Occurrence and Potential Biological Effects of ... - ACS Publications

Aug 11, 2016 - We examined the fate and ecological effects of amphetamine on biofilm, seston, and aquatic insect communities in artificial streams exp...
1 downloads 4 Views 789KB Size
Subscriber access provided by United Arab Emirates University | Libraries Deanship

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

Environmental Science & Technology

1 2

Occurrence and potential biological effects of

3

amphetamine on stream communities

4 5

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

6

Marshall1, and John J. Kelly4

7 8

1

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

9

2

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

10

States

11

3

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

12

4

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

13

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

14

Agency, Arlington, Virginia, 22202, United States

15

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

16 17

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

18

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

19

1 ACS Paragon Plus Environment

Environmental Science & Technology

20 21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

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

37

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

38

communities.

2 ACS Paragon Plus Environment

Page 2 of 33

Page 3 of 33

39 40

Environmental Science & Technology

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

41

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

42

pharmaceuticals originating from human consumption and excretion, manufacturing processes,

43

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

44

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

45

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

46

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

47

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

48

compounds have ecological consequences.8

49

Stimulants such as methamphetamine and other amphetamines increase dopamine levels

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

environments across the globe may be on the rise.

60 61

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

3 ACS Paragon Plus Environment

Environmental Science & Technology

62

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

63

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

64

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

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

ecological consequences for stream communities.

75 76

MATERIALS AND METHODS

77

Study Area

78

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

79

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

80

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

81

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

82

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

83

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

84

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

4 ACS Paragon Plus Environment

Page 4 of 33

Page 5 of 33

Environmental Science & Technology

85

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

86

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

87

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

88

in June 2013 and one day in July 2014.

89 90 91

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

92

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

93

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

94

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

95

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

96

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

97

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

98

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

99

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

100

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

101

with 100 ng of internal standards.

102

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

103

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

104

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

105

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

106

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

107

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

5 ACS Paragon Plus Environment

Environmental Science & Technology

108

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

109

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

110

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

111

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

112

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

113

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

114 115 116

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

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

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

125

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

126

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

127

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

128

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

129

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

130

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

6 ACS Paragon Plus Environment

Page 6 of 33

Page 7 of 33

Environmental Science & Technology

131

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

132

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

133

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

134

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

135 136 137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

AFDM-1 to standardize metabolism measurements by biomass.

151 152 153

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

7 ACS Paragon Plus Environment

Environmental Science & Technology

154

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

155

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

156

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

157

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

158

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

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

effects showed non-negligible treatment × day interactions (p