Influence of streambed substratum composition on stream
microbial communities exposed to the fungicide
tebuconazole
F L O R E N C E D O N N A D I E U *,†, P A S C A L E B E S S E - H O G G A N‡ , §, C H R I S T I A N E F O R E S T I E R *,† A N D J O A N A R T I G A S *,†
*CNRS, UMR 6023, LMGE, Aubiere, France
†Clermont Universite, Universite Blaise Pascal-Universite d’Auvergne, Laboratoire Microorganismes : Genome et Environnement, Clermont-Ferrand, France
‡CNRS, UMR 6296, Institut de Chimie de Clermont-Ferrand, Aubiere, France §Clermont Universite, Universite Blaise Pascal, ICCF, Clermont-Ferrand, France
S U M M A R Y
1. Fungicides reaching stream ecosystems have different capacities to adsorb to benthic substrata and thus cause varying effects on benthic microbial communities. We evaluated the potential of a triazole fungicide, tebuconazole (TBZ), to adsorb to submerged leaves and sand and assessed TBZ effects on sand- and leaf-associated microbial communities.
2. An indoor stream channel experiment was designed to test TBZ effects slightly above concentra-tions measured in agricultural streams (10.7 0.3 lg L 1) on the microbial communities associated with either submerged leaves alone, sand alone or a mixed substratum of leaves+ sand over 5 weeks. Weekly samples were taken to determine TBZ effects on the biomass and activity (litter breakdown rate, fungal sporulation rate and extracellular enzyme activities) of fungi and bacteria as well as the dissipation of TBZ in stream channels.
3. TBZ significantly reduced fungal biomass (17–20% relative to controls) but increased bacterial bio-mass (34–50%) on leaves and sand incubated separately. TBZ also significantly inhibited phenol oxi-dase activity (36%) in sand but not in leaves. Differences in TBZ effects between leaves and sand communities were not explained by differences between substrata in TBZ adsorption but more likely were related to differences in biomass accrual and structure of the microbial communities.
4. Mixing leaves+sand tended to attenuate TBZ effects on microbial communities, which was proba-bly explained by a greater surface area available for TBZ adsorption to the substrata. This hypothesis is supported by the greater TBZ dissipation in stream channels receiving both leaves and sand. How-ever, leaf and sand mixtures not only diluted but also modified TBZ effects on sand communities (14% increase in fungal biomass and 82% increase in the sporulation of aquatic hyphomycetes). This result suggests that sand could serve as a refuge for fungi during TBZ-contamination episodes. 5. Our study points to the importance of substratum heterogeneity for the ecotoxicological effects of TBZ on agricultural streams and highlights the contrasting responses of fungi and bacteria to con-tamination by a widespread fungicide.
Keywords: aquatic hyphomycetes, bacteria, experimental stream, leaf litter, stream sediment
Introduction
Fungicide application in agriculture has increased over the last decade, including in North America and Europe
(Geiger et al., 2010; Battaglin et al., 2011), which has been reflected in the surface and groundwater quality of agri-cultural catchments. Systemic fungicides such as tebu-conazole (TBZ, a triazole fungicide) have been detected
in streams at concentrations ranging from 0.1 to 9.1lg L 1 (Berenzen et al., 2005; Battaglin et al., 2011), especially during late summer to early autumn when summer storms follow fungicide applications (Rabiet et al., 2010; Battaglin et al., 2011). Fungicides are likely to be toxic also to freshwater fungi, which are important components of stream food webs and instrumental in decomposing plant litter. This has led to calls that toxic-ity to freshwater fungi should be included in risk assess-ments of agricultural fungicides (Sch€afer et al., 2011). In addition, the low specificity in the mode of action of sys-temic fungicides such as TBZ has the potential to impact a wide range of nontarget organisms (Maltby, Brock & van den Brink, 2009).
An important consideration when assessing fungicides in streams is their association with submerged substrata and how this can influence effects on microbial commu-nities. Streambeds are composed of a variety of soft (i.e. decomposing leaves) and hard (i.e. sand, rocks) sub-strata colonised by specific microbial communities. Fungicides adsorb onto these substrata or are trans-ported downstream, depending on their physicochemi-cal characteristics. The fungicides currently used in agriculture are moderately hydrophobic (i.e. log Kow= 3.7 for TBZ; Rabiet et al., 2010) and hence tend to
adsorb rapidly. TBZ is expected to adsorb mostly to par-ticulate organic matter such as decomposing leaves, although sorption to mineral surfaces can also occur ( Cadkova et al., 2012). Measurements of TBZ sorption in streambeds are rare (Smalling et al., 2013), but results from soils suggest it adsorbs mostly to humic sub-stances, though differences in sorption behaviour have been detected between the commercial form of TBZ and the analytical-grade pure chemical ( Cadkova et al., 2012). Fungicide concentrations in sediments and suspended solids in streams varied over time and across different geographical areas (0.3–198 lg kg 1 dry mass; Smalling
et al., 2013). Other studies suggest that pesticides, including fungicides, are typically more concentrated in suspended solids than in bed sediments (i.e. Kuivila & Hladik, 2008), which could be partly explained by the typically greater organic carbon content of suspended solids (Pereira et al., 1996). Therefore, TBZ would be expected to have a greater impact on microbial commu-nities colonising leaves than on microbes associated with sand.
