Occurrence of Triclocarban and Triclosan in an Agro-ecosystem

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Occurrence of triclocarban and triclosan in an agro-ecosystem following application of biosolids Jessica J. Sherburne, Amanda M. Anaya, Kim J. Fernie, Jennifer S. Forbey, Edward T. Furlong, Dana Ward Kolpin, Alfred M. Dufty, and Chad Alan Kinney Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01834 • Publication Date (Web): 07 Nov 2016 Downloaded from http://pubs.acs.org on November 14, 2016

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Environmental Science & Technology

119x81mm (150 x 150 DPI)

ACS Paragon Plus Environment


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Occurrence of triclocarban and triclosan in an agro-ecosystem

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following application of biosolids

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Jessica J. Sherburne §, Amanda M. Anaya ҂, Kim J. Fernie ∞,§, Jennifer S. Forbey §, Edward T.

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Furlong †, Dana W. Kolpin ≡, Alfred M. Dufty §, Chad A. Kinney* , ҂.

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§

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83725

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҂

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∞

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and Climate Change Canada, 867 Lakeshore Rd., Burlington, ON CANADA L7S 1A1

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Department of Biological Sciences, Boise State University, 1900 University Dr., Boise, ID

Department of Chemistry, Colorado State University, 2200 Bonforte Blvd., Pueblo, CO 81001 Ecotoxicology and Wildlife Health Division, Science and Technology Branch, Environment

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†

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95, Denver, CO 80225

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≡

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* Correspondance:

U.S. Geological Survey, National Water Quality Laboratory, Denver Federal Center, Building

U.S. Geological Survey, 400 S. Clinton St., Iowa City, IA 52240 Email : [email protected]

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Tel : (719) 549-2600

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Mail : Colorado State University-Pueblo

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Chemistry Department

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2200 Bonforte Blvd

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Pueblo, CO 81001

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ABSTRACT

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Triclocarban (TCC) and triclosan (TCS), two of the most used antimicrobial compounds, can be

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introduced into ecosystems by applying wastewater treatment plant biosolids to agricultural

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fields. Concentrations of TCC and TCS were measured in different trophic levels within a

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terrestrial food web encompassing land-applied biosolids, soil, earthworms (Lumbricus), deer

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mice (Peromyscus maniculatus), and eggs of European starlings (Sturnus vulgaris) and

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American kestrels (Falco sparverius), at an experimental site amended with biosolids for the

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previous seven years. The samples from this site were compared to the same types of samples

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from a reference (biosolids-free) agricultural site. Inter-site comparisons showed that

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concentrations of both antimicrobials were higher on the experimental site in the soil,

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earthworms, mice (livers), and European starling eggs (TCC: 15.4-31.4 ng/g), but not American

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kestrel eggs (TCC: 3.6 ng/g) compared to the control site. Inter-species comparisons on the

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experimental site indicated significantly higher TCC concentrations in mice (TCC: 12.6-33.3

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ng/g) and in starling eggs (TCC: 15.4-31.4 ng/g) than in kestrel eggs (TCC: 3.6 ng/g). Nesting

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success of kestrels only was significantly lower on the experimental site compared to the

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reference site due to nest abandonment. This study demonstrates that biosolids-derived TCC and

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TCS are present throughout the terrestrial food web, including secondary (e.g., starlings) and

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tertiary (i.e., kestrels) consumers, after repeated, long-term biosolids application.

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INTRODUCTION Triclocarban (TCC) and triclosan (TCS) are two widely used antimicrobial compounds

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that have been used in personal care products for decades.1 The accumulation of these

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antimicrobials in biosolids produced at wastewater treatment plants (i.e. treated sewage sludge),

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and subsequently environmental systems, raises concern for both humans and wildlife. In the

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terrestrial environment, TCC and TCS have been observed to accumulate in plants and

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earthworms inhabiting soils amended with biosolids, and the overuse of antimicrobial products is

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reported to result in the presence of antibiotic resistant bacteria in soil.2, 3, 4,5

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Both TCC and TCS are antimicrobial chemicals introduced into the environment

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primarily through human activities. When products containing TCC or TCS are used, the

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antimicrobials wash into sewer systems and enter the wastewater treatment process. TCC and

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TCS are incompletely removed during such wastewater treatment processes.6 Much of the

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removal can be attributed to partitioning of TCC and TCS to carbon-rich biosolids during

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treatment.1,4 As a result, relatively high concentrations of TCS (30,000 ± 11,000 ng/g) and TCC

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(50,000 ± 15,000 ng/g) are frequently detected in biosolids.7,8

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In the United States, approximately 8 million dry metric tons of biosolids are produced

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annually, about half of which is applied to land as fertilizer, and the other half is either

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incinerated or deposited in landfills.9 Biosolids are classified as treated municipal solid waste

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that must meet regulatory standards primarily for pathogen and metal content, but are not

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regulated or monitored for TCC and TCS concentrations.10 When biosolids containing TCC and

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TCS are applied to agricultural fields, both antimicrobial compounds have been detected in the

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soil.4 Several studies demonstrate that the fate of TCC and TCS includes accumulation at lower

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trophic levels in the ecosystems exposed to biosolids including food crops (e.g., soybean), 3


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meadow fescue (Festuca pratense) and earthworms (Lumbricus).3,11,12 TCC and TCS have been

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detected in samples from organisms at higher trophic levels, including aquatic vertebrates and in

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the breast milk, plasma, and urine of humans.13,14

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In addition to concerns raised by the detection of antimicrobials across trophic levels,

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elevated concentrations of antimicrobials can alter the physiology of animals. Exposure to TCS

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resulted in a decrease in total serum thyroxine concentrations in Long-Evans female rats (Rattus

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norvegicus) and altered skeletal muscle function in fat head minnows (Pimephales

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promelas).15,16 Elevated TCC and TCS concentrations have also been shown to be highly toxic to

