Dissolved Organic Nitrogen Inputs from Wastewater Treatment Plant

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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

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Environmental Science & Technology

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Dissolved organic nitrogen inputs from wastewater

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treatment plant effluents increase responses of

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planktonic metabolic rates to warming.

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Raquel Vaquer-Sunyer*†, Daniel J. Conley†, Saraladevi Muthusamy‡, Markus V. Lindh‡, Jarone

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Pinhassi‡ and Emma S. Kritzberg§

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AUTHOR ADDRESS:

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† Department of Geology, Lund University, Sölvegatan 12, SE-223 62, Lund, Sweden

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‡ Centre for Ecology and Evolution in Microbial model Systems – EEMiS, Linnaeus University,

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SE-39182 Kalmar, Sweden

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§ Department of Biology, Lund University, Sölvegatan 37, SE-223 62, Lund, Sweden

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*Corresponding author: Raquel Vaquer-Sunyer. New address: Interdisciplinary Ecology group,

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Department of Biology, University of the Balearic Islands (UIB). Crta. Valdemossa km 7.5, CP:

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07122 Palma de Mallorca, Spain. Telephone: (+34) 971172525, e-mail: [email protected]

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KEYWORDS: Warming, Eutrophication, planktonic metabolic rates, activation energies, Q10,

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dissolved organic nitrogen (DON), dissolved organic matter (DOM), Baltic Sea.

ACS Paragon Plus Environment


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ABSTRACT

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Increased anthropogenic pressures on coastal marine ecosystems in the last century are

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threatening their biodiversity and functioning. Two major stressors affecting these systems are

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global warming and increases in nutrient loadings. Global warming is expected to increase both

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atmospheric and water temperatures and increase precipitation and terrestrial runoff, further

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increasing organic matter and nutrient inputs to coastal areas. Dissolved organic nitrogen (DON)

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concentrations frequently exceed that of dissolved inorganic nitrogen in aquatic systems. Many

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components of the DON pool have been shown to supply nitrogen nutrition to phytoplankton and

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bacteria. Predictions of how global warming and eutrophication will affect metabolic rates and

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dissolved oxygen dynamics in the future are needed to elucidate its impacts on biodiversity and

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ecosystem functioning. Here, we experimentally determine effects of simultaneous DON

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additions and warming on planktonic community metabolism in the Baltic Sea, the largest

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coastal area suffering from eutrophication-driven hypoxia. Both bacterioplankton community

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composition and metabolic rates changed in relation to temperature. DON additions from

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wastewater treatment plant effluents significantly increased activation energies for community

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respiration and gross primary production. Activation energies for community respiration were

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higher than those for gross primary production. Results support the prediction that warming of

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the Baltic Sea will enhance planktonic respiration rates faster than planktonic primary

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production. Higher increases in respiration rates than in production may lead to depletion of the

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oxygen pool, further aggravating hypoxia in the Baltic Sea.

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INTRODUCTION The coastal ocean plays a major role in the global biogeochemical cycles of carbon,

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nitrogen and oxygen1. The coastal zone, representing about 10% of the ocean surface, supports

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~20% of oceanic primary production and ~10% of global primary production2. This provides the

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resource base and habitat for diverse coastal communities. Increasing anthropogenic pressures

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are threatening the biodiversity and functioning of these coastal ecosystems3, 4. Ocean warming

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due to climate change is becoming a critical stressor. Metabolic theory predicts that warming

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will enhance respiration more than primary production5, which could result in a consumption of

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oxygen. Moreover, warming affects multiple interacting processes affecting oxygen dynamics

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such as intensifying stratification, decreasing oxygen solubility, rising sea level, and intensifying

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coastal upwelling, among others. In all, this points to the risk that warming may generate

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excessive oxygen consumption leading to hypoxia (< 2 mg O2/l) or anoxia (undetectable levels

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of oxygen) if oxygen is not replenished.

