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History matters: pre-exposure to wastewater enhances pesticide toxicity in invertebrates Jochen Peter Zubrod, Dominic Englert, Simon Lüderwald, Sandra Poganiuch, Mirco Bundschuh, and Ralf Schulz Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b01303 • Publication Date (Web): 06 Jul 2017 Downloaded from http://pubs.acs.org on July 10, 2017
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Environmental Science & Technology
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History matters: pre-exposure to wastewater
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enhances pesticide toxicity in invertebrates
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Jochen P. Zubrod†,*, Dominic Englert†, Simon Lüderwald†, Sandra Poganiuch†, Ralf Schulz†,
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Mirco Bundschuh‡
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†
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Landau, Germany
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‡
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Institute for Environmental Sciences, University of Koblenz-Landau, Fortstraße 7, D-76829
Department of Aquatic Sciences and Assessment, Swedish University of Agricultural
Sciences, Lennart Hjelms väg 9, SWE-75007 Uppsala, Sweden
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KEYWORDS: agriculture – multiple stress – shredder – neonicotinoid – wastewater
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WORD COUNT (limit is 7,000):
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Abstract: 194; MS body: 4,123; Acknowledgments: 109; Description of Supporting
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Information: 42; Figures: 3 x 600; Tables: 1 x 300 Total: 6,568
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ABSTRACT (150-200 words)
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Disturbance regimes determine communities’ structure and functioning. Nonetheless, little
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effort has been undertaken to understand interactions of press and pulse disturbances. In this
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context, leaf-shredding macroinvertebrates can be chronically exposed to wastewater
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treatment plant effluents (i.e., press disturbance) before experiencing pesticide exposure
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following agricultural run-off (i.e., pulse disturbance). It is assumed that wastewater pre-
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exposure alters animals’ sensitivity to pesticides. To test this hypothesis, we exposed model-
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populations of the shredder Gammarus fossarum to wastewater at three field-relevant dilution
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levels (i.e., 0%, 50%, and 100%). After 2, 4, and 6 weeks, survival, leaf consumption, dry
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weight, and energy reserves were monitored. Additionally, animals were assessed for their
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sensitivity towards the neonicotinoid insecticide thiacloprid using their feeding rate as
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response variable. Both wastewater treatments reduced gammarids’ survival, leaf
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consumption, dry weight, and energy reserves. Moreover, both wastewater pre-exposure
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scenarios increased animals’ sensitivity towards thiacloprid by up to 2.5 times compared to
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the control. Our results thus demonstrate that press disturbance as posed by wastewater pre-
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exposure can enhance susceptability of key players in ecosystem functioning to further (pulse)
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disturbances. Therefore, applying mitigation measures such as advanced treatment
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technologies seems sensible to support functional integrity in the multiple-stress situation.
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Environmental Science & Technology
INTRODUCTION
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Prevailing disturbance regimes determine community structure and functioning in
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ecosystems. Based on their temporal patterns they may be categorized as press (constant,
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maintained level) or pulse (short-term, sharply delineated) disturbances.1,2 Although the so-
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called multiple-stress situation is generally acknowledged and research on the interaction of a
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diverse range of natural and anthropogenic stressors is available,3,4 little effort has been
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undertaken to understand the interactions of press and pulse disturbances (but see for instance
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Parkyn and Collier5 and Bruder et al.6).
