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Agricultural and Environmental Chemistry

At the Intersection of Urbanization, Water, and Food Security: Determination of Select Contaminants of Emerging Concern in Mussels and Oysters S. Rebekah Burket, Yelena Sapozhnikova, Shan S. Chung, and Bryan W Brooks J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05730 • Publication Date (Web): 10 Mar 2018 Downloaded from http://pubs.acs.org on March 11, 2018

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Journal of Agricultural and Food Chemistry

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At the Intersection of Urbanization, Water, and Food Security: Determination of Select

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Contaminants of Emerging Concern in Mussels and Oysters from Hong Kong

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S. Rebekah Burket,1 Yelena Sapozhnikova,2 J.S. Zheng3, Shan Shan Chung,3 Bryan W. Brooks1*

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Baylor University, Waco, Texas, USA; 2USDA Agricultural Research Service, Wyndmoor, PA,

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USA; 3Hong Kong Baptist University, Kowloon Tong, Hong Kong. *[email protected]

Department of Environmental Science, Center for Reservoir and Aquatic Systems Research,

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Table of Contents Graphic

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1 ACS Paragon Plus Environment


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ABSTRACT

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Aquaculture, which is growing 3-5 times faster than terrestrial agriculture, will play an important

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role to meet future global food production needs. However, over 80% of global sewage

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production is returned to the environment untreated or poorly treated. In developing nations,

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these non-traditional waters of diverse quality are being recycled for aquaculture, yet chemical

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residues are differentially studied. Here, we examined pharmaceuticals, pesticides, PCBs,

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PBDEs, PAHs and flame retardants in marine bivalves using isotope dilution LC-MS/MS and

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low pressure (LP)GC-MS/MS. Green-lipped mussels from the field and oysters from aquaculture

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net pens, which are harvested as food products, were collected adjacent to point source municipal

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wastewater and landfill leachate effluent discharges, respectively, in Hong Kong, the fourth most

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densely populated country in the world. Multiple classes of pharmaceutical, pesticides, PAHs

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and phosphorus-based flame retardants were detected at low µg/kg levels. Acceptable servings

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per week indicated minimal risk for a number of chemicals; however, such calculations could not

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be performed for a number of contaminants of emerging concern. Future efforts are needed to

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better understand contaminant influences on marine bivalve populations and aquaculture product

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safety, particularly in rapidly urbanizing regions of developing countries with limited

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infrastructure.

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Keywords: aquaculture, pharmaceuticals, contaminants of emerging concern, bivalves,

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bioaccumulation, urbanization

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INTRODUCTION

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By 2050, global population is expected to surpass 9.7 billion, with more than 6.3 billion people

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living in urban areas.1 Coupled with climate change, global economic and trade uncertainties,

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and competition for natural resources, food security for more than 9 billion people is listed as a

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high priority goal in the United Nations (UN) 2030 Agenda for Sustainable Development.2

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Aquaculture and fisheries will contribute greatly to global food security as important sources of

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food and nutrition. Capture fishery production has remained relatively constant since 1985, but

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marine fish stocks continue to decline due to overfishing, with over 30 percent of fish stocks

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estimated at biologically unsustainable levels in 2013.3 In 2014, aquaculture surpassed wild-

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caught fisheries for production of fish for human consumption the first time.3

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In fact, between 1961-2013, growth in global supplies of fish for human consumption

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have increased at an annual rate of 3.2 percent, which is faster than the 2016 population growth

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rate estimate of approximately 1.2 percent.3 4 World per capita fish consumption has increased

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from 9.9 kg in the 1960s to more than 20 kg presently due to increased production, reduced

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waste, improved distribution, and growing demand associated with population growth, rising

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incomes, and urbanization. With rapid urbanization occurring globally, the UN estimates predict

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nearly 70 percent of the population will live in urban areas by 2050.1 However, rates of

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urbanization vary by region, with Africa and Asia urbanizing more rapidly than other areas at 1.5

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and 1.1 percent per year, respectively.1 Urban environments are also variable ranging from

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smaller municipalities to megacities (pop. > 10 million). Close to half of all people in urban areas

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reside in peri-urban settlements, while just 12 percent live in one of 28 global megacities (pop. >

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10 million).1 Almost all future rapid urbanization is expected in medium-sized cities located in

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Africa and Asia.