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Oxygen deficiencies have increased in frequency, duration, and severity in the world’s

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coastal areas during the last decades6, and hypoxia is emerging as a major threat to marine

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coastal biodiversity7. The Baltic Sea has the largest area suffering from eutrophication-driven

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hypoxia8 and it has increased by 10-fold during the last 115 years9. Recently, warming is

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worsening oxygen conditions in the Baltic Sea, due to increased respiration rates with higher

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temperatures9, and temperature is one of the key factors controlling the extent of hypoxia10, 11.

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The decline in dissolved oxygen can cause death of marine organisms and catastrophic changes

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of ecosystems7. Species vulnerable to low oxygen, in particular fishes and crustaceans, are

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removed and replaced by tolerant ones. Species diversity and the number of trophic levels are

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reduced. The deleterious effects of oxygen depletion can be further aggravated by ocean


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warming12.

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A primary driver of hypoxia is eutrophication. Nutrient loading to rivers and coastal

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waters have increased over the last century, leading to accelerated primary production, algal

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blooms, accumulation of organic matter, and excessive oxygen consumption. Managerial efforts

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to prevent and mitigate hypoxia are focused on reducing inorganic nutrient loading (e.g. Nitrates

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Directive (91/676/EEC)), and disregard the organic fraction of the nutrient pool. However, the

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concentration of dissolved organic nitrogen (DON) frequently exceeds that of inorganic nitrogen

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in both marine and freshwaters13, 14. Studies indicate that many components of the DON pool are

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available to phytoplankton and bacteria13, 15, being a dynamic actor in the North Sea16 and in the

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Gulf of Riga17, 18. For instance, in the Gulf of Riga DON has been shown to be a major source of

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nitrogen to phytoplankton17. Nevertheless, the possible influence of DON on eutrophication and

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hypoxia has been neglected in most studies. Climate change may further enhance DON loading

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to coastal areas by increasing atmospheric deposition, runoff19, flooding and ice melting. How

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climate change will affect dissolved oxygen dynamics, through temperature effects as well as

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altered DON loading, remains an open question, with potential consequences to the biodiversity,

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structure and function of coastal systems.

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Sea-surface temperatures in the Baltic Sea are forecasted to increase between 2 and 4 ºC

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by the end of the Century20. The primary objective of this study is to assess how metabolic rates

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(gross primary production (GPP), community respiration (CR) and bacterial production (BP))

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respond to warming, and how DON loading may modulate that response. Two sources of DON

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were used – water from a boreal river and effluents from a wastewater treatment plant (WWTP).

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The response in metabolic rates was evaluated with and without DON enrichment both by

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experimental warming, and seasonal variability.


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METHODS

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Sampling

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A natural marine planktonic community from the Baltic Proper was collected 10 km off the

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east coast of Öland, Sweden, at the Linnaeus Microbial Observatory (LMO, N 56°55.851, E

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17°03.640). Water was sampled from 2 m depth and filtered through a 150-µm net to remove

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large grazers.

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For DON enrichment, we collected river water from Emån River that drains into the Baltic

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Proper, and effluent from the WWTP in Kalmar. The Emån catchment area is dominated by

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boreal forest (69 %) and 2 % is peat-land21. Samples for DON enrichment were filtered using

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pre-combusted (450ºC, 4 h) glass-fiber (GF/F Whatman) filters and 0.2 µm membrane filters and

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frozen until the start of the experiment. All equipment used for handling the samples was acid

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washed.

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Warming experiments

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Treatments

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Two warming experiments were performed, one in summer (beginning of September 2013)

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and one in winter (March 2014). Each experiment had two DON treatments – one amended with

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river water and one amended with WWTP effluent - and one control (seawater).

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treatments were incubated at 4 different temperatures (-2, 0, +2 and +4 ºC from sampling

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temperature, 18 ºC in summer and 4 ºC in winter) leading to a total of 12 treatments (Fig. S1).

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The DON treatments consisted of 1:10 volume:volume of DON source to seawater. An

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autoclaved sea salt solution22 was added with the DON to keep salinity constant across

These


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treatments22.

