Occurrence and Potential Biological Effects of Amphetamine on

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Occurrence and potential biological effects of amphetamine on stream communities Sylvia S. Lee, Alexis M. Paspalof, Daniel Snow, Erinn Kate Richmond, Emma J Rosi-Marshall, and John J. Kelly Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03717 • Publication Date (Web): 11 Aug 2016 Downloaded from http://pubs.acs.org on August 16, 2016

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Environmental Science & Technology

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Occurrence and potential biological effects of

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amphetamine on stream communities

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Sylvia S. Lee1*, Alexis M. Paspalof2, Daniel D. Snow2, Erinn K. Richmond3, Emma J. Rosi-

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Marshall1, and John J. Kelly4

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Cary Institute of Ecosystem Studies, Millbrook, New York, 12545, United States

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Water Sciences Laboratory, University of Nebraska–Lincoln, Lincoln, Nebraska, 68583, United

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States

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Water Studies Centre, Monash University, Victoria, 3800, Australia

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Department of Biology, Loyola University Chicago, Chicago, Illinois, 60660, United States

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*Current affiliation: Office of Research and Development, U.S. Environmental Protection

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Agency, Arlington, Virginia, 22202, United States

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Corresponding author email: [email protected] phone: (703)347-8058

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KEYWORDS: amphetamine, illicit drugs, urban streams, artificial streams, Baltimore, 16S

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rRNA, MiSeq, biofilm, seston, diatoms, emergence, aquatic insects

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1 ACS Paragon Plus Environment


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ABSTRACT The presence of pharmaceuticals, including illicit drugs in aquatic systems, is a topic of

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environmental significance because of their global occurrence and potential effects on aquatic

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ecosystems and human health, but few studies have examined the ecological effects of illicit

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drugs. We conducted a survey of several drug residues, including the potentially illicit drug

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amphetamine, at 6 stream sites along an urban to rural gradient in Baltimore, Maryland, USA.

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We detected numerous drugs, including amphetamine (3 to 630 ng L-1), in all stream sites. We

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examined the fate and ecological effects of amphetamine on biofilm, seston, and aquatic insect

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communities in artificial streams exposed to an environmentally relevant concentration (1 µg L-1)

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of amphetamine. The amphetamine parent compound decreased in the artificial streams from less

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than 1 µg L-1 on day 1 to 0.11 µg L-1 on day 22. In artificial streams treated with amphetamine,

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there was up to 45% lower biofilm chlorophyll a per ash-free dry mass, 85% lower biofilm gross

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primary production, 24% greater seston ash-free dry mass, and 30% lower seston community

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respiration compared to control streams. Exposing streams to amphetamine also changed the

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composition of bacterial and diatom communities in biofilms at day 21 and increased cumulative

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dipteran emergence by 65% and 89% during the first and third weeks of the experiment,

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respectively. This study demonstrates that amphetamine and other biologically active drugs are

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present in urban streams and have the potential to affect both structure and function of stream

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communities.


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INTRODUCTION Pharmaceuticals and their breakdown products occur in surface waters around the world

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primarily from inputs of treated or untreated human wastewater, which can contain

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pharmaceuticals originating from human consumption and excretion, manufacturing processes,

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or improper disposal. 1–3 In addition, the use and misuse of various narcotics result in the

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presence of detectable concentrations of illicit stimulants, such as methamphetamine and

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amphetamine (AMPH), in surface waters, which has made it possible to track drug usage by

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analyzing the concentrations of these compounds in surface waters.4–7 Although illicit and

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legally-used stimulants are detected in surface waters, it is not currently known whether these

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compounds have ecological consequences.8

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Stimulants such as methamphetamine and other amphetamines increase dopamine levels

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in the human brain, a neurotransmitter associated with pleasure, movement and attention. Other

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pharmacological effects of stimulants include loss of appetite, as well as increased wakefulness

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and attentiveness. These desired biological effects have led to the increased use of stimulant

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medications in the treatment of diseases such as attention-deficit hyperactivity disorder (ADHD)

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and narcolepsy.9,10 Unfortunately, many of the same chemicals are also used illicitly as

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narcotics.11,12 After ingestion of AMPH approximately 30-40% of the parent compound plus its

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metabolites are excreted in human urine and feces,4 and these can be transported into surface

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waters directly or through wastewater treatment facilities. Based on increases in both medical

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and illicit usage, there is cause to speculate that the release of stimulants to various aquatic

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environments across the globe may be on the rise.