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Megacities, along with rapidly urbanizing medium (pop. between 500,000 and 1 million)

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and large-sized (pop. between 5 and 10 million) cities represent unique challenges for sustainable

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development and food security, while protecting public health and the environment.5 In 2014,

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nearly half of the 28 global megacities were located in Asia.1 Rapid urbanization in these areas

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can strain infrastructure, resulting in variable delivery of essential environmental public health

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services, including access to potable water, solid waste disposal and wastewater treatment.5

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Annual municipal solid waste (MSW) generation varies widely based on income, with higher

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income countries producing the most waste annually. However, the rate of MSW generation is

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increasing most rapidly in urbanizing environments, with Asia predicted as the top producer of

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MSW by 2030.6 Organized and reliable MSW collection is important for the protection of public

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health and the environment; however, collection coverage varies greatly. In Asia, MSW

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collection coverage ranges from 50 to 90 percent, with varying rates of collection and techniques

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of disposal, storage and treatment of landfill leachates.6 Municipal wastewater treatment also

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varies globally, with 80 percent of all generated sewage released to ecosystems without any

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treatment or responsible reuse.7, 8 In 2013, 68 percent of generated sewage in Asia was

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untreated.7

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Growing demand for water and food in rapidly urbanizing environments can be mitigated

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with sustainable and responsible wastewater reuse.9 For example, both treated and untreated

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wastewater has been used in China for agriculture. In 2007, China reported wastewater reuse at

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over 14.8 million m3/day.10 Such observations are important for food safety because Asia

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accounts for nearly 89 percent of global aquaculture production, with over 61 percent occurring

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in mainland China alone.3 Demand for seafood and aquaculture products is increasing,

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particularly in Asia, and cultivation methods are often adjusted to accommodate higher organism


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density via the addition of antibiotics to feed stock. Wastewater reuse for agriculture and other

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purposes is driven by water scarcity and increased demand in densely populated areas. In

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developing and rapidly urbanizing areas, treated wastewater effluent from municipal wastewater

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treatment plants (WWTPs) and MSW landfill leachate are likely utilized in aquaculture, though

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the complete extent of use of these “nontraditional waters” for aquaculture is not known.

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Wastewaters and landfill leachates are important sources of anthropogenic contaminants

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like pharmaceuticals and personal care products, pesticides, polychlorinated biphenyls (PCBs),

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polycyclic aromatic hydrocarbons (PAHs), polybrominated diphenyl ethers (PBDEs), and other

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flame retardants.11, 12 This represents a critical consideration because the majority of global

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aquaculture production, and increasingly consumption, takes place in developing countries, yet

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research focused on aquaculture product safety, public health and the environment are often

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reported from developed countries, where aquaculture practices differ greatly. Herein,

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contaminant residues in aquaculture food products are not well studied, particularly in rapidly

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developing regions of Asia. The primary objective of the present study was to examine

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accumulation of traditional pollutants and contaminants of emerging concern in mollusks from

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along the coast of Hong Kong, the fourth most densely populated country in the world, near

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effluent discharges from municipal and landfill leachate treatment plants. We selected green-

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lipped mussels (Perna viridis) from Victoria Harbor and oysters (Crassostrea hongkongensis)

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from aquaculture operations in Deep Bay for a novel study of pharmaceuticals, pesticides, PCBs,

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PAHs, PBDEs and other flame retardants.

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METHODS



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Study site and sample collection

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Hong Kong is a densely populated megacity, with population densities as high as 57,250

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people/km2.13 Stonecutters Island Sewage Treatment Plant, a chemically enhanced primary

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treatment system, services 93% of the Hong Kong population, discharging up to 2.65 million

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m3/d to Victoria Harbor. P. viridis (n = 17) were collected dockside in Victoria Harbor, adjacent

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to the Stonecutters Island discharge location. The West New Territories (WENT) Landfill, one of

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the largest active landfills in Asia, is located on the Northwest side of Hong Kong and began

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operation in 1993. WENT landfill, one of three active landfills in Hong Kong, receives more

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than 7300 tons of municipal and other waste daily. Leachate is collected for treatment on site

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prior to submarine discharge to the North Western Water Control Zone adjacent to Deep Bay. C.

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hongkongensis (n = 7) were collected from an aquaculture platform adjacent to this point source

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landfill leachate effluent discharge.

