Subscriber access provided by Kaohsiung Medical University
Characterization of Natural and Affected Environments
Internal concentrations in gammarids reveal increased risk of organic micropollutants in wastewater-impacted streams Nicole Alessandra Munz, Qiuguo Fu, Christian Stamm, and Juliane Hollender Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b03632 • Publication Date (Web): 17 Aug 2018 Downloaded from http://pubs.acs.org on August 17, 2018
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 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 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.
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 30
Environmental Science & Technology
1
Internal concentrations in gammarids reveal increased risk of organic
2
micropollutants in wastewater-impacted streams
3
Nicole A. Munz1,2, Qiuguo Fu1, Christian Stamm2, Juliane Hollender1,2,*
4
1
5
2
Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland Institute of Biogeochemistry and Pollutant Dynamics, ETH Zürich, 8092 Zürich, Switzerland
6 7 8 9
*Corresponding author. Present address:
10
Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland.
11
Tel: +41 58 765 54 93. E-mail:
[email protected] 12 13 14
TOC:
15 16 17 18 19 1 ACS Paragon Plus Environment
Environmental Science & Technology
Page 2 of 30
20
Abstract
21
Internal concentrations link external exposure to the potential effect, as they reflect what the
22
organisms actually take up and experience physiologically. In this study, we investigated whether
23
frequently detected risk-driving substances in water were found in the exposed organisms and if they
24
are classified the same based on the whole body internal concentrations. Field gammarids were
25
collected upstream and downstream of ten wastewater treatment plants in mixed land use catchments.
26
The sampling was conducted in autumn and winter, during low flow conditions when diffuse
27
agricultural input was reduced. The field study was complemented with laboratory and flume
28
experiments to determine the bioaccumulation potentials of selected substances. For 32 substances,
29
apparent bioaccumulation factors in gammarids were determined for the first time. With a sensitive
30
multi-residue method based on online-solid phase extraction followed by liquid chromatography
31
coupled to high resolution mass spectrometry, we detected 63 (semi-) polar organic substances in the
32
field gammarids, showing higher concentrations downstream than upstream. Interestingly,
33
neonicotinoids, which are particularly toxic towards invertebrates, were frequently detected and were
34
further determined as major contributors to the toxic pressure based on the toxic unit approach
35
integrating internal concentration and toxic potency. The total toxic pressure based on internal
36
concentrations was substantially higher compared to when external concentrations were used. Thus,
37
internal concentrations may add more value to the current environmental risk assessment that is
38
typically based solely on external exposure.
39 40
Introduction
41
Thousands of chemicals are regularly released into the environment from various sources (e.g.
42
households, agriculture) and can enter streams through different pathways (e.g. wastewater effluent,
43
urban and agricultural runoff). Consequently, numerous compounds are detected in freshwater
44
ecosystems as complex mixtures, mostly at trace concentrations in the ng/L to µg/L range.1 Some of
45
these compounds are designed to be biologically active (i.e., pesticides, pharmaceuticals) and when
46
taken up, they may adversely affect non-target organisms.2-4
47
While environmental risk assessment at present is typically based on exposure levels in water, the
48
inclusion of the internal concentrations of chemicals is increasingly regarded as indispensable for the
2 ACS Paragon Plus Environment
Page 3 of 30
Environmental Science & Technology
49
effects assessment of chemicals in aquatic organisms.2, 5-6 Compared to water concentrations, internal
50
concentrations do not only assess what the aquatic organisms “see”, but also reflect what they actually
51
take up and could therefore provide indications of potential effects. This is especially true if only short-
52
term sampling of water is conducted. While grab samples represent snapshots of the exposure,
53
internal concentrations integrate the concentrations over time. Sometimes, passive sampling is applied
54
to biomimic integrative sampling of organisms , but processes such as active uptake and metabolism
55
cannot be represented with passive samplers. In biomonitoring studies, fish are often considered as
56
most appropriate organisms because they are not only relevant for the environment but also for
57
humans as a food source. However, besides ethical considerations, one of their limitations is their
58
relatively high mobility. In contrast, invertebrates (e.g. gammarids) have a much lower mobility and can
59
serve as good site-specific bioindicators.4 Gammarids are important keystone species for detritus
60
processing (shredders) and form an important link to higher trophic level organisms as they are preyed
61
on by fish.8-9 These amphipods are highly sensitive towards a range of different stressors and are thus
62
often used as model organisms in ecotoxicological studies.
63
freshwater systems8, 10, they are suitable for investigations of internal exposures in field biomonitoring
64
studies.
65
However, information on bioaccumulation of organic compounds in aquatic organisms other than fish
66
is rather limited, especially for chemicals with many polar functional groups wherein prediction of
67
bioaccumulation is difficult.12 Although non-polar compounds have a higher bioaccumulation potential,
68
polar compounds are usually present in surface water at much higher bioavailable concentrations and
69
thus they can be considered as equally relevant for toxicity.13 Studies investigating the
70
bioaccumulation of selected polar substances in gammarids under controlled laboratory conditions
71
showed that many polar compounds are taken up and can contribute to possible adverse effects.14-19
72
In only a few field studies, the presence of polar organic substances in gammarids was observed in
73
experiments with caged organisms20-21 and gammarids directly collected from streams22-24.