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The goals of this study were to measure the concentrations of these and other drugs in urban streams in Baltimore, and to examine the potential ecological effects of these compounds



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on stream communities. In addition to measuring the concentration of AMPH in urban streams,

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we used an artificial stream experiment to determine whether AMPH affects biofilm, seston, and

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aquatic insect communities. Using 8 artificial streams, we exposed stream communities to 1 µg

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L-1 AMPH, an environmentally relevant concentration based on our measurement of AMPH in

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urban streams. We measured the concentrations of AMPH at the beginning and end of the 3

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week experiment to examine its persistence. We also measured the effects of AMPH exposure on

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biofilm and seston biomass and metabolism (chlorophyll a, ash-free dry mass, gross primary

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production and community respiration), biofilm bacterial and diatom species composition, and

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aquatic insect emergence. Recent studies suggest that other drugs can affect some of these

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ecological endpoints.13–15 The occurrence of AMPH and other illicit drugs in stream

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environments has been the subject of research around the world.5,16–18 However, this study is one

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of the first to test whether this biologically active, highly addictive, and widely used drug has

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ecological consequences for stream communities.

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MATERIALS AND METHODS

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Study Area

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The Gwynns Falls watershed is part of the Baltimore Ecosystem Study Long-Term

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Ecological Research program (beslter.org). The sites sampled in this study that were located in

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the Gwynns Falls watershed include Gwynnbrook (a suburban stream that drains an area with a

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combination of sewers and septic systems), Dead Run (a more urbanized stream), Gwynns Run

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(a highly urbanized stream with a history of sewage leaks due to failing infrastructure), and

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Gwynns Run at Carroll Park (the most downstream site near the confluence of Baltimore

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Harbor). Two additional streams sampled in this study, Pond Branch (a forested stream) and


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Baisman Run (a forested stream with low residential development and septic systems), are not

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within the Gwynns Falls watershed, but are located within the Oregon Ridge watershed, the

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closest remaining intact forested region. We collected water samples at all locations on one day

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in June 2013 and one day in July 2014.

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Sample Collection and Extraction, Reagents, and Analysis Surface water samples were collected in pre-cleaned 250 mL amber glass jars from each

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site and chilled on ice until they could be frozen upon return to the laboratory. Mesocosm

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samples were collected and processed using the same procedures. Thawed samples were filtered

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using a Whatman 25 mm GF/F glass fiber filter, and then a 100 mL portion weighed for

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polymeric solid phase extraction (SPE) with Oasis 200 mg HLB sorbent (Waters Corporation,

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Milford, MA). Cartridges were conditioned with 6 mL each of high purity acetone and methanol

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(OptimaTM grade, Fisher Scientific, St. Louis, MO, USA), followed by 6 mL of purified reagent

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water (Barnstead Nanopure, Dubuque, IA, USA). Cartridges were eluted with 6 mL acetone,

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followed by 6 mL of methanol, and the eluates were concentrated under vacuum and constant

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stream of nitrogen gas. Residues were dissolved in 200 µL methanol:water (50:50) and fortified

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with 100 ng of internal standards.

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Standard compounds, purchased from Sigma Aldrich (St. Louis, MO) and Cerilliant,

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included D-amphetamine (AMPH), methamphetamine, MDMA (3,4-methylenedioxy-

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methamphetamine), acetaminophen, caffeine, 1,7-dimethylxanthine, diphenhydramine,

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cimetidine, sulfamethoxazole, sulfadimethoxine, cotinine, morphine, carbamazepine, and

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thiabendazole. Labeled internal standards (13C3-caffeine, methamphetamine-d8, MDMA-d8,

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morphine-d3, and 13C6-sulfamethazine) were purchased from Cerilliant (Round Rock, TX) and



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Cambridge Isotopes (Tewksbury, MA). Extracts were analyzed using multiple reaction

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monitoring (MRM) using liquid chromatography tandem mass spectrometry (LC-MS/MS) on a

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Quattro MicroTM (Waters Corporation, Milford, MA) triple quadrupole mass spectrometer

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interfaced with a Waters 2695 HPLC. Method detection limits, determined from repeated

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analysis of a low-level (0.005 µgL-1) fortified water sample, ranged from 0.001 to 0.017 µg L-1.