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Japanese rice fish (Oryzias latipes) during their early life stages, and in the presence of

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testosterone, can enhance androgenic effects in these fish.17

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Despite known accumulation in ecosystems and the negative physiological consequences

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of exposure to TCC and TCS in some organisms, these antimicrobials have not been adequately

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studied in higher trophic levels in natural systems. For the purposes of this study, we investigated

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a terrestrial food web encompassing biosolids; soil; earthworms (primary consumer); deer mice

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(Peromyscus maniculatus) (secondary consumer); European starlings (Sturnus vulgaris), a

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secondary consumer of invertebrates; and American kestrels (Falco sparverius), a tertiary

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consumer of rodents, small birds, and invertebrates.18,19 The objectives of this study were to (a)

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determine the concentrations of the antimicrobials, TCC and TCS, in biosolids, soil, earthworms,

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deer mice, and eggs of the European starling and the American kestrel collected from a

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biosolids-amended experimental agricultural site and a reference, biosolids-free, agricultural site,

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and (b) determine if there were correlative relationships between concentrations of TCC, TCS,

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and reproductive endpoints, specifically egg viability and nesting success, of the two avian

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species examined. We hypothesized that substrates and organisms at the experimental site would 4



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contain higher concentrations of TCC and TCS than those from the reference site due to the

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application of biosolids. We also hypothesized that kestrels would have higher concentrations of

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TCC and TCS than starlings because they occupy a higher trophic level than starlings. In

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addition, we hypothesized that exposure to TCC and TCS would be associated with lower egg

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viability and lower nesting success by these birds. To our knowledge, this is the first study that

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focuses on the presence and potential consequences of TCC and TCS in higher trophic levels of a

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terrestrial food web.

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EXPERIMENTAL

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Study Areas

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The study area consisted of an experimental (i.e. biosolids applied site) and reference site

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(i.e biosolids free site). These sites were separated by approximately 10 km. The reference site is

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located near Nampa, Idaho in Ada and Canyon Counties (43°32’83” N, 116°27’31”W, elevation

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797-m, Fig. S1a and b). This reference site consisted of an area of 85-km² including agricultural

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and residential development that has never received biosolids according to county records.

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Within the reference site, we monitored 74 wooden nest boxes (Fig. S1b) mounted at

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approximately 2.5-m high on poles, 20 of which were used by American kestrels and 22 by

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European starlings over two field seasons. The experimental site was a 16-km², municipal-owned farm located near Kuna, Idaho in

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Ada County (43°23’38”N, 116°17’87”W, elevation 866-m, Fig. S1a and c). The experimental

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site currently uses dewatered and anaerobically digested municipal biosolids from the Ada

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County Wastewater Treatment Facility (WWTF) as fertilizer for alfalfa, corn, and winter wheat 5


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used for livestock feed. The Ada County WWTF treats wastewater primarily by secondary

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treatment processes from Boise, Idaho (population: 205,671), a city that produces approximately

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1.1 x 108 L of wastewater per day. When this study was conducted, the experimental site had a

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continuous, seven-year history of biosolids application for certain plots within the site. All fields

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within the experimental site were tested for heavy metals. When concentrations of heavy metals

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were below county standards, biosolids were applied to these fields. Biosolids were not applied

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to all fields every year, in response to annual variation in heavy metal content measured in soil.

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Biosolids were applied at a rate of approximately 3 tons per acre (673 kg/1000 m2, 1/4 inch-thick

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layer covering approximately 50% of the soil surface), and then were incorporated via disc

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harrow into the soil to a depth of 20 cm. Given the variation in history of biosolids application

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on the experimental site, we selected and sampled fields receiving biosolids representative of the

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median historical number of applications for the experimental site. There were 20 nest boxes

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located on the experimental site that were within 1-km of the plots amended with biosolids. Of

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the 20 nest boxes, five were used by American kestrels and six were used by European starlings.

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Biosolids. A composite biosolids sample, consisting of nine independent samples weighing 225-

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g each, was collected in March 2012 from one of the two open-air concrete holding areas at the

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experimental site prior to application of the biosolids to the agricultural fields (Fig. S1c).

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Biosolids holding areas store large volumes of biosolids, and each holding area contained

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biosolids from the same source. Biosolids sampling involved using a clean shovel to break the

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thick outer-crust on the biosolids, and then using a hexane-rinsed stainless steel spoon to collect

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each sample. Each collected sample was spaced two meters apart. Samples were pooled,

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homogenized to be representative of that applied on the fields, and stored in glass jars sealed

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with foil-lined plastic lids at -20°C until analysis. This protocol is in accordance with the 6



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regulated collection protocol used by the municipal-owned farm.20

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Soil. Soil from the experimental site was also sampled in March 2012 prior to application of the

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biosolids, and was resampled in May 2012 approximately two weeks after the biosolids had been

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applied and incorporated into the soil. Soil from the reference site was sampled in March 2012,

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and was resampled in June 2012 to correspond with the sample collections from the experimental

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site. Areas where soil was sampled at each site were chosen primarily to maintain a consistent

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soil type, (fine, silty, mixed-mesic, Xerollic Haplargids)21, and secondarily were within 1-km of

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nest boxes used by breeding American kestrels and European starlings (Fig. S1b and c). Three

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samples within the experimental site (N = 3) or reference site (N = 3) were collected at a depth of

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20-cm and approximately 40-m apart (Fig. S1b and c).20 The three samples within each site were

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subsequently pooled and mixed with a hexane-rinsed stainless steel spoon to create a single

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sample that was representative of the soil throughout each site. A total of 225-g of each pooled

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sample was retained and placed in a glass jar with a foil-lined plastic lid and was stored at -20°C

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prior to analysis.

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Earthworms. Earthworms (Lumbricus), a common food source for European starlings and deer

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mice, were collected from the experimental and reference sites in June 2012 after biosolids

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application had occurred and following a suitable exposure period (Fig. S1b and c). We used a

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composite sample of earthworms as representative of that consumed by the starlings.