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In this context, the continuous discharge of wastewater from municipal wastewater
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treatment plants represents a press disturbance regime in many low-order streams. This is
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because conventional wastewater treatment (i.e., without use of advanced treatment
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technologies) is not capable of removing all chemicals of anthropogenic origin resulting in a
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continuous exposure of the receiving ecosystems to a wide array of inorganic and organic
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micropollutants and high concentrations of potentially toxic nutrients.7,8 In many of these
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streams the breakdown of leaf litter is a critical ecosystem-level process with leaf-shredding
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macroinvertebrates acting as key players.9,10 However, treated wastewater triggers detrimental
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effects in the physiological fitness and functional performance of shredders such as members
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of the genus Gammarus (Crustacea; Amphipoda)11,12 that frequently inhabit low-order
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streams into which wastewater is discharged.8,13 In consequence, treated wastewater can
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negatively affect in-stream leaf processing rates, while the strength of individual- and
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ecosystem-level effects depends inter alia on the dilution factor of wastewater in the
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receiving stream.8 Low-order streams, however, often also receive pesticides (e.g.,
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insecticides) from adjacent agricultural areas via spray drift and surface runoff.14,15 Since the
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exposure to treated wastewater can reduce the physiological fitness (e.g., energy reserves) of
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aquatic invertebrates (e.g., gammarids12), their sensitivity towards pulse disturbances due to
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pesticide exposure might be modified.16 3 ACS Paragon Plus Environment
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To empirically test this hypothesis, we exposed the amphipod shredder Gammarus
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fossarum KOCH for up to 6 weeks in stream microcosms to wastewater simulating three
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dilution levels (i.e., 0%, 50%, and 100%). After 2, 4, and 6 weeks subsamples of the exposed
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populations were assessed for their sensitivity – employing dose-response experiments –
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towards the model insecticide thiacloprid using the feeding rate of G. fossarum as sublethal
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response variable. During pre-exposure, animals’ survival, energy uptake (i.e., leaf feeding),
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and physiological fitness (judged by animals’ dry weight and energy reserves) were
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monitored to facilitate a mechanistic understanding of a potential wastewater-induced
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modification of their senstivity towards the model insecticide. We hypothesized that
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wastewater exposure would result in a lower energy uptake and physiological fitness of
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Gammarus11 and, at the same time, to trigger a higher senstivity towards the model
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insecticide.16 Moreover, we anticipated these effects to be more pronounced with an
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increasing share of the wastewater during the pre-exposure,17 while genetic erosion due to the
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selective removal of wastewater-sensitive genotypes from the Gammarus populations was
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expected to increase18 or decrease19 insecticide sensitivity over time.
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MATERIAL AND METHODS
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Leaf material
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Leaves of Alnus glutinosa (L.) GAERTN. (black alder) were collected in October 2014 near
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Landau, Germany (49°11’N; 8°05’E) and stored at -20°C until further processing. Leaves
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were deployed for 14 days in fine-mesh bags in the Rodenbach, Germany (49°33’N, 8°02’E),
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upstream of any agricultural activity, settlement, and wastewater inlet, a method known to
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establish a natural microbial community on the leaf material.20 Back in the laboratory,
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unconditioned leaves were added to the retrieved leaf material and the mixed leaves were kept
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in aerated conditioning medium21 at 16±1°C in total darkness for another 14 days before
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being used as microbial inoculum. 4 ACS Paragon Plus Environment
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Leaf strips of 3 to 5 cm × 5 to 9 cm and discs of 2 cm diameter that served as food in the
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stream microcosms and during the dose-response experiments (see below), respectively, were
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cut from unconditioned black alder leaves using scissors and a cork borer, respectively, and
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placed in aerated circular aquaria (150 strips or 660 discs per aquarium). These aquaria were
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filled with 14 L of conditioning medium together with 50 g fresh weight of the microbial
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inoculum. Conditioning took place at 16±1°C in total darkness and lasted 12 days with a
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complete medium exchange after 6 days. Afterwards, leaf strips and discs were sterilized by
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autoclaving to prevent microbial re-colonization of leaf material and thus uncontrollable
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indirect effects during the experiment.22 Subsequently, strips and discs were dried at 60 °C for
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48 and 24 h, respectively, and weighed in portions of 24 (to the nearest 0.1 mg) and two (to
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the nearest 0.01 mg), respectively. Before use in the experiment, leaf material was re-soaked
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in test medium (i.e., SAM-5S23 containing 147 mg CaCl2×2H2O, 85.5 mg NaHCO3, 61.5 mg
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MgSO4×7H2O, 3.8 mg KCl, and 1.03 mg NaBr per liter) for 48 h to reduce buoyancy.