74
The goal of this study was to determine the chemical risk of polar and semi-polar organic
75
micropollutants, which are frequently analyzed and detected in water, based on whole body internal
76
concentrations (hereafter named internal concentrations) in gammarids collected in the field. In a
77
previous study on the external exposure of micropollutants we identified pesticides as main drivers of
78
the risk.
7
25
8-11
Because of their high abundance in
Thus, we wanted to investigate whether i) the identified risk drivers in water are actually
3 ACS Paragon Plus Environment
Environmental Science & Technology
Page 4 of 30
79
detected in gammarids collected at the same time, and ii) if yes, whether they would also be classified
80
as risk drivers based on the internal exposure. Laboratory experiments and artificial flume experiments
81
supplemented the field data for a more comprehensive understanding of the bioaccumulation potential
82
under controlled and semi-realistic exposure conditions. Gammarids were collected at ten sites in
83
mixed land use catchments upstream and downstream of wastewater treatment plants (WWTP),
84
where our previous study on the external exposure was also conducted.
85
pressure on invertebrates was estimated based on the internal concentrations and the laboratory
86
determined bioaccumulation potentials as well as EC50 values for invertebrates, and was then
87
compared to the toxic pressure calculated with the external water concentrations.
25
The potential mixture toxic
88 89
Material & Methods
90
Field samples (gammarids and water)
91
Gammarids (mainly G.fossarum) were collected at nine sites upstream and downstream of WWTPs
92
(Aadorf, Elgg, Ellikon, Knonau, Marthalen, Unterehrendingen, Val-de-Ruz, Villeret, Zullwil in
93
September 2014 and January 2015 and at the site Ellikon also in October 2015 (Figure S1)). Herisau
94
WWTP underwent a major process upgrade in 2015 with the addition of an advanced treatment based
95
on sorption to powdered activated carbon for the enhanced elimination of micropollutants. Hence,
96
additional gammarids collection was completed in May 2015 and June 2016 to assess the impact of
97
treatment upgrades. Water grab samples were previously taken at the same sites and time points
98
using the methodology described in Munz et al. (2017) . As controls, uncontaminated gammarids
99
were collected at a pristine site near Zurich, Switzerland.19 At each site, approximately 100 individuals,
25
100
without visible eggs, were collected using kick-nets. They were transferred to the lab in buckets filled
101
with stream water and leaves. In the lab, gammarids were transferred to Falcon tubes, frozen at -20
102
°C and then stored at -80 °C until analysis. Replicate samples were analyzed for each site whenever
103
possible. To determine recovery, thawed gammarids from two sites (upstream and downstream) were
104
spiked with each target substance at a level of 250 ng/L; the area (for absolute recovery) or
105
concentration (for relative recovery) was subtracted by the value of the unspiked sample and divided
106
by the value of the corresponding calibration standard. Number, weight and major species of
107
gammarids are provided in Table S1.
4 ACS Paragon Plus Environment
Page 5 of 30
Environmental Science & Technology
108
Laboratory exposure experiment
109
Gammarids (G.pulex) were collected at the pristine site mentioned above and acclimatized to the
110
laboratory conditions (12 °C, 12 h light/12 h dark cycle) in an aquarium using artificial pond water
111
(APW) and fed with alder leaves collected in the creek. Details on the preparation of APW are
112
provided in Rösch et al. (2016).19 Gammarids were exposed to i) APW spiked with a mixture of 52
113
(semi-) polar organic substances (e.g. pesticides, pharmaceuticals) at nominal concentrations of 5
114
µg/L each, and ii) to wastewater diluted in APW (30%, 60%, 90% of wastewater). The list of
115
substances was selected based on the priority mixture described in Munz et al. (2017) . For details on
116
the substance selection and the wastewater sample see Section S1. Gammarids without visible eggs
117
(13 - 16 organisms) were added to 600 mL-glass beakers filled in triplicate with 500 mL of the
118
exposure media and 5 alder leaves, and were incubated for 48 h at 12 °C. An incubation period of 48
119
h was regarded as sufficient since other studies have shown that steady state was reached within this
120
time frame for chemicals with comparable logKow values.15, 17, 19, 26 Controls i) without chemicals or
121
wastewater (triplicates), ii) without organisms (duplicates), and iii) without leaves and organisms
122
(duplicates) were performed. The exposure media was sampled at the beginning (t0), after 24 hours
123
(t24) and at the end (t48) of the experiment. Gammarids were also collected directly from the aquarium
124
to determine recoveries and background concentrations. All samples were stored at -20 °C until
125
analysis. Number and weight of gammarids per replicate are listed in Table S2.