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Approximately 30 g of earthworms were collected from the experimental and reference sites in

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the same general areas that soil samples were collected. Earthworms were pooled within each

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site, brought back to the lab, and rinsed with deionized water to remove soil residues from the

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surface of the earthworms. The earthworms were not allowed to void their guts prior to storage

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and analysis so as to be representative of earthworms consumed by higher trophic organisms. 7


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The pooled earthworms were then weighed, placed in glass jars with foil-lined plastic lids, and

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stored at -20°C prior to analysis.

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Deer Mice. Deer mice, a common food source for American kestrels, were collected in June of

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2012 (Boise State University IACUC permit no. #006-AC11-005).19 Kill-traps, located

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approximately 100 m from the nearest nest box, were used to collect 11 deer mice from the

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reference site and 28 deer mice from the experimental site. Mice were collected and stored intact

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in glass jars with foil-lined plastic lids at -20°C until dissection. Deer mice were thawed,

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weighed, and sexed before dissection. The liver and skeletal muscles were excised from each

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individual. Each tissue was individually weighed, wrapped in aluminum foil, placed in glass

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vials, and stored at -20°C prior to analysis. Of the 39 mice samples collected, matched liver and

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muscle tissues from the same 10 mice (N = 7 experimental site; N = 3 reference site) were

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analyzed for TCC and TCS concentrations.

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Eggs. As it is difficult to determine the order in which eggs have been laid, one egg was selected

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at random from each complete clutch of ≥ 3 eggs from randomly selected nests of the starlings

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and kestrels on the experimental and reference sites during the spring of 2011 and 2012 (Boise

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State University IACUC permit no. #006-AC11-005). All egg collections occurred during the

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same time period using the same protocol on both the experimental and reference sites. The eggs

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were stored at 5°C until they were prepared for freezing. Of the eggs collected, only eggs

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collected in 2012 were used for the measurement of TCC and TCS content to coincide with

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collection of other samples. Ten kestrel eggs (5 per site) and 12 starling eggs (6 per site) were

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analyzed for TCC and TCS concentrations.

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Egg Measurements. Each egg was weighed to the nearest g, and the length and breadth

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measured to the nearest mm using a Reid digital caliper (model # K309A.W-1230). Eggs were 8



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dissected along the equator and the mass of the egg contents was measured. Egg contents were

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stored at -60°C in sterilized glass vials with aluminum foil lined lids. After these egg

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measurements were collected, egg shells were boiled (7 mins for American kestrel eggs, 4-mins

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for European starling eggs) in deionized water to remove the inner membrane. Egg shells were

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allowed to dry and then egg shell mass was measured. Egg shell thickness was measured using a

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dial indicator gage mounted on a bench comparator.22 Egg volume of the American kestrel was

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calculated using length (L) and breadth (B) measurements in the equation:

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(0.6057 − 0.0018B)* L(B²)

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(1)

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Egg volume of the European starling was calculated using length (L) and breadth (B)

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measurements in the equation:

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0.035+ 0.530* (L*(B²)) 24

(2)

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Nest Success. All nests were monitored on a weekly basis and the number of eggs and nestlings

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were recorded. Nest box occupancy rates by species were calculated and compared between the

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two study sites. We used the standard nesting success assessment protocol to classify nest

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success.25 A nest was considered to be successful when at least one nestling within the brood had

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reached 80% of the average age of when nestlings fledge from their nest; for American kestrels,

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this would be when at least one chick within the brood had reached 22 d of age, while for

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European starlings, it would be 16-17 d of age.

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Biosolids, Soil, and Earthworm Extraction of TCC and TCS. Following Kinney et al. 12,26,

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biosolids, soil, and earthworm samples were prepared in triplicate for TCC and TCS

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quantification using pressurized liquid extraction (PLE; Dionex-ASE100, Dionex Corp.,

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Sunnyvale, CA) followed by quantitative analysis by liquid chromatography/mass spectrometry 9


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(LC/MS, Shimadzu LCMS 2010A). Method performance data are available in Table S1. TCC

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and TCS concentrations reported for earthworms, mice liver and muscle, and bird eggs are

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reported on a wet weight basis.

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Deer Mouse Tissue Extraction for TCC and TCS. The method for extracting TCC and TCS

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from liver and muscle tissue was developed to incorporate sample extraction and clean up into a

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single pressurized liquid extraction (PLE) step27 and analyzed using isotope dilution LC/MS

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analysis. For the extraction of liver and muscle tissue, the high pressure extraction cell (66-mL)

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was loaded with silica (7 g, 7:1 silica:tissue ratio by mass). Tissue samples were first

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homogenized with Na2SO4. The homogenate tissue sample was then placed on top of the sorbent

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and the isotope-labeled internal standards (50-µL each of 2-µg/mL 13C13-TCC and 13C12-TCS

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solutions) were added. Any remaining void volume was filled with ashed Ottawa sand (400 °C, 4

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hr). TCC and TCS were extracted from the liver homogenate using a 1:1 dichloromethane:ethyl

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acetate solution. TCC and TCS was extracted from the muscle homogenate using a 3:1:1

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dichloromethane:acetonitrile:methanol solution. All tissue homogenates were extracted by PLE

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at 100°C and 10,300 kPa for 1 static cycle at 5 min. The resulting extracts were evaporated to

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dryness under nitrogen at 70°C (Labconco RapidVap N2), and then reconstituted with 1 mL of

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acetonitrile:water (75:25). The samples were stored at 5°C until analysis by LC/MS. As a

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measure of quality control, spiked (TCC and TCS fortified ashed sand) and blank (ashed sand

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only) samples were extracted and included with each set of 10 tissue samples.