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Chemicals

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All chemicals and their corresponding isotopically-labeled analogs were obtained from various

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vendors. Acetaminophen, acetaminophen-d4, amitriptyline, amitriptyline-d3, aripiprazole,

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aripiprazole-d8, benzoylecgonine, benzoylecgonine-d3, buprenorphine, buprenorphine-d4,

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caffeine, carbamazepine, carbamazepine-d10, diclofenac, diltiazem, diphenhydramine,

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diphenhydramine-d3, fluoxetine, fluoxetine-d6, methylphenidate, methylphenidate-d9,

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norfluoxetine, norfluoxetine-d6, promethazine, promethazine-d3, and sertraline were purchased

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as certified analytical standards from Cerilliant (Round Rock, TX, USA). Amlodipine,

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amlodipine-d4, caffeine-d9, desmethylsertraline, desmethylsertraline-d4, diclofenac-d4, diltiazem-

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d3, 13C-erythromycin, sertraline-d3, sulfamethoxazole-d4, trimethoprim and trimethoprim-d9


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were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). Erythromycin,

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sucralose, and sulfamethoxazole were purchased from Sigma-Aldrich (St. Louis, MO, USA) and

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sucralose-d6 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All

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chemicals were reagent grade and used as received. HPLC grade methanol (MeOH) was

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obtained from Fisher Scientific (Fair Lawn, NJ, USA), formic acid was purchased from VWR

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Scientific (Radnor, PA, USA), and a Thermo Barnstead™ Nanopure™ (Dubuque, IA, USA)

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Diamond UV water purification system was used throughout sample analysis to provide 18 MΩ

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water. Chemical standards and vendors for pesticides, PCBs, PAHs, PBDEs and other flame

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retardants have been previously described.14-16

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Pharmaceutical analysis - Sample preparation

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Tissue extraction methods in this study were similar to previously reported methods.17-19

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Collected bivalves were immediately separated from their shells, homogenized, and frozen at -

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20°C prior to shipment to Baylor University for extraction. Approximately 1 g of tissue

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homogenate (wet weight, ww) was transferred to a 20 mL borosilicate glass vial. Homogenizing

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solution (8 mL of a 1:1 mixture of 0.1 M acetic acid and MeOH) was added, along with 50 µL of

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deuterated internal standard mix. Samples were shaken and spun on a rotary extractor for 20 min

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at 25°C. Samples were transferred to 50 mL polypropylene copolymer round-bottomed

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centrifuge tubes (Nalgene Co., Nalgene Brand Products, Rochester, NY, USA) and centrifuged

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at 20000 rpm for 45 min at 4°C. The supernatant was transferred into 18 mL borosilicate glass

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culture tubes (VWR Scientific), and the solvent was evaporated under N2 gas at 45°C in a

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Turbovap evaporator. Once dried (approx. 4 h), samples were reconstituted with 1 mL 95:5 0.1%

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(v/v) formic acid:MeOH and filtered using Pall Acrodisc® hydrophobic Teflon Supor membrane



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syringe filters (13-mm diameter; 0.2-µm pore size; VWR Scientific, Suwanee, GA, USA) before

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analysis.

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Pharmaceutical analysis - Instrumental analysis

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Samples were analyzed for wastewater and landfill leachate indicators, including targeted

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pharmaceuticals, using isotope-dilution liquid chromatography-tandem mass spectrometry (LC-

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MS/MS) with an Agilent Infinity 1260 autosampler/quaternary pumping system, jet stream

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thermal gradient electrospray ionization source, and model 6420 triple quadrupole mass

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analyzer. A binary gradient method consisting of aqueous 0.1% formic acid as solvent A, and

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MeOH as solvent B, was used. Separation was performed using a 10 cm × 2.1 mm Poroshell 120

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SB-AQ column (120Å, 2.7 µm, Agilent Technologies, Santa Clara, CA, USA) preceded by a 5

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mm × 2.1 mm Poroshell 120 SB-C18 attachable guard column. The flow rate was held constant

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at 0.5 mL/min and column temperature was maintained at 60°C. The injection volume was 10

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µL. Analytes were ionized in positive and negative mode using electrospray ionization. MRM

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transitions for the target analytes and associated instrument parameters were automatically

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determined using MassHunter Optimizer Software by flow injection analysis.

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In the present study, method detection limits (MDLs) represented the lowest

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concentrations of an analyte that were reported with 99% confidence that the concentration is

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different from zero in a given matrix. The EPA guideline (40 CFR Part 136, Appendix B) for

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generating method detection limits was followed to generate MDLs. Corresponding MDLs and

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instrument limit of detection (LOD) can be found in SI Table 1. Quantitation was performed

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using an isotope dilution calibration method, as previously described.19 Calibration data were fit

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to a linear regression, and correlation coefficients (r2) for all analytes were ≥ 0.98. Quality


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assurance and quality control measures included running a continued calibration verification

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(CCV) sample every five samples to check calibration validity during the run, with an

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acceptability criterion of ± 20%. Multiple blanks (field, reagent, method) and duplicate matrix

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spikes were included in each analytical sample batch. All matrix spike recoveries fell between

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80% and 120%.

