Subscriber access provided by UNIV OF NEBRASKA - LINCOLN
Article
Dissolved organic nitrogen inputs from wastewater treatment plant effluents increase responses of planktonic metabolic rates to warming Raquel Vaquer-Sunyer, Daniel J. Conley, Saraladevi Muthusamy, Markus V Lindh, Jarone Pinhassi, and Emma S Kritzberg Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00674 • Publication Date (Web): 10 Sep 2015 Downloaded from http://pubs.acs.org on September 14, 2015
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 36
Environmental Science & Technology
1
2
Dissolved organic nitrogen inputs from wastewater
3
treatment plant effluents increase responses of
4
planktonic metabolic rates to warming.
5 6
Raquel Vaquer-Sunyer*†, Daniel J. Conley†, Saraladevi Muthusamy‡, Markus V. Lindh‡, Jarone
7
Pinhassi‡ and Emma S. Kritzberg§
8 9
AUTHOR ADDRESS:
10
† Department of Geology, Lund University, Sölvegatan 12, SE-223 62, Lund, Sweden
11
‡ Centre for Ecology and Evolution in Microbial model Systems – EEMiS, Linnaeus University,
12
SE-39182 Kalmar, Sweden
13
§ Department of Biology, Lund University, Sölvegatan 37, SE-223 62, Lund, Sweden
14 15
*Corresponding author: Raquel Vaquer-Sunyer. New address: Interdisciplinary Ecology group,
16
Department of Biology, University of the Balearic Islands (UIB). Crta. Valdemossa km 7.5, CP:
17
07122 Palma de Mallorca, Spain. Telephone: (+34) 971172525, e-mail:
[email protected] 18 19
KEYWORDS: Warming, Eutrophication, planktonic metabolic rates, activation energies, Q10,
20
dissolved organic nitrogen (DON), dissolved organic matter (DOM), Baltic Sea.
ACS Paragon Plus Environment
Environmental Science & Technology
21
Page 2 of 36
ABSTRACT
22
Increased anthropogenic pressures on coastal marine ecosystems in the last century are
23
threatening their biodiversity and functioning. Two major stressors affecting these systems are
24
global warming and increases in nutrient loadings. Global warming is expected to increase both
25
atmospheric and water temperatures and increase precipitation and terrestrial runoff, further
26
increasing organic matter and nutrient inputs to coastal areas. Dissolved organic nitrogen (DON)
27
concentrations frequently exceed that of dissolved inorganic nitrogen in aquatic systems. Many
28
components of the DON pool have been shown to supply nitrogen nutrition to phytoplankton and
29
bacteria. Predictions of how global warming and eutrophication will affect metabolic rates and
30
dissolved oxygen dynamics in the future are needed to elucidate its impacts on biodiversity and
31
ecosystem functioning. Here, we experimentally determine effects of simultaneous DON
32
additions and warming on planktonic community metabolism in the Baltic Sea, the largest
33
coastal area suffering from eutrophication-driven hypoxia. Both bacterioplankton community
34
composition and metabolic rates changed in relation to temperature. DON additions from
35
wastewater treatment plant effluents significantly increased activation energies for community
36
respiration and gross primary production. Activation energies for community respiration were
37
higher than those for gross primary production. Results support the prediction that warming of
38
the Baltic Sea will enhance planktonic respiration rates faster than planktonic primary
39
production. Higher increases in respiration rates than in production may lead to depletion of the
40
oxygen pool, further aggravating hypoxia in the Baltic Sea.
41 42 43
ACS Paragon Plus Environment
2
Page 3 of 36
44 45
Environmental Science & Technology
INTRODUCTION The coastal ocean plays a major role in the global biogeochemical cycles of carbon,
46
nitrogen and oxygen1. The coastal zone, representing about 10% of the ocean surface, supports
47
~20% of oceanic primary production and ~10% of global primary production2. This provides the
48
resource base and habitat for diverse coastal communities. Increasing anthropogenic pressures
49
are threatening the biodiversity and functioning of these coastal ecosystems3, 4. Ocean warming
50
due to climate change is becoming a critical stressor. Metabolic theory predicts that warming
51
will enhance respiration more than primary production5, which could result in a consumption of
52
oxygen. Moreover, warming affects multiple interacting processes affecting oxygen dynamics
53
such as intensifying stratification, decreasing oxygen solubility, rising sea level, and intensifying
54
coastal upwelling, among others. In all, this points to the risk that warming may generate
55
excessive oxygen consumption leading to hypoxia (< 2 mg O2/l) or anoxia (undetectable levels
56
of oxygen) if oxygen is not replenished.