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Test organisms
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Gammarus fossarum were kick-sampled in the near-natural stream Hainbach, Germany
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(49°14’N; 8°03’E; the population inhabiting this sample site is exclusively composed of
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animals belonging to cryptic lineage B24) 7 days prior to the start of the experiment. In the
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laboratory, gammarids were subjected to a passive underwater separation technique as
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described by Franke25: animals were placed on top of a stack of sieves situated in an aerated
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tank filled with SAM-5S with sieves’ mesh sizes progressively becoming smaller towards the
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bottom of the tank. Making use of gammarids’ negative phototaxis, by illuminating the tank
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from above (~400 lx), a downward movement was initiated that divided them into different
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size classes. Only adult males – identified by their position in precopula pairs – of
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approximately 6 to 8 mm body length, which were visually free from acanthocephalan
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parasites, were used in order to reduce variability in inter alia the feeding behavior of 5 ACS Paragon Plus Environment
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Gammarus.26,27 Throughout the acclimation phase in the laboratory (i.e., 7 days), animals
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were kept in aerated stream water from the collection site in a climate-controlled chamber at
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16±1 °C in total darkness, while they were fed ad libitum with pre-conditioned black alder
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leaves. At the start of the experiment 30 animals were shock-frozen in liquid nitrogen and
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stored
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-80 °C for determining gammarids’ dry weights and energy reserves at test initiation (i.e.,
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before exposure to wastewater).
in
glass
tubes
at
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Wastewater exposure
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Time-proportional composite samples (24-hours) of treated wastewater were collected in
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stainless steel containers in biweekly intervals from April to May 2015 at the effluent of the
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wastewater treatment plant Landau-Mörlheim. This plant treats wastewater of a population
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equivalent of approximately 90,000 (~40,000 from industry) using mechanical, biological,
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and
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landau.de/index.phtml?sNavID=1804.65&La=1). Sampling of wastewater was finalized two
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days before the start of the experiment and the medium renewals after 2 and 4 weeks. At the
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same time, stream water from the Hainbach was sampled in stainless steel containers. Both
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types of water were transported to the laboratory, where fine particulate organic matter was
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removed using an external aquarium filtration system (pore density=10 ppi; ecco pro 2034;
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Eheim, Deizisau, Germany). Afterwards, the water was continuously aerated and stored at
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16±1 °C until use (for water quality parameters see Table S1).
chemical
(i.e.,
phosphorus
elimination)
treatment
steps
(http://www.ew-
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Pre-exposure of the Gammarus populations was conducted in the Landau laboratory stream
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microcosm facility (http://uni-ko-ld.de/f7), involving 18 independent stainless steel artificial
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streams (120×30×20 cm3; water volume 40 L) serving as experimental units (for a schematic
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representation see Figure 1A). A pedal wheel in each microcosm facilitated a continuous
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water movement (flow velocity ~0.1 m/s). All experimental units were situated in a water 6 ACS Paragon Plus Environment
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bath set at 16±1 °C, while the day/night rhythm was set at 12/12 h (illuminance=90-130 lx).
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At test initiation, six microcosms were filled with 40 L of stream water from the Hainbach, a
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second set of six microcosms received a 50:50 mixture of stream and wastewater (40 L total),
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and a third set of six microcosms contained 40 L of wastewater (for a schematic
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representation of the timeline of events during the experiment see Figure 1B). Thereby, two
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realistic dilution potentials were realized that roughly mirrored the two extremes determined
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for the receiving stream (i.e., the Queich) of the wastewater treatment plant Landau-Mörlheim
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(i.e., 90% and 35% of wastewater during summer and winter, respectively, over the first ~350
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m after wastewater enters the stream).8 Recently, it was shown that nearly 90% of small
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German streams (mean low flow< 1m3/s) receiving treated wastewater have a low dilution
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potential (