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Pesticide and other environmental contaminants analysis

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Sample preparation for pesticides, PCBs, PAHs, PBDEs and other flame retardants was based on

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QuEChERS extraction with acetonitrile (MeCN) as previously reported.14 Samples of oysters

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and mussels (0.7-2 g) were placed in 15 mL polypropylene tubes, internal standards were added,

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and the tubes were allowed to stand for 15 min. Reagent blank and reagent spikes (1.6 mL of

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water), replicated samples, replicated spiked samples (matrix spikes), and NIST Standard

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Reference Materials 1946 and 1947 were included in the batch. For extraction, 2 mL of

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acetonitrile was added, and the tubes were shaken on a vortex platform shaker for 10 min at 80%

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setting with maximum pulsing. For phase separation, MgSO4 (0.8 g) and NaCl (0.2 g) were

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added to the tubes, following by shaking for 1 min. The tubes were centrifuged for 3 min at 4150

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rpm. Aliquot of the extract (0.6 mL) was transferred to autosampler vials for cleanup with mini-

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SPE columns using robotic Instrument Top Sample Preparation (ITSP) installed on the top GC-

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MS/MS instrument. ITSP was performed as previously described,15 followed by low pressure

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(LP)GC-MS/MS analysis for 211 analytes, including pesticides, PCBs, PAHs, PBDEs and other

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flame retardants. Another aliquot of the extract (0.5 mL) was filtered through 0.2 µm PVDF

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filter vial and subjected to UPLC-MS/MS analysis for 107 pesticides (SI Table 2) as previously

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described.16



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Statistical Analyses

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Results were analyzed using SigmaPlot (Systat Software, San Jose, CA, USA). Tissue

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concentrations between green-lipped mussels and oysters were compared with a t test. If

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normality or equal variance assumptions were not met, a Mann-Whitney rank sum test was used.

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When the analyte concentration was below the MDL, a value of half of the MDL was assigned

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for statistical analysis.20 The Grubb’s test identified one green-lipped mussel sample as an

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outlier. Elevated data for this outlier was not included in statistical analyses, but is listed in SI

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Table 3.

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RESULTS AND DISCUSSION

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We selected Hong Kong for a unique study at the intersection of urbanization, water, and food

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security. We examined green-lipped mussels collected from the field near the discharge of a

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large wastewater effluent discharge. Unlike many regions in Asia where wastewater treatment is

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limited or absent, the Stonecutter Island wastewater treatment plant employs chemically

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enhanced primary treatment technology, which was not intended to significantly remove

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contaminants of emerging concern. We further examined oysters collected at an aquaculture

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facility located adjacent to leachate effluent discharge from one of the largest active landfills in

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Asia. Responsible management of solid wastes and associated leachates to groundwater and

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surface waters are particularly relevant in Southeast Asia. For example, China alone doubled e-

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waste production between 2010 and 2015.21 We thus chose a diverse group of target analytes

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representing consumer products and industrial chemicals for this novel shellfish accumulation

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study in the fourth most densely populated country in the world.


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In the present study, green-lipped mussels and oysters accumulated low levels of

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pharmaceuticals, including antidepressants (amitriptyline, fluoxetine, and sertraline),

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antihypertensives (amlodipine, diltiazem, and propranolol), antibiotics (erythromycin and

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trimethoprim), stimulants (caffeine and methylphenidate), an anesthetic and potential drug of

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abuse (ketamine), an anticonvulsant (carbamazepine), a nonsteroidal anti-inflammatory drug

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(diclofenac), and antihistamines (diphenhydramine and promethazine) (Table 1). Among

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pharmaceutical analytes examined, significantly (p < 0.05) higher levels of several compounds

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were observed in oysters from Deep Bay compared to green-lipped mussels from Victoria

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Harbor (Figure 1). For example, the antidepressants (mean ±SD) amitriptyline (0.2 ± 0.1 µg/kg

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ww), fluoxetine (0.2 ± 0.1 µg/kg ww) and sertraline (1.4 ± 0.4 µg/kg ww) were significantly (p

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