57
Oxygen deficiencies have increased in frequency, duration, and severity in the world’s
58
coastal areas during the last decades6, and hypoxia is emerging as a major threat to marine
59
coastal biodiversity7. The Baltic Sea has the largest area suffering from eutrophication-driven
60
hypoxia8 and it has increased by 10-fold during the last 115 years9. Recently, warming is
61
worsening oxygen conditions in the Baltic Sea, due to increased respiration rates with higher
62
temperatures9, and temperature is one of the key factors controlling the extent of hypoxia10, 11.
63
The decline in dissolved oxygen can cause death of marine organisms and catastrophic changes
64
of ecosystems7. Species vulnerable to low oxygen, in particular fishes and crustaceans, are
65
removed and replaced by tolerant ones. Species diversity and the number of trophic levels are
66
reduced. The deleterious effects of oxygen depletion can be further aggravated by ocean
ACS Paragon Plus Environment
3
Environmental Science & Technology
67
Page 4 of 36
warming12.
68
A primary driver of hypoxia is eutrophication. Nutrient loading to rivers and coastal
69
waters have increased over the last century, leading to accelerated primary production, algal
70
blooms, accumulation of organic matter, and excessive oxygen consumption. Managerial efforts
71
to prevent and mitigate hypoxia are focused on reducing inorganic nutrient loading (e.g. Nitrates
72
Directive (91/676/EEC)), and disregard the organic fraction of the nutrient pool. However, the
73
concentration of dissolved organic nitrogen (DON) frequently exceeds that of inorganic nitrogen
74
in both marine and freshwaters13, 14. Studies indicate that many components of the DON pool are
75
available to phytoplankton and bacteria13, 15, being a dynamic actor in the North Sea16 and in the
76
Gulf of Riga17, 18. For instance, in the Gulf of Riga DON has been shown to be a major source of
77
nitrogen to phytoplankton17. Nevertheless, the possible influence of DON on eutrophication and
78
hypoxia has been neglected in most studies. Climate change may further enhance DON loading
79
to coastal areas by increasing atmospheric deposition, runoff19, flooding and ice melting. How
80
climate change will affect dissolved oxygen dynamics, through temperature effects as well as
81
altered DON loading, remains an open question, with potential consequences to the biodiversity,
82
structure and function of coastal systems.
83
Sea-surface temperatures in the Baltic Sea are forecasted to increase between 2 and 4 ºC
84
by the end of the Century20. The primary objective of this study is to assess how metabolic rates
85
(gross primary production (GPP), community respiration (CR) and bacterial production (BP))
86
respond to warming, and how DON loading may modulate that response. Two sources of DON
87
were used – water from a boreal river and effluents from a wastewater treatment plant (WWTP).
88
The response in metabolic rates was evaluated with and without DON enrichment both by
89
experimental warming, and seasonal variability.
ACS Paragon Plus Environment
4
Page 5 of 36
Environmental Science & Technology
90 91
METHODS
92
Sampling
93
A natural marine planktonic community from the Baltic Proper was collected 10 km off the
94
east coast of Öland, Sweden, at the Linnaeus Microbial Observatory (LMO, N 56°55.851, E
95
17°03.640). Water was sampled from 2 m depth and filtered through a 150-µm net to remove
96
large grazers.
97
For DON enrichment, we collected river water from Emån River that drains into the Baltic
98
Proper, and effluent from the WWTP in Kalmar. The Emån catchment area is dominated by
99
boreal forest (69 %) and 2 % is peat-land21. Samples for DON enrichment were filtered using
100
pre-combusted (450ºC, 4 h) glass-fiber (GF/F Whatman) filters and 0.2 µm membrane filters and
101
frozen until the start of the experiment. All equipment used for handling the samples was acid
102
washed.
103 104
Warming experiments
105
Treatments
106
Two warming experiments were performed, one in summer (beginning of September 2013)
107
and one in winter (March 2014). Each experiment had two DON treatments – one amended with
108
river water and one amended with WWTP effluent - and one control (seawater).
109
treatments were incubated at 4 different temperatures (-2, 0, +2 and +4 ºC from sampling
110
temperature, 18 ºC in summer and 4 ºC in winter) leading to a total of 12 treatments (Fig. S1).
