as well as highway flares. There is also potential contamination from fertilizers (e.g., Chilean nitrate, where perchlorate co-occurs naturally), and new work has revealed other natural sources of perchlorate. In addition, perchlorate can be a contaminant in sodium hypochlorite (liquid bleach) that is used in drinking water treatment. Perchlorate is an anion that is very water-soluble and environmentally stable. It can accumulate in plants (including lettuce, wheat, and alfalfa), which can contribute to exposure in humans and animals. In addition, perchlorate is not removed by conventional water treatment processes, so human exposure can also occur through drinking water. Health concerns arise from perchlorate’s ability to displace iodide in the thyroid gland, which can affect metabolism, growth, and development. Due to these concerns and due to the proportion of the U.S. population exposed to it, the U.S. EPA has now decided to regulate perchlorate under the Safe Drinking Water Act (http://water.epa. gov/drink/contaminants/unregulated/perchlorate.cfm). The regulation is currently being developed, and there is not a proposed maximum contaminant level (MCL) as of yet. Perchlorate was previously on the U.S. EPA’s earlier CCL lists (CCL-1 and CCL-2) and is now on the CCL-3. (http://water.epa.gov/ scitech/drinkingwater/dws/ccl/ccl3.cfm). Perchlorate was also included in the first UCMR (http://water.epa.gov/lawsregs/ rulesregs/sdwa/ucmr/data.cfm). The U.S. EPA established a reference dose of 0.0007 mg/kg/day, which translates to a drinking water equivalent level (DWEL) of 24.5 μg/L.1 Prior to this decision to regulate on a national basis, California had already issued a state regulation of 6 μg/L (in 2007) (www.cdph.ca.gov/ certlic/drinkingwater/Pages/Perchlorate.aspx), and several states had issued advisory levels, ranging from 1 to 18 μg/L (www.epa. gov/fedfac/documents/perchlorate_links.htm#state_adv). There are several EPA Methods for measuring perchlorate in water, including EPA Method 314.2 (2-dimensional ion chromatography (IC) with suppressed conductivity detection), EPA Method 331 (LC/ESI-MS/MS), and EPA Method 332 (IC/ESI-MS/MS). (www.epa.gov/safewater/methods/analyticalmethods_ogwdw. html; www.epa.gov/nerlcwww/ordmeth.htm). English et al. published an intriguing study of perchlorate exposure biomarkers in a highly exposed human population.228 Perchlorate contamination is particularly high in the Lower Colorado River, which serves as the sole source of irrigation water for California’s Imperial Valley and can be an important 771

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source of exposure to people living in this region. Urine was measured from people consuming locally grown produce in this region, as well as their drinking water and local produce. All but two of the water samples tested negative for perchlorate, but it was detected up to 1816 ppb in produce. Estimated doses ranged from 0.02 to 0.51 μg/kg body weight/day. The geometric mean was 70% higher than for the reference population. Although none of the exposures exceeded the U.S. EPA reference dose, 3 participants exceeded the acceptable daily dose used by the California Office of Environmental Health Hazard Assessment. Recent studies address perchlorate occurrence in drinking water. For example, Blount et al. investigated perchlorate, nitrate, and iodide intake through direct and indirect consumption of tap water.229 Median perchlorate levels measured in tap water were 1.16 μg/L, which were below the U.S. EPA’s DWEL of 24.5 μg/L. Significant correlations were found between perchlorate and nitrate. Using individual tap water consumption data and body weight, the median perchlorate dose attributable to tap water was 9.11 ng/kg-day. In another study, Wu et al. measured perchlorate in tap water, groundwater, surface waters, and bottled water from China.230 LC/MS/MS was used for measurement. Perchlorate was detected in 86% of the samples, with mean levels of 2.5, 3.0, 2.8, and 0.22 μg/L in tap water, groundwater, surface water, and bottled water, respectively. Infant formula was the focus of another study by Schier et al., who measured perchlorate in commercially available powdered infant formulas.231 Levels ranged up to 5.1 μg/L (bovine-based milk), 0.44 μg/L (soy-based milk), 0.93 μg/L (lactose-free milk), and 0.4 μg/L (“elemental milk”, consisting of amino acids). Results showed that the perchlorate reference dose can be exceeded when certain bovine-based milk formulas are ingested or when powdered infant formulas are reconstituted with perchlorate-contaminated water. Soybean sprouts, lotus root, and water dropwort were the focus of another perchlorate uptake study by Yang and Her in South Korea.232 IC/MS/MS was used for measurement. Perchlorate was detected in 91% of the soybean samples, up to 78.4 μg/kg dry weight. The highest perchlorate level in lotus root was 17.3 μg/kg and for water dropwort was 39.9 μg/kg. Jackson et al. evaluated the isotopic composition of natural perchlorate in the southwestern U.S. to understand its origins.233 Stable isotope ratios were measured for perchlorate (δ18O, Δ17O, δ37Cl) and associated nitrate in groundwater from the southern High Plains of Texas and New Mexico, the Middle Rio Grande Basin in New Mexico, unsaturated subsoil in the southern High Plains, and nitrate-rich deposits near Death Valley, California. Natural perchlorate in the southwestern U.S. displayed a wide range of isotopic compositions that are distinct from those reported previously from the Atacama Desert of Chile or for synthetic perchlorate. Death Valley samples indicated partial atmospheric formation via reaction with ozone. In contrast, perchlorate isotope ratios from western Texas and New Mexico indicated that they were affected by postdepositional oxygen isotope exchange. This study provides important new information on the possibility of divergent perchlorate formation mechanisms and isotopic exchange in biologically active environments.