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Egg Extraction for TCC and TCS. Egg samples were extracted using a modified liquid-liquid

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extraction method prior to quantification using isotope-dilution LC/MS analysis. Approximately

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3-g of egg was homogenized by blender and transferred to an ashed glass centrifuge tube.

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Isotope-labeled internal standards (50-mL each of 13C13 TCC, 2 µg/mL-1; 13C12TCS, 2 µg mL-1) 10



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were added to the homogenized egg sample. TCC and TCS were extracted from the egg

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homogenate using, 6 mL of a 75:25 dichloromethane:methanol solution. The sample-solvent

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mixture was vigorously mixed by hand. After mixing, the sample was centrifuged (5000 rpm for

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5-min) to facilitate separation of the solvent and egg. The solvent layer was removed and

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transferred to ashed evaporation glassware and evaporated to dryness at 70°C under a gentle

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stream of nitrogen (Labconco RapidVap N2). The extract was reconstituted with 6 mL of

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acetonitrile. To limit the presence of lipids in the final extract, the sample was washed with 3.5

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mL of n-hexane. Following addition of the n-hexane, the sample was placed on a vortex mixer

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for 30 s. The hexane layer was removed and discarded. The extract was again evaporated to

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dryness at 70°C under nitrogen and reconstituted with 1 mL of 75:25 acetonitrile:water. As a

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measure of quality control, spiked (“organic” chicken egg fortified with TCC and TCS) and

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blank (“organic” chicken egg) samples were prepared and analyzed with every set of 10 egg

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samples.

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Analytical QA/QC. At least one method spike and method blank sample were evaluated for

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each set of extractions and quantifications.12,26 No detectable quantitites of TCC and TCS were

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observed in any of the method blanks analyzed in this study. Isotope-labeled internal standards

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were added to all samples, spikes, and blanks to correct for any differences in sample volume, as

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a marker of retention time for TCC and TCS, and to correct for deviations in method recoveries.

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In addition, the ratio of multiple characteristic ions for each compound and chromatographic

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retention times were compared to authentic standards for compound verification. The method

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performance for soil, biosolids, and earthworms samples had been previously assessed, including

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recovery of spiked sample (method and matrix spikes) as well as statistically determined method

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detection limits for each method.5,12,26 Method recovery and method detection limits for existing

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methods and those developed as part of this project are included in Table S1.

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Liquid Chromatography/Mass Spectrometry Instrumental Analysis. TCC and TCS were

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quantified by high-performance liquid chromatography coupled with electrospray

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ionization/quadrupole mass spectrometry (HPLC/ESI/MS, Shimadzu 2010A LCMS, Shimadzu

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Scientific, Columbia, MD 21046) operated in the negative ion mode using selected-ion

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monitoring to improve sensitivity and minimize chemical interferences. Sample components

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were separated using a Synergi 4-um hydro-RP 80A 50-mm x 4.6-mm column (Phenomenex).

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Biota-to-Soil Accumulation Factors. Biota-to-soil accumulation factors (BSAFs) for TCC and

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TCS in the organisms included in this study were calculated as the ratio of the lipid normalized

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concentration of TCC or TCS in the organism to the carbon normalized concentration of TCC or

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TCS in the soil at the experimental site. The total lipid content in the earthworms and deer mice

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liver and muscle tissues were determined using a method described previously.28 Briefly,

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homogenized samples were extracted using 2:1(v:v) chloroform:methanol. The extract mixture

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was centrifuged to promote solvent separation. The chloroform layer was removed and filtered

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through a 0.45 µm syringe filter. The total lipid content was determined gravimetrically. The

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total lipid content of the starling and kestrel eggs was determined using a method described

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previously29, which is similar to that used for the earthworms and deer mice tissues. Briefly,

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about 1 g of homogenized egg sample was extracted using 2:1 (v:v) chloroform:methanol

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overnight at 4°C. The extract was filtered through a 0.45 µm syringe filter, and 4 mL of a 0.88%

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NaCl solution was added. Following phase separation the chloroform layer was collected and the

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total lipid content was determined gravimetrically.

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Statistical Analysis. Positive detection of TCC and TCS measured in starling eggs, and kestrel

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eggs at concentrations that were less than the method detection limit (< MDL) were assigned a

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value of ½ the MDL (TCC: 1.35 ng/g ww for eggs; TCS: 1.8 ng/g ww for mice liver and 1.65

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ng/g ww for eggs) for statistical purposes only.30 Residuals were analyzed using a Shapiro-Wilks

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Normality test and were not normally distributed, nor could be log-transformed. Therefore,

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Kruskal-Wallis non-parametric ANOVA tests were performed comparing TCC and TCS

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concentrations between the reference and experimental sites within each bird species (intra-

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species comparisons) to identify differences relating to the applications of the biosolids (e.g.,

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comparing TCC concentrations in starling eggs from the reference site to starling eggs from the

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experimental site). Kruskal-Wallis ANOVAs were also used to compare TCC or TCS

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concentrations among species on the same site (e.g., kestrel eggs vs. starling eggs on the

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experimental site), to identify possible inter-species differences in exposure to these chemicals.

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When significant differences were found (p < 0.05) among the three species (mice, starlings,

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kestrels), Wilcoxon post hoc comparisons were performed. A contingency table was used to test

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for site differences in occupancy rates and nesting success of each species in 2011 (both avian

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species), and repeated for kestrels breeding in 2012. Spearman’s rank correlations were used to

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identify significant correlations between TCC or TCS egg concentrations and egg size

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parameters collected in 2012. All statistical analyses were performed using SAS 9.4® except for

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the contingency tables that were analyzed using RStudio® (version 2013, R Core Development

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Team). We report median, mean, range, and standard error of mean (SEM), when appropriate,

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and statistical significance was considered to be p ≤ 0.05.

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RESULTS AND DISCUSSION

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TCC and TCS Concentrations.