126
Flume experiments
127
A description of the setup of the flume experiments can be found in Stamm et al. (2016) . Briefly, 16
128
channels arranged in four blocks (four replicates of each treatment) were setup directly at the WWTP
129
Bachwis (Fällanden, Switzerland). They were fed with upstream water of the Glatt River and treated
130
wastewater (experiment 1) or spiked chemicals in river water (experiment 3) (experiment numbering
131
according to Stamm et al. (2016) ). The gammarids were placed in cages in the channels during four
132
(experiment 3, G.fossarum) to five weeks (experiment 1, G.pulex). The experiments were conducted in
133
August 2014 (experiment 1) and June 2015 (experiment 3). The gammarids were collected at the end
134
of the experiments and directly frozen at -20°C and stored at -80°C until analysis. To obtain a
135
sufficient mass of gammarids (~500 mg) for bioanalysis, different replicates from the four blocks were
136
pooled, if necessary. Further details on the two flume experiments are described in Section S2.
137
Number and weight of gammarids per channel are listed in Table S3. Water samples of the flumes
25
27
27
5 ACS Paragon Plus Environment
Environmental Science & Technology
Page 6 of 30
138
were taken at different time points of the experiment. The analysis of 57 organic micropollutants in the
139
flume water was done using online solid phase extraction (SPE) followed by liquid chromatography
140
coupled to high resolution tandem mass spectrometry (LC-HRMS/MS) as previously described in
141
Munz et al. (2017) .
25
142 143
Sample preparation, enrichment and clean-up
144
Thawed gammarids were quickly rinsed with nanopure water, blotted dry with tissue and weighed into
145
a 2-mL microcentrifuge tube to a final weight of approximately 500 mg (on average 40 organisms
146
depending on size; in the exposure experiment all exposed gammarids were used). After the addition
147
of 80 µL internal standard (1 mg/L) they were stored overnight at 4°C. The remaining solvent was
148
shortly evaporated with a gentle stream of nitrogen, then 500 mg of 1 mm zirconia/silica beads
149
(BioSpec Products, Inc., U.S.A.), 500 µL of acetonitrile (ACN) and 500 µL of nanopure water were
150
added. Extraction and homogenization were carried out using a Fast Prep bead beater (MP
151
Biomedicals, Switzerland) in two cycles of 15 s at 6 m/s with cooling on ice in between. Afterwards,
152
samples were centrifuged (6 min, 10 000 rpm, 20 °C) and 800 µL of the supernatant was transferred
153
into a tube containing 500 mg QuEChERS salts (4:1, MgSO4:NaCl, Agilent Technologies),
154
immediately vortexed and then centrifuged again (6 min, 10 000 rpm, 20 °C). ACN (500 µL) was
155
added to the first homogenate with the already used QuEChERS salts and all the steps were repeated
156
to increase recoveries. For a further clean-up step, especially for the elimination of lipids, the
157
combined supernatant (approx. 800 µL) was transferred to a tube containing heptane (500 µL),
158
vortexed and centrifuged (6 min, 10 000 rpm, 20 °C). Heptane (400 µL) was removed and a second
159
heptane extraction (500 µL) was carried out. Finally, 700 µL of the ACN phase (bottom layer) was
160
transferred to a clean HPLC glass vial and was filled with methanol to a final volume of 2 mL. The
161
extracts were stored at 4 °C until analysis.
162
Chemical analysis of gammarid extracts
163
All gammarid extracts were analyzed using the online SPE LC-HRMS/MS setup similar to the water
164
samples described in Munz et al. (2017)25 (see Section S3). For the gammarid extracts, 200 µL of the
165
extract was spiked into an online vial filled with 20 mL nanopure water. Quantification of up to 84
166
target substances was done with internal standard calibration using the software TraceFinder
6 ACS Paragon Plus Environment
Page 7 of 30
Environmental Science & Technology
167
v3.2/v4.1 (Thermo Scientific; Table S4). In addition to the selected substances in the lab experiment
168
(n=52), the field gammarids were screened for further target substances (spiked in the calibration
169
standards) using CompoundDiscoverer 2.1 (Thermo Scientific). A mass list of the additional 395 target
170
substances spiked in the calibration standards (Table S5) was processed over the five most polluted
171
samples (>= 10 detected substances). Only substances analyzed in positive ionization mode were
172
considered in this step because of higher sensitivity and larger number of analytes. After filtering out
173
the background, only targets which showed an acceptable signal (i.e., peak shape, retention time) in
174
the calibration curve and in at least one of the selected samples were considered for further
175
quantification in TraceFinder.
176
For the reported internal concentration, the average of the concentrations in the replicates was
177
calculated. If the concentration of one replicate was >LOQ and the concentration of the other replicate
178
was LOD, half the LOQ value was used. If the concentration of one replicate was >LOQ
179
and the concentration of the other replicate was
