Dissolved Organic Nitrogen Inputs from Wastewater Treatment Plant

Sep 10, 2015 - (31) All statistical tests were made using R statistical software. ..... This research is a contribution to the “The Role of Dissolve...
0 downloads 10 Views 2MB Size
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