111
The DON treatments consisted of 1:10 volume:volume of DON source to seawater. An
112
autoclaved sea salt solution22 was added with the DON to keep salinity constant across
These
ACS Paragon Plus Environment
5
Environmental Science & Technology
113
Page 6 of 36
treatments22.
114 115 116
Metabolic rates Community respiration (CR) was assessed by oxygen consumption as measured by
117
Winkler titrations, i.e. CR was calculated as the difference between the initial oxygen
118
concentration and the oxygen concentration after incubation in darkness and reported in mmol
119
O2 m−3 day−1. Water was carefully siphoned into 55 mL narrow-mouth Winkler bottles. Bottles
120
for measuring initial oxygen concentration were fixed immediately (9-10 bottles per treatment).
121
Another 5-6 bottles for each treatment were incubated in the dark at the four different
122
temperature regimes in controlled temperature chambers. Incubations lasted for 24 and 48h in the
123
summer experiment and 48 and 72h in winter. Dissolved oxygen in the bottles was fixed
124
immediately and analyzed by high-precision Winkler titration, following Carritt and Carpenter23,
125
using a precise automated titration system with potentiometric (redox electrode) endpoint
126
detection (Mettler Toledo, DL28 titrator)24.
127
Additional 2 L bottles were incubated to determine nutrient content and bacterial
128
production and bacterial community composition. Bacterial production (BP) was measured for
129
initial samples as well as after 24 and 48h in the summer experiment and after 48 and 72h in
130
winter. BP was estimated by measuring incorporation of 3H-leucine following Smith and
131
Azam25. Water samples (1.5 ml, 3 replicates and 1 killed control (sample with 5% trichloroacetic
132
acid (TCA)) were incubated 60 minutes with 98.8 nM of 3H-leucine (13.4 Ci mmol-1). The
133
incubation was terminated by adding TCA (5% final concentration). The samples were then
134
centrifuged at 16000g for 10 minutes and the bacterial pellet was washed once with 5% TCA and
135
once with 80% ethanol. After the supernatant was discarded, 0.5 ml of scintillation cocktail
ACS Paragon Plus Environment
6
Page 7 of 36
Environmental Science & Technology
136
(Ecoscint A, Kimberly Research) was added and 3H -activity measured on a Beckman LS 6500
137
scintillation counter. BP was calculated assuming a leucine to carbon conversion factor of 1.5 kg
138
C mol-1 leucine26.
139 140
Experiments conducted at in situ temperature.
141
Treatments
142
A total of eight experiments were performed during the seasons of summer (3), spring (2),
143
autumn (2) and winter (1). Each experiment consisted of 5 different treatments, with additions of
144
DON amendments (4 experiments with river water and 4 experiments with WWTP effluents).
145
One DON treatment consisted in DON source to a proportion of 1:10 in seawater (1:10) and a
146
second DON treatment was seawater with a 1:5 portion of DON source (1:5). There was also a
147
treatment with the addition of the same concentration of inorganic nutrients (nitrate, nitrite and
148
phosphate) that were contained in the DON 1:5 treatment (IN). Finally there was a control (C)
149
treatment with only seawater, and a diluted control (CD) consisting of seawater diluted with
150
autoclaved milli-Q water to have the same portion of community that the 1:10, 1:5 and IN
151
treatments (Fig. S1). To keep salinity constant in all treatments, a salt solution was added with
152
the amendments/dilutions.
153
When evaluating the experiments, treatments were grouped in three different types: No
154
addition, including both C and CD; DON addition, including 1:10 and 1:5 treatments; and IN.
155
DON addition treatments were divided in river additions and WWTP effluent additions.
156
Metabolic rates
157
Changes in dissolved oxygen (DO) in closed bottles were assumed to result from
158
biological metabolic processes and to represent net community production (NCP = GPP − CR).
ACS Paragon Plus Environment
7
Environmental Science & Technology
Page 8 of 36
159
Water from the respective treatments was siphoned carefully into 2.3 L glass bottles sealed with
160
gas tight stoppers. Bottles were incubated at the in situ temperature in temperature-controlled
161
chambers during one week. Oxygen was measured every minute using optical oxygen sensors
162
(optodes) and a 10-channel fiber optic oxygen transmitter (Oxy-10, PreSens®).