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In general, the application of biosolids to agricultural fields on the experimental site

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yielded higher concentrations of TCC and/or TCS in soil, earthworms, the liver of deer mice, and

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starling eggs than the reference site (Table 1); this pattern, however, was not evident with the

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kestrel eggs. In the current study, the concentrations of TCC (1026 – 1472 ng/g ww) and TCS

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(1114 – 1350 ng/g ww) in the biosolids collected prior to application were generally lower than

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concentrations reported in most comparable studies where biosolids were applied to agricultural

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soil.12,31,32 Although it is unknown if these concentrations are representative of TCC and TCS in

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the biosolids applied at the experimental site in previous years, Pycke et al.33 demonstrated little

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variation in TCC and TCS concentrations in biosolids from by a single WWTF over more than

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one year of sample collection. Correspondingly, the concentrations of TCC (14.8-27.3 ng/g ww)

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and TCS (2.7-4.4 ng/g ww) in the soil collected from the experimental site for this study were at

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the lower range of concentrations reported for biosolids-amended soils. 12,31,32 The lower

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concentrations of TCC and TCS in the biosolids and soils in this study compared to other similar

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studies may result from differing inputs of antimicrobials and treatment methodologies among

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WWTFs, differential field application rates of biosolids, conditions of uptake or degradation, and

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different environmental conditions among studies.

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Concentrations of TCS detected in earthworms from the experimental site in this study

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(5.9 – 42.8 ng/g ww) were 34-fold lower than concentrations detected in earthworms in a

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previous study,12 which likely reflects the lower concentrations of TCS in the biosolids and soils

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in this study compared to previous research. TCC concentrations in earthworms at the

308

experimental site in this study (5.1 ng/g ww) was 5 to 26 times lower than TCC previously 14



Page 16 of 41

309

reported in Eisenia foetida in a laboratory exposure study likely reflects lower TCC in the

310

biosolids applied at the experimental site in this study compared to the laboratory exposure

311

study.4 In this study, the earthworms were not depurated as to represent the potential exposure to

312

TCC and TCS available to predators that consume earthworms (e.g., starlings). Here, the

313

concentrations of TCS in the earthworms exceeded what was measured in the soil at the

314

experimental site, while the opposite pattern occurred with TCC.

315

Both TCC (20.0 ± 4.6 ng/g) and TCS (5.0 ± 1.2 ng/g) were detected in the deer mice

316

livers but not their muscle, and only in mice from the experimental site (Table 1). Since TCC

317

and TCS are lipophilic, it is probable that very low, undetectable concentrations of both

318

antimicrobials occurred in the muscle of these mice, but at concentrations below the limits of

319

detection. The lipid content of deer mice liver was greater than deer mice muscle tissue (Table

320

S2). There is limited, if any, information available for TCC or TCS concentrations measured in

321

the tissues of free-ranging small mammals, including mice exposed to biosolids. Assuming

322

toxicity thresholds would be similar between free-ranging mice and laboratory rodents, the TCS

323

hepatic concentrations measured in the deer mice during this study (6.6-10.7 ng/g) are likely

324

below the threshold for disrupting thyroid function that occurred in laboratory rats dosed with

325

TCS concentrations of 35.6 to 300 mg/kg.15,34 To our knowledge, this is the first study to detect

326

TCC and TCS in wild rodents and suggests that accumulation of these antimicrobials by small

327

mammals is a potential route of exposure to predators foraging on them (e.g., American kestrels).

328

Overall, it is likely that total exposure to TCC and TCS accumulation of these compounds is

329

underestimated in this work because common metabolites/degradation products of TCC and TCS

330

known to be present in biosolids were not included as analytes in this project.33

331 15


Page 17 of 41

332


Bioaccumulation of TCC and TCS.

333

In an effort to assess the bioaccumulation of TCC and TCS in the biological samples

334

included in this study, biota-to-soil accumulation factors (BSAFs) were calculated (Table 2).

335

With the exception of the BSAF for TCS in earthworms and starling eggs, all of the BSAFs are

336

below a value of 1. This may suggest that direct exposure to soil is not a major source of TCC

337

and TCS to organisms in this study other than earthworms and possibly starlings. The BSAFs for

338

TCC (0.79) and TCS (67) in earthworms are estimates because the earthworms in this study were

339

not depurated prior to TCC and TCS analysis. However, the concentration reported for TCS in

340

the earthworms is substantially greater than that reported for the soil, which suggests any TCS in

341

the gut contents are a minor component of TCS reported for earthworms. These estimated

342

BSAFs for TCC and TCS in earthworms suggests greater bioavailability of TCS in soils

343

compared to TCC. A comparison of BSAFs for TCC and TCS for the organisms analyzed in this

344

study to other studies is limited. BSAFs for TCC in earthworm Eisenia foetida exposed to

345

biosolids amended soils was previously reported to rage from 0.22 to 0.71.4 The BSAF for TCC

346

in earthworms of 0.79 in this study is in similar to those previous reported. In addition, a

347

comparison of the bioaccumulation factor (BAF; ratio of concentration in the organism to the

348

concentration in the soil) for TCS in earthworms can be made with previously reported values. In

349

this study, the BAF for TCS in earthworms can only be estimated because the earthworms by

350

design of this study were not depurated. The estimated BAF for TCS (15.9) is within the range

351

previously reported (BAF = 10.9 – 40.1) for earthworms in biosolids amended soils and

352

demonstrates a similar relative uptake of TCS by earthworms at this field site.35

353 354

Assessing Intra-Species Differences in TCC and TCS concentrations: Avian eggs. 16



Page 18 of 41

355

Generally, concentrations of TCC and TCS were detected in the eggs of both avian

356

species collected from the experimental and reference sites; notably, TCS occurred at measurable

357

concentrations in only one starling egg from the reference site, and TCC concentrations were

358

below measureable concentrations in most of the kestrel eggs from both sites (Table 1). The

359

detection of TCC and TCS in the starling and kestrel eggs from the reference site may reflect the

360

mobility of the birds when foraging and/or the general ubiquitous occurrence of these

361

antimicrobials in the environment.