Most research on TBZ effects has focused on soil microbial communities (Cycon et al., 2006; Bending, Rodriguez-Cruz & Lincoln, 2007). TBZ clearly has the potential to decrease soil microbial biomass and activity, though effects vary with application dose and frequency
and the influence of adjuvants in commercial formula-tions (Mu~noz-Leoz et al., 2011). In aquatic ecosystems, TBZ effects have been documented for aquatic hypho-mycetes and detritivorous invertebrates (Bundschuh et al., 2011; Artigas et al., 2012). TBZ prevents ergosterol biosynthesis by inhibiting the sterol C14-a-demethylation (Copping & Hewitt, 1998). Accordingly, a 25–45% reduc-tion in fungal biomass measured as ergosterol was found on leaves exposed to TBZ, depending on fungi-cide concentration (50–500 lg L 1; Bundschuh et al.,
2011). The fungicide also reduced species richness of aquatic hyphomycetes at all concentrations tested and modified the kinetics of b-glucosidase, b-xylosidase and cellobiohydrolase, resulting in slow cellulose degrada-tion by leaf-associated microorganisms (Artigas et al., 2012). TBZ effects on aquatic ecosystems have been also studied in biofilm and plankton microbial communities (Artigas et al., 2014; Pascault et al., 2014), where different functional indicators showed variable responses to TBZ, whereas bacterial community structure was unaffected. Bacteria associated with decomposing leaves are also inhibited by TBZ, although less than fungi (Bundschuh et al., 2011; Artigas et al., 2012). Information is lacking, however, on the response to TBZ of sand-associated microbial communities in streams and how a mixture of leaves and sand can modulate fungicide effects on microbial communities.
The objectives of this study were to determine the adsorption of TBZ to decomposing leaves and sand and to assess how sorption can affect the response of stream fungal and bacterial communities. An indoor-channel experiment was designed to assess TBZ effects on leaves and sand both separately and in combination (leaves + sand) to mimic a realistic field exposure. We hypothe-sised that TBZ will adsorb more to leaves than to sand and that fungicide effects will be greater on leaf-associ-ated microbial communities dominleaf-associ-ated by fungi.
Methods
Experimental design
Eighteen indoor stream channels (63 cm9 11 cm 9 4 cm= l 9 w 9 d) were used to determine TBZ toxicity to microbial communities associated with decomposing leaves and sand. Channels received leaves alone, sand alone or a mix of leaves and sand. The channels with leaves contained seven packs (1.5 g each) of freshly fal-len and air-dried Fagus leaves collected in the streamside forest in the autumn of 2012. Channels containing sand received 8 kg of sand fresh mass. A third set of channels
received both leaves (seven packs) and sand (8 kg). Half of the streams, three of each substratum treatment, were contaminated with TBZ (see below). The 18 channels each had a separate 15-L tank which supplied water by individual aquarium pumps (NJ 1200, Newa, Italy; 6.6 L min 1). Temperature in the channels was kept at 19°C and the photoperiod was 13 : 11 h, similar to field conditions. Dechlorinated tap water (On Line Active, Brita GmbH, Taunusstein, Germany) was continuously recirculated in the channels.
Microbial communities to inoculate the channels were obtained in spring 2013 from a third-order forest stream located in central France (Artiere stream, Puy-de-D^ome region). A well-developed riparian forest, mostly com-posed of Fagus sylvatica L., Castanea sativa Mill. and some Alnus glutinosa (L.) Gaertn., surrounded the selected reach. The streambed was composed of similar propor-tions of rocks and cobbles (riffles) and sand and gravel (pools). Particulate organic matter (leaves of Fagus and other woody plant debris) was mostly accumulated between rocks and cobbles. Sand and gravel were par-tially covered by fine-particulate organic matter, espe-cially at the stream margin.
Sand-associated microbial communities were collected from the top 2 cm of sediment of the Artiere stream. The sand was placed in three 0.04 m3 plastic containers
filled with stream water, transported to the laboratory and immediately transferred to the experimental chan-nels 1 week before starting the experiment. A mix of freshly fallen and partially decomposed leaves of Fagus sylvatica was collected from the same stream 3 days prior to the experiment and transported to the labora-tory in plastic bags. The leaves were cleaned with fil-tered stream water before cutting discs of 2 cm diameter, which were placed in sterile 200-mL flasks (10 in total) to induce sporulation of aquatic hyphomycetes. Each flask received 15 leaf discs and 50 mL of 0.2-lm fil-tered stream water and was incubated at 10°C on a sha-ker (180 rpm). After 48 h, the contents of the flasks including leaf discs were ground with a Bosch coffee grinder (180 W, Nazarje, Slovenia), pooled in a single sterile flask and immediately used to inoculate the experimental channels containing leaves (40 mL inocu-lum/channel).
A TBZ stock solution (10 mg L 1 dissolved in
ultra-pure water; Sigma Aldrich, Germany) was used to con-taminate the experimental channels at a nominal concentration of 12lg L 1, whereas control channels were not contaminated. This concentration was chosen according to TBZ-contamination peaks detected in the Artiere stream (Phyt’Auvergne, 2014) and other rivers
from a neighbouring region in France (Rh^one-Alpes; Rabiet et al., 2010). Water in the experimental channels was replaced weekly to maintain TBZ concentrations close to 12lg L 1 and prevent nutrient limitation of the microbial communities. Leaves, sand and mixed substra-tum (leaves+ sand) for microbial analyses were sampled 0, 7, 14, 21, 28 and 35 days after TBZ contamination. Water samples (100 mL) were taken at the same times, filled in glass flasks and frozen at 20°C for later analy-sis of dissolved nutrient and TBZ concentrations.
Water analyses
TBZ concentrations were measured 30 min before and after each water exchange to determine TBZ dissipation in the channels. TBZ was quantified by high-pressure liquid chromatography (HPLC) after concentration on conditioned (5 mL methanol+ 5 mL deionised water) tC18 cartridges (Sep-Pakâ Vac RC, 500 mg, Waters, Mil-ford, MA, U.S.A.) and elution with 8 mL methanol. Methanol