163
Incubations were performed under natural light regime, illuminated by artificial light
164
(OSRAM L36W/865 Lumilux Daylight), with a mean photosynthetically active radiation (PAR)
165
intensity of 1373.2 µW/cm2. Light hours ranged from 7h 20m in the autumn experiment
166
performed in December 2013 to 17h 10m on the spring experiment in June 2013. Due to the
167
large difference in light hours between experiments, GPP was standardized by light hours
168
(GPP/light hours).
169
During the night changes in DO are produced by respiration, as in the absence of light no
170
photosynthetic production occurs. CR was calculated from the rate of change in oxygen at night -
171
from half an hour after lights went off to half an hour before light went on (NCP in darkness =
172
CR). NCP was calculated from the rate of change in DO at one-minute intervals accumulated
173
over each 24 h period. Assuming that daytime CR equals that during the night, GPP was
174
estimated as the sum of NCP and CR. In order to derive daily metabolic rates, individual
175
estimates of GPP, NCP and CR resolved at one-minute intervals were accumulated over each 24-
176
h period during experiments and reported in mmol O2 m−3 day−1. BP was measured on days 0, 1,
177
3, 5 and 7.
178
Methods for water chemistry and bacterial community composition are given as Supporting
179
Information (SI).
180 181
Calculation of activation energies (Ea) and Q10 values
ACS Paragon Plus Environment
8
Page 9 of 36
Environmental Science & Technology
182
Arrhenius plots of the natural logarithm of the given metabolic rate against the inverse of
183
the temperature (Kelvin) multiplied by the Boltzmann’s constant (8.62 x 10-5 eV k-1) were used
184
to calculate activation energies. An estimation of the activation energy for GPP, CR and BP (Ea,
185
units eV) was derived from the slope of the Arrhenius plot. Ea was converted from eV to J mol-1
186
using a conversion factor of 96486.9.
187
The Q10 (the relative rate of change in a given metabolic rate expected for a 10ºC
188
temperature increase) was calculated by fitting the equation proposed by Raven and Geider27:
189 190
where R is the gas constant (8.314472 mol-1 K-1), T is the mean absolute temperature across the
191
range over which Q10 was measured (K) and Ea is the activation energy (J mol-1)27.
192
Statistics
193
The data from both warming experiments were combined in a single analysis to test for
194
the relationship between metabolic rates (the natural logarithm) and the inverse of temperature
195
(1/kT) by mixed effects model. To account for temporal pseudo-replication we used sampling
196
date as random effect. We used sampling treatments (addition of DON from rivers (River), from
197
effluent (WWTP) and control (Seawater)) as fixed factors.
198
Additionally, linear mixed effects models were used to test for relationships between
199
temperature and metabolic rates using data from experiments conducted at in situ temperature.
200
To account for temporal pseudo-replication in the statistical model, we included sampling dates
201
as random effects and jar identity to account for community pseudo-replication. In addition DON
202
treatments (addition of DON from rivers (River) and from effluent (WWTP)), no addition
203
(Seawater) or inorganic nutrients additions (IN)) were used as fixed factors to check differences
204
between treatments. We used mixed effects models to test for relationships between temperature
ACS Paragon Plus Environment
9
Environmental Science & Technology
Page 10 of 36
205
and metabolic rates as these models allow accounting for temporal and community pseudo-
206
replication by using sampling dates and jar identity as random effects. Mixed effects models
207
have been used in similar studies previously 28-30. We used Post-hoc comparison using
208
generalized linear hypothesis test (glht) with “Tukey” to test for significant differences between
209
treatments. Pseudo-R2 of the models were calculated following Xu 200331. All statistical tests
210
were made using R statistical software.
211 212
RESULTS
213
Water chemistry
214
Nutrients content in seawater and rivers and WWTP effluent are shown in table S1.
215
Nutrient content in WWTP effluent waters was much higher than in the river water (table S1).
216
Conversely, dissolved organic carbon (DOC) content in river waters was higher than in WWTP
217
effluents. Nutrient content in seawater tended to be higher during winter months and decreased
218
in summer (table S1).
219 220 221
Warming experiments Incubation temperatures in the warming experiments ranged from 1.7ºC (-2ºC treatment
222
in the warming experiment performed in winter) to 22.6 ºC (+4ºC treatment in the summer
223
experiment). Different treatments showed a different dependence with temperature: Mixed effect
224
model results showed significant differences in the slope (p < 0.03) and in the intercept (p