362

Intra-specific site comparisons indicated that the starling eggs from the experimental site

363

had significantly higher concentrations of TCC than those on the reference site (X2 = 3.7 df = 1 p

364

= 0.054, Fig. 1). Although the concentration of TCS in starling eggs at the experimental site

365

exceeds the concentration of TCS detected in a single egg from the reference site by a factor of

366

1.6 to 6.5 times, the difference in concentrations is not statistically significant (p = 0.32) (Table

367

1). The eggs of American kestrels collected from the experimental site had similar concentrations

368

of TCS (p = 0.71), and very low levels of TCC (p = 1.0), as the kestrel eggs from the reference

369

site (Fig. 1). The fact that starling eggs, but not kestrel eggs, had measurable and higher TCC and

370

TCS concentrations on the experimental site may be related to differences in feeding strategies

371

and prey choice between the two species. Starlings tend to forage in the same general area for

372

most of the year, including directly in agricultural fields.34 In contrast, kestrels forage more

373

frequently in fallow land adjacent to agricultural fields, and their territory size is more variable.19

374

Consequently starlings may more consistently feed in agricultural areas amended with biosolids

375

throughout the year compared to kestrels.

376 377

Assessing Inter-Species Differences in TCC and TCS concentrations 17


Page 19 of 41

378


TCC and TCS concentrations were also compared between the two avian species on the

379

experimental site, and then similarly on the reference site. The concentrations of TCS were

380

similar in the kestrel and starling eggs on both study sites (p ≥ 0.12). The concentrations of TCC

381

in the kestrel eggs were significantly lower than the starling eggs collected on the experimental

382

site (X2 = 7.54 df = 1 p = 0.02) (Fig. 1), and marginally lower on the reference site (X2 = 3.68 df

383

= 1 p = 0.06). Starlings insert their beak into the ground to retrieve various invertebrates and

384

other prey36, and in so doing, they may simultaneously ingest soil particles containing TCC. In

385

contrast, when feeding or capturing prey, kestrels do not come into direct contact or ingest soil.

386

That a similar, clear pattern was not observed with TCS may reflect the lower TCS

387

concentrations in the soil at the experimental site compared to TCC. Alternatively, because

388

species can vary in rates of metabolism,37 it is also possible that kestrels metabolize or eliminate

389

antimicrobials more efficiently than European starlings.

390

The inter-species comparisons were expanded to include mice as well as the starling and

391

kestrel eggs, but this was only possible on the experimental site since TCC and TCS were not

392

detected in mice on the reference site (Fig. 1; Table 1). On the experimental study site, TCS

393

concentrations remained similar among the mice, starling eggs, and kestrel eggs (p = 0.13), while

394

concentrations of TCC differed significantly among the three species overall (X2 = 7.54 df = 2 P

395

= 0.023). The TCC concentrations were similar between the mice and starling eggs (p = 0.669),

396

but significantly higher in both species (mice: X2 = 6.55 df = 1 p = 0.010; starling eggs: X2 =

397

6.55 df = 1 p = 0.010) when compared to the kestrel eggs (Fig. 1; Table 1). In contrast to

398

kestrels, starlings and mice come into direct contact with biosolids-amended soil: starlings

399

through inserting their beaks to capture soil invertebrates, and mice by digging and burrowing in

400

soil. This inter-species difference in direct contact with the soil may explain the greater 18



Page 20 of 41

401

concentrations of TCC in the mice and starling eggs compared to the kestrel eggs. More research

402

is required to characterize and determine the relative importance of soil contact and diet as routes

403

of exposure to TCC by vertebrates.

404

Concentrations of antimicrobials in relation to egg and reproductive measures.

405

The results of this study indicate that both bird species were exposed to and accumulated

406

TCC and TCS since both antimicrobials were found in detectable concentrations in their eggs.

407

However, there was no evidence to suggest that the concentrations of TCC or TCS were

408

correlated with the egg size or egg shell thickness variables of either species (all p-values ≥ 0.21,

409

Table S3), which were similar to measurements previously reported for these species.18,19 Nor

410

was there a significant difference in nest success between experimental and reference sites for

411

European starlings (p = 0.30) (Fig. 1; Table 3). However, in 2012, nesting success of American

412

kestrels was significantly lower on the experimental site (10% and 17% success rate in 2011 and

413

2012, respectively) compared to the kestrels on the reference site (50% success rate, p = 0.015,

414

Table 3). The sample size in our study was small so caution should be used when interpreting

415

these results. Nevertheless, nesting success rates of 10% and 17% at the experimental site are

416

substantially lower than the average rate of 51% determined in a concurrent study of 102

417

American kestrel pairs conducted in the same county as our study.38

418

A combination of multiple stressors may have contributed to the reduced nesting success

419

of the kestrels observed at the experimental site in our study.39 The kestrels’ exposure to the low

420

concentrations of TCC and TCS observed is unlikely to be a contributing factor, since they were

421

far below known toxic levels for aquatic invertebrates and fish.40-42 The possible toxicity of TCC

422

and TCS to terrestrial wildlife species remains unknown and warrants further investigation. The

423

reduced rate of nesting success by kestrels on the experimental site was principally due to nest 19


Page 21 of 41


424

abandonment. The underlying causes of nest abandonment are unclear but could be influenced

425

by exposure to other contaminants that are known to be commonly present in biosolids but not

426

analyzed in this study, disturbance caused by different farming practices, or other experimental

427

site-specific conditions that determine nesting success, e.g., the availability and quality of prey,

428

thus determining foraging behavior.