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Metabolic rates Community respiration (CR) was assessed by oxygen consumption as measured by

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Winkler titrations, i.e. CR was calculated as the difference between the initial oxygen

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concentration and the oxygen concentration after incubation in darkness and reported in mmol

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O2 m−3 day−1. Water was carefully siphoned into 55 mL narrow-mouth Winkler bottles. Bottles

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for measuring initial oxygen concentration were fixed immediately (9-10 bottles per treatment).

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Another 5-6 bottles for each treatment were incubated in the dark at the four different

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temperature regimes in controlled temperature chambers. Incubations lasted for 24 and 48h in the

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summer experiment and 48 and 72h in winter. Dissolved oxygen in the bottles was fixed

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immediately and analyzed by high-precision Winkler titration, following Carritt and Carpenter23,

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using a precise automated titration system with potentiometric (redox electrode) endpoint

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detection (Mettler Toledo, DL28 titrator)24.

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Additional 2 L bottles were incubated to determine nutrient content and bacterial

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production and bacterial community composition. Bacterial production (BP) was measured for

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initial samples as well as after 24 and 48h in the summer experiment and after 48 and 72h in

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winter. BP was estimated by measuring incorporation of 3H-leucine following Smith and

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Azam25. Water samples (1.5 ml, 3 replicates and 1 killed control (sample with 5% trichloroacetic

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acid (TCA)) were incubated 60 minutes with 98.8 nM of 3H-leucine (13.4 Ci mmol-1). The

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incubation was terminated by adding TCA (5% final concentration). The samples were then

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centrifuged at 16000g for 10 minutes and the bacterial pellet was washed once with 5% TCA and

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once with 80% ethanol. After the supernatant was discarded, 0.5 ml of scintillation cocktail


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(Ecoscint A, Kimberly Research) was added and 3H -activity measured on a Beckman LS 6500

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scintillation counter. BP was calculated assuming a leucine to carbon conversion factor of 1.5 kg

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C mol-1 leucine26.

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Experiments conducted at in situ temperature.

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Treatments

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A total of eight experiments were performed during the seasons of summer (3), spring (2),

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autumn (2) and winter (1). Each experiment consisted of 5 different treatments, with additions of

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DON amendments (4 experiments with river water and 4 experiments with WWTP effluents).

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One DON treatment consisted in DON source to a proportion of 1:10 in seawater (1:10) and a

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second DON treatment was seawater with a 1:5 portion of DON source (1:5). There was also a

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treatment with the addition of the same concentration of inorganic nutrients (nitrate, nitrite and

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phosphate) that were contained in the DON 1:5 treatment (IN). Finally there was a control (C)

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treatment with only seawater, and a diluted control (CD) consisting of seawater diluted with

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autoclaved milli-Q water to have the same portion of community that the 1:10, 1:5 and IN

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treatments (Fig. S1). To keep salinity constant in all treatments, a salt solution was added with

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the amendments/dilutions.

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When evaluating the experiments, treatments were grouped in three different types: No

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addition, including both C and CD; DON addition, including 1:10 and 1:5 treatments; and IN.

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DON addition treatments were divided in river additions and WWTP effluent additions.

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Metabolic rates

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Changes in dissolved oxygen (DO) in closed bottles were assumed to result from

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biological metabolic processes and to represent net community production (NCP = GPP − CR).


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Water from the respective treatments was siphoned carefully into 2.3 L glass bottles sealed with

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gas tight stoppers. Bottles were incubated at the in situ temperature in temperature-controlled

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chambers during one week. Oxygen was measured every minute using optical oxygen sensors

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(optodes) and a 10-channel fiber optic oxygen transmitter (Oxy-10, PreSens®).