nutrients (from agricultural runoff and wastewater discharges) have led to increased algal blooms and an accompanying increased incidence of shellfish poisoning, large fish kills, and deaths of livestock and wildlife, as well as illness and death in humans. Toxins produced by these algae have been implicated in the adverse effects. The most commonly occurring algal toxins are microcystins, nodularins, anatoxins, cylindrospermopsin, and saxitoxins. “Red tide” toxins are also often found in coastal waters. Microcystins and nodularins are hepatotoxic high molecular weight, cyclic peptide structures. Anatoxins, cylindrospermopsin, and saxitoxins are heterocyclic alkaloids; anatoxins and saxitoxins are neurotoxic, and cylindrospermopsin is hepatotoxic. “Red tide” toxins include brevetoxins, which have heterocyclic polyether structures and are neurotoxic. Microcystins (of which, more than 70 different variants have been isolated and characterized) are the most frequently reported of the algal toxins. The most common microcystins are cyclic heptapeptides that contain the amino acids leucine and arginine in their structures. Nearly every part of the world that uses surface water as a drinking water source has encountered problems with cyanobacteria and their toxins. Algal toxins were on the U.S. EPA’s previous CCLs (CCL-1 and CCL-2) in a general way, “cyanobacteria (blue-green algae, other freshwater algae, and their toxins”, and now, the CCL3 has specifically named three cyanobacterial toxins: anatoxin-a, microcystin-LR, and cylindrospermopsin for the new list (http:// water.epa.gov/scitech/drinkingwater/dws/ccl/ccl3.cfm). Several countries, including Australia, Brazil, Canada, France, Italy, Poland, and New Zealand, have guideline values for microcystins, anatoxin-a, and cylindrospermopsin (ranging from 1.0 to 1.5 μg/L). Many of these toxins have relatively high molecular weights and are highly polar. In 2010, a special issue of the journal Toxicon, on “Harmful Algal Blooms and Natural Toxins in Fresh and Marine Waters” included 14 papers on the exposure, occurrence, detection, toxicity, control, management, and policy.234 This issue is a must-read for the latest state-of-the science for algae and their toxins. Dorr et al. reviewed the occurrence, toxicity, and toxicological assays for microcystins in South American aquatic ecosystems.235 The acute poisoning of patients receiving hemodialysis in 1996 in Brazil was highlighted as the catalyst for the discovery of microcystins in South America. A dog poisoning in New Zealand was the focus of an investigation by Wood et al., who found microcystin-LR and other variants to be responsible.236 This was the first report of a benthic microcystin-producing species causing an animal death in New Zealand. LC/MS was used to measure the microcystins; no cylindrospermopsin, saxitoxins, or anatoxins were detected. Berry et al. examined the bioaccumulation of microcystins in fish following a cyanobacterial bloom in Mexico.237 Fish were obtained from local markets and small commercial catches during the bloom. LC/MS was used for measurement. All three species of fish bioaccumulated the microcystins, and toxin content correlated with trophic level. Detection in silversides and Goodea species was particularly relevant because both are consumed in their entirety, including the livers, which accumulate the microcystins. New methods continue to be developed for algal toxins. For example, Li et al. reported a new LC/ESI-MS/MS method for measuring the neurotoxin beta-N-methylamino-L-alanine (BMAA) in cyanobacterial isolates.238 Detection limits of 2 pg could be achieved. A new solid phase adsorption toxin tracking