429

With reports of antimicrobial concentrations of 30,000 ± 11,000 ng/g dw in biosolids

430

from the mid-Atlantic United States8, together with the potential for toxicity of these

431

antimicrobials to invertebrates and vertebrates, it is important to continue to monitor TCC and

432

TCS in biosolids and food webs receiving biosolids.12-14 This study demonstrates that following

433

extended application of municipal biosolids containing TCC and TCS to an agricultural field (at

434

concentrations lower than reported in other studies12,31.32), concentrations of these two

435

antimicrobials were measured in each trophic level of the terrestrial food web examined.

436

Antimicrobials were detected in soil as well as in primary (earthworms), secondary (deer mice,

437

starlings), and tertiary (kestrels) consumers. Furthermore, the TCC and TCS concentrations in

438

these trophic levels reflect extended application of biosolids to agricultural soils for seven years:

439

concentrations were higher in biosolids, soil, deer mice livers, and European starling eggs at the

440

experimental site than at the reference site. Trophic level differences in concentrations of these

441

two antimicrobials may reflect differences in feeding behavior and strategies between species.

442

The concentrations of TCC and TCS measured in the current study were not correlated with egg

443

parameters of either avian species, although the kestrels demonstrated lower reproductive

444

success on the experimental site. Understanding the toxicokinetics of these and other

445

antimicrobials, and identifying possible effects of TCC and TCS, in wild animals (e.g., mice,

446

birds) requires further investigation. 20



Page 22 of 41

447 448

ACKNOWLEDGMENT

449

It is with great sadness that we note that our colleague and coauthor, Dr. Alfred M. Dufty, Jr.,

450

passed away before the publication of this article. This work was supported by funding from a

451

Colorado State University–Pueblo Seed Grant and funding from the College of Science and

452

Mathematics; support from the U.S. Geological Survey Toxics Substances Hydrology Program,

453

Sigma Xi Grant-In-Aid of Research, Raptor Research Center and the Department of Biological

454

Sciences at Boise State University, and Environment and Climate Change Canada. The use of

455

trade, firm, or brand names in this paper is for identification purposes only and does not

456

constitute endorsement by the authors or the U.S. Geological Survey.

457 458

SUPPORTING INFORMATION

459

Supporting information, Tables S1-S3 and Fig. S1a-c, is available free of charge via the Internet

460

at http://pubs.acs.org/.

21


Page 23 of 41


461

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592

28



593 594 595 596

Page 30 of 41

Figure 1: Arithmetic mean concentrations ± standard errors about the means (SEM) of triclocarban (TCC) in American kestrel (AMKE) and European Starling (EUST) eggs, and mice tissue (liver) collected in 2012. Different letters depict significant differences between means using Wilcoxon non-parametric test.

597

598 599 600 601 602 603 604 605 606 607

29


Page 31 of 41

608 609 610 611 612


Table 1: Concentrations (ng/g) of triclocarban (TCC) and triclosan (TCC) in abiotic and biotic samples collected at different trophic levels from the experimental site amended with muncipal biosolids for seven years and the reference site, in 2012. Medians (bolded), (min-max), and [mean ± SEM] are presented. When appropriate, means and standard errors about the mean (SEM) are presented in Figures 1 and 2.

Sample

TCC (ng/g ww) †

TCS (ng/g ww)

Biosolids (n=1) 1250 1230† Soil – Reference site* pre-application (n=1)* ND† ND† post-application (n=1)* ND† ND† Soil – Experimental site pre-application (n=1) 14.8† 4.4† post-application (n=1) 27.3† 2.7† Earthworms‡ Reference (n=1) ND† 5.9† Experimental (n=1) 5.1† 42.8† Deer mice - liver‡ Reference (n = 3) ND ND Experimental (n = 7) 17.0 (12.6 - 33.3) [20.0 ± 4.6] 3.3 (6.6-10.7) [5.0 ± 1.2]§ Deer mice - muscle‡ Reference (n = 3) ND ND Experimental (n = 7) ND ND ‡ European starling eggs Reference (n = 6) 4.6 (3.6-5.0) [3.9 ± 0.7]§ 5.8 § Experimental (n = 6) 23.2 (15.4 - 31.4) [20.4 ± 4.4] 13.7 (9.4-37.9) [16.3 ± 5.1]§ American kestrel eggs‡ Reference (n=5) 4.6 (4.1-5.4) [4.0 ± 0.8]§ 3.3º Experimental (n=5) 4.2 (4.2-13.4) [5.4 ± 2.2]§ 3.6º 613 ND = Not Detected 614 *Biosolids were not applied to the reference site. † 615 Values represent the average of triplicate measurements of a composite sample from three 616 independent locations. ‡ 617 Reported values are on a wet weight basis. 618 § For the purposes of statistical analysis, values that were positively detected (correct retention 619 time, ions, and ion ratios) but below the method detection limit (MDL) were assigned ½ MDL 620 (TCC: 1.35 ng/g ww for eggs; TCS: 1.8 ng/g ww for mice liver and 1.65 ng/g ww). 621 One out of 6 eggs had a detectable level of TCS, the others had nondetectable concentrations. 622 º Reference site eggs had 4 out of 5 detections of TCC. Only one was above the MDL. 623 Experimental site eggs had 5 out of 5 detections of TCC. Only one was above the MDL. 30



624

Table 2: Biota-to-soil accumulation factors (BSAFs) for organisms from the experimental site

625 626

Sample TCC TCS Earthworms 0.79* 67* Deer Mouse (liver) 0.20 0.50 Starling eggs 0.25 2.0 Kestrel eggs 0.05 0.77 * BSAF estimated for earthworms because earthworms were not depurated as part of the experimental design.