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Incubations were performed under natural light regime, illuminated by artificial light

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(OSRAM L36W/865 Lumilux Daylight), with a mean photosynthetically active radiation (PAR)

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intensity of 1373.2 µW/cm2. Light hours ranged from 7h 20m in the autumn experiment

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performed in December 2013 to 17h 10m on the spring experiment in June 2013. Due to the

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large difference in light hours between experiments, GPP was standardized by light hours

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(GPP/light hours).

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During the night changes in DO are produced by respiration, as in the absence of light no

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photosynthetic production occurs. CR was calculated from the rate of change in oxygen at night -

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from half an hour after lights went off to half an hour before light went on (NCP in darkness =

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CR). NCP was calculated from the rate of change in DO at one-minute intervals accumulated

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over each 24 h period. Assuming that daytime CR equals that during the night, GPP was

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estimated as the sum of NCP and CR. In order to derive daily metabolic rates, individual

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estimates of GPP, NCP and CR resolved at one-minute intervals were accumulated over each 24-

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h period during experiments and reported in mmol O2 m−3 day−1. BP was measured on days 0, 1,

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3, 5 and 7.

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Methods for water chemistry and bacterial community composition are given as Supporting

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Information (SI).

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Calculation of activation energies (Ea) and Q10 values


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Arrhenius plots of the natural logarithm of the given metabolic rate against the inverse of

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the temperature (Kelvin) multiplied by the Boltzmann’s constant (8.62 x 10-5 eV k-1) were used

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to calculate activation energies. An estimation of the activation energy for GPP, CR and BP (Ea,

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units eV) was derived from the slope of the Arrhenius plot. Ea was converted from eV to J mol-1

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using a conversion factor of 96486.9.

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The Q10 (the relative rate of change in a given metabolic rate expected for a 10ºC

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temperature increase) was calculated by fitting the equation proposed by Raven and Geider27:

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where R is the gas constant (8.314472 mol-1 K-1), T is the mean absolute temperature across the

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range over which Q10 was measured (K) and Ea is the activation energy (J mol-1)27.

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Statistics

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The data from both warming experiments were combined in a single analysis to test for

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the relationship between metabolic rates (the natural logarithm) and the inverse of temperature

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(1/kT) by mixed effects model. To account for temporal pseudo-replication we used sampling

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date as random effect. We used sampling treatments (addition of DON from rivers (River), from

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effluent (WWTP) and control (Seawater)) as fixed factors.

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Additionally, linear mixed effects models were used to test for relationships between

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temperature and metabolic rates using data from experiments conducted at in situ temperature.

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To account for temporal pseudo-replication in the statistical model, we included sampling dates

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as random effects and jar identity to account for community pseudo-replication. In addition DON

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treatments (addition of DON from rivers (River) and from effluent (WWTP)), no addition

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(Seawater) or inorganic nutrients additions (IN)) were used as fixed factors to check differences

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between treatments. We used mixed effects models to test for relationships between temperature


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and metabolic rates as these models allow accounting for temporal and community pseudo-

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replication by using sampling dates and jar identity as random effects. Mixed effects models

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have been used in similar studies previously 28-30. We used Post-hoc comparison using

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generalized linear hypothesis test (glht) with “Tukey” to test for significant differences between

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treatments. Pseudo-R2 of the models were calculated following Xu 200331. All statistical tests

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were made using R statistical software.

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RESULTS

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Water chemistry

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Nutrients content in seawater and rivers and WWTP effluent are shown in table S1.

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Nutrient content in WWTP effluent waters was much higher than in the river water (table S1).

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Conversely, dissolved organic carbon (DOC) content in river waters was higher than in WWTP

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effluents. Nutrient content in seawater tended to be higher during winter months and decreased

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in summer (table S1).

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Warming experiments Incubation temperatures in the warming experiments ranged from 1.7ºC (-2ºC treatment

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in the warming experiment performed in winter) to 22.6 ºC (+4ºC treatment in the summer

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experiment). Different treatments showed a different dependence with temperature: Mixed effect

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model results showed significant differences in the slope (p < 0.03) and in the intercept (p

Dissolved Organic Nitrogen Inputs from Wastewater Treatment Plant

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