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For more details on instrumental conditions and method validation, see S1.

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Artificial Streams Artificial stream experiments were conducted in recirculating mesocosms at the Cary

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Institute for 3 weeks in June to July 2014. Detailed descriptions of the facility have been reported

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previously.19 Replicates of 4 control and 4 AMPH-exposed streams were used. To mimic natural

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attributes of streams, a known volume of landscaping rock was added to each stream. Benthic

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microbes were obtained by scrubbing rocks from a nearby forested stream (East Branch

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Wappinger Creek, Millbrook, NY, USA) to form a slurry that was added to artificial streams to

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allow microbes to colonize substrates for 2 months. Nutrients were added to the streams twice a

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week to aid biofilm growth. Nitrogen was added in the form of ammonium (NH4+) and

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phosphorus as phosphate (PO4-3) at target concentrations of 40 µg L-1 and 2.5 µg L-1,

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respectively. In addition, we supplemented each artificial stream with 20 more rocks of similar

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sizes from Wappinger Creek to add additional algae, bacteria, and aquatic insects. We filled each

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stream with 60 L of low nutrient groundwater sourced from a forested area. Additional water was

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added daily to compensate for evaporative losses and to ensure a constant volume of 60 L.

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Velocity of the streams was kept at a constant speed of 0.26 m s-1. At the beginning of the

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experiment, AMPH was added to 4 streams at a target concentration of 1 µg L-1 and a second set


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of 4 streams was maintained at 0 µg L-1 as controls. We collected one replicate water sample

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from each stream on day 1 (20 minutes after AMPH additions) and day 22 (the end of the

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experiment) to measure AMPH concentrations. Artificial stream water samples were treated in

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the same fashion as described above for stream water samples collected in Baltimore.

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Stream Community Responses to AMPH Effects of AMPH exposure were measured on biofilm and seston communities on days 1,

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4, 7, 14 and 21 as chlorophyll a (chl a), ash-free dry mass (AFDM), chl a per AFDM, gross

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primary production (GPP), and community respiration (CR). Seston samples were collected by

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sampling the water column of the artificial streams. Biofilms were collected for chl a and AFDM

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analysis by collecting one rock from each stream and scrubbing biofilms to form a slurry. Known

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amounts of the seston or biofilm slurries were filtered through 0.7um GF/F glass fiber filters

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(Whatman®) and analyzed for chl a and AFDM. Extraction of Chl a was done by freezing the

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filters for at least 24 hours and immersing filters in basic methanol in the dark for 24 hours.20 Chl

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a concentrations were measured using a Turner Designs Model TD-700 fluorometer (Turner

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Designs, Sunnyvale, California, USA). To measure AFDM, samples were dried in an oven at

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60°C for 24 hours. These samples were then weighed, combusted at 500°C for 1 hour and then

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reweighed to obtain AFDM.21 GPP and CR were measured in light and dark chambers as

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previously described.13 The units of GPP and CR measurements were adjusted to mg O2 h-1 mg

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AFDM-1 to standardize metabolism measurements by biomass.

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Statistical Analysis Response variable distributions were examined using histograms, QQ-plots, residual



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diagnostics, and goodness-of-fit tests available in the proc univariate procedure in SAS. The

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lognormal probability distribution adequately fit the distributions of all response variables,

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except GPP. The lognormal distribution is right-skewed, with all values >0, and has been used to

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estimate stream metabolism data.22,23 The gamma distribution was chosen for GPP because of

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improved distribution of residuals. Generalized linear mixed models (GLMMs) were used to test

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the effects of AMPH using the proc glimmix procedure in SAS. The maximum likelihood

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method was used for all GLMMs. The GLMMs were used to test the main fixed effects of

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treatment, day, and the interaction of treatment × day on stream biofilms and a random effect of

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stream ID to account for errors associated with within-treatment variability among artificial

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streams.24,25 Because of the repeated measures experimental design, inclusion of temporal

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autocorrelation structure in the GLMMs was attempted, but was only included if the addition of

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this factor did not result in over-specified or non-converging models. When tests of main fixed

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effects showed non-negligible treatment × day interactions (p

Occurrence and Potential Biological Effects of Amphetamine on

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