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31


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627 628 629 630 631 632

Table 3. Number of nests for American kestrels and European starlings breeding on the experimental site and reference site, 2011 and 2012. A nesting attempt was classified as unsuccessful if a full clutch was observed but no nestlings grew to be 20 days old (the equivalent of 80% of the average age at first flight). A nesting attempt was deemed successful only if at least one nestling reached 20 days or 80% of the average age at first flight. Nesting success information was not collected for European starling nests in 2012. Species Site Year Unsuccessful Successful Nesting Mean # nesting nesting attempt Success fledglings per attempt (%) successful nesting attempt American kestrel Reference 2011 12 6 50 3.2 American kestrel

Experimental 2011

10

1

10

2.0

American kestrel

Reference

2012

12

6

50

3.3

American kestrel

Experimental 2012

6

1

17

2.0

European starling

Reference

2011

6

6

100

2.5

European starling

Experimental 2011

6

5

83

2.5

633 634

32



635

SUPPORTING INFORMATION

636

Occurrence of triclocarban and triclosan in an agro-ecosystem following

637

application of biosolids

638

Jessica J. Sherburne §, Amanda M. Anaya ҂, Kim J. Fernie ∞,§, Jennifer S. Forbey §, Edward T.

639

Furlong †, Dana W. Kolpin ≡, Alfred M. Dufty §, Chad A. Kinney* , ҂.

640

§

641

83725

642

҂

643

∞

644

and Climate Change Canada, 867 Lakeshore Rd., Burlington, ON CANADA L7S 1A1

645

†

646

95, Denver, CO 80225

647

≡

648

Number of Pages: 6

649

Number of Figures: 3

650

Number of Tables: 2

651

* Correspondance:

Page 34 of 41

Department of Biological Sciences, Boise State University, 1900 University Dr., Boise, ID

Department of Chemistry, Colorado State University, 2200 Bonforte Blvd., Pueblo, CO 81001 Ecotoxicology and Wildlife Health Division, Science and Technology Branch, Environment

U.S. Geological Survey, National Water Quality Laboratory, Denver Federal Center, Building

U.S. Geological Survey, 400 S. Clinton St., Iowa City, IA 52240

Email :

652

Tel : (719) 549-2600

653

Mail : Colorado State University-Pueblo

654

Chemistry Department

655

2200 Bonforte Blvd

656 33


Page 35 of 41


657 658

Introduction

659

In the Supplemental Information section, the analytical performance parameters and detection

660

limits are presented. In addition, data are presented that describe the egg morphometric

661

measurements of American kestrel eggs and European starling eggs that were collected in 2012

662

from the experimental site that had been amended with municipal biosolids for seven years, and

663

the reference site. Finally, maps of the study sites are provided.

664

34



Page 36 of 41

Table S1: Analytical Method Performance Parameters for triclocarban (TCC) and triclosan (TCS). A. Absolute method recovery (%)* for triclocarban (TCC) and triclosan (TCS) TCC

TCS

Biosolids

83.9 ± 2.5

87.6 ± 4.5

Soil

84.4 ± 5.4

86.3 ± 4.6

Earthworms

72.5 ± 4.2

61.0 ± 6.7†

Deer mice - Liver

54.9 ± 13

99.2 ± 4.2

Deer mice - Muscle

51.2 ± 4.2

55.1 ± 5.2

Eggs

41.0 ± 7.1

53.0 ± 5.0

* Low absolute method recovery are overcome by the use of isotope dilution (isotope-labeled internal standards added at the beginning of extraction process). † Method performance parameters previously reported 12 B. Method detection limits* for triclocarban (TCC) and triclosan (TCS) concentration (ng/g) TCC

TCS

5.5

2.8

3.4

2.6

3.1

3.3

Deer mice - Liver‡

3.2

3.6

Deer mice - Muscle‡

4.2

14.6

Eggs‡

2.7

3.3

Biosolids† Soil† Earthworms

‡

*Concentrations detected above the Method Detection Limit (MDL) were used in analysis. Any positive detection below the MDL was assigned a value of half of the MDL for statistical analysis.26 † Dry weight basis ‡ Fresh weight basis

35


Page 37 of 41

665


Table S2: Total lipids in biological samples and organic matter in soils

Sample Earthworms (control) Earthworms (experimental) Deer Mouse (liver) Deer Mouse (muscle) Starling egg Kestrel egg

Soil (control) soil (experimental)

Total Lipids (% wet wt) 0.73 ± 0.07

Total Lipids (% dry wt) 3.6 ± 0.4

0.71 ± 0.05 11.1 ± 1.2 8.8 ± 1.0 9.0 ± 0.1 7.8 ± 0.4

3.7 ± 0.3 23.5 ± 3.4 15.7 ± 2.0 NA NA

SOM (%) 2.8 ± 0.6 3.0 ± 0.2

666

36



Page 38 of 41

667

Table S3: Egg size measurements for American kestrel eggs and European starling eggs

668

collected in 2012 from the experimental site amended with municipal biosolids for seven years

669

and the reference site. Arithmetic means ± standard errors about the means (SEM) are presented.

670 American kestrels

European starlings

Reference

Experimental Reference

Experimental

Egg length (mm)

35.7 ± 0.4

35.4 ± 0.3

30.7 ± 0.5

30.4 ± 0.9

Egg breadth (mm)

29.1 ± 0.4

28.8 ± 0.3

21.7 ± 0.1

21.4 ± 0.2

Egg volume (mL3)

16.7 ± 0.6

16.2 ± 0.4

7.7 ± 0.1

7.4 ± 0.3

Egg mass (g)

13.7 ± 0.7

13.5 ± 0.4

6.5 ± 0.1

6.1 ± 0.3

Shell mass (g)

1.1 ± 0.06

1.1 ± 0.03

0.5 ± 0.01

0.5 ± 0.02

Shell thickness (mm)

0.2 ± 0.006

0.2 ± 0.005

0.1 ± 0.004

0.1 ± 0.003

671 672 673 674 675 676 677

37


Page 39 of 41

678


Figure S1a: Overall map of Reference and Experimental field sites

679 680 38



Page 40 of 41

681 682

Figure S1b: Reference Site

683 684 685 686 39


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687 688 689

Figure S1c: Experimental Site

690 691 692

40


Occurrence of Triclocarban and Triclosan in an Agro-ecosystem

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