’ ALGAL TOXINS Algal toxins (mostly cyanobacterial toxins produced from blue-green algae) are of increasing interest in the United States and in other countries around the world. Increased discharges of 772

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method called SPATT was created by Wood et al. to measure anatoxin-a and homoanatoxin-a in river water.239 Fifteen different adsorption substrates were screened for integrated, in situ extraction. Nine of the sorbents retained anatoxin-a at >70%, with powdered activated carbon (PAC) and Strata-X the best phases for the extraction bags. A 3-day field study in a river containing toxic benthic cyanobacterial mats was undertaken using PAC and Strata-X SPATT bags. Anatoxin-a and homoanatoxin-a were detected in all SPATT bags, whereas surface grab samples only allowed detection of these toxins in one of the samples collected. Ionic-liquid supported cloud point extraction was used for extracting microcystin-LR from water in a new method by Pavagadhi et al.240 1-Butyl-3-methylimidazolium hexafluorophosphate was used as the ionic liquid, and detection limits of 0.03 μg/L could be achieved. This method was subsequently tested on waters from reservoirs. Deleuze et al. created a new MALDI-TOF-MS method for microcystins.241 This method utilizes the reductive properties of the matrix 1,5-diaminonaphthalene, which can selectively reduce the carbon
carbon double bond of the seventh amino acid. A new UPLC/MS/MS method was created by Oehrle et al. for measuring the CCL cyanotoxins (microcystin-LR, anatoxin-a, and cylindrospermopsin) in a single analysis, in less than 8 min.242

’ MICROORGANISMS Outbreaks of waterborne illness in the United States and other parts of the world have necessitated improved analytical methods for detecting and identifying microorganisms in water and other environmental samples. Several microorganisms are included on the new CCL-3 (http://water.epa.gov/scitech/drinkingwater/ dws/ccl/ccl3.cfm). The U.S. EPA’s National Exposure Research Laboratory in Cincinnati has developed several methods for measuring microorganisms in water (www.epa.gov/nerlcwww). These include methods for Cryptosporidium, Giardia, E. coli, Aeromonas, coliphages, viruses, total coliforms, and enterococci. E. coli O157:H7 and H1N1 (swine flu) have captured a lot of attention recently because they have caused a number of outbreaks and deaths around the world. Traditional biological methods are often used for detection of microorganisms, including cell culture, immunological methods, polymerase chain reaction (PCR), and microscopic identification, but ESI and MALDI-MS methods are also often used. In a new review article, Sauer and Kliem outlined mass spectrometry tools for classifying and identifying bacteria.243 Seng et al. reviewed the use of MALDI-TOF-MS techniques for identifying microorganisms.244 In another review, Schneider and Riedel trace the historical development of environmental proteomics and summarize milestone developments for analyzing the structure and function of microbial communities.245 New methods include one by Whitehouse et al., who used PCR with ESI-MS to identify pathogenic Vibrio species in the aquatic environment of the former Soviet Republic of Georgia.246 Using this method, 9 different Vibrio species were detected in 41% of the 248 water samples collected in freshwater lakes. Topdown proteomics was used in another method by Wynne et al. for measuring bacteria with capillary-LC/Orbitrap-MS/MS.247 Using this method, not only were the representative proteins identified but also the target bacteria could also be placed in their correct phylogeny. In addition, this method provided strong

experimental evidence for correct, missing, and misannotated bacterial protein sequences. Fenselau et al. created a microwaveassisted acid cleavage method to improve the detection of human adenovirus by MALDI-MS.248 With this method, denaturization and proteolysis could be done in a single reaction and allowed peptide products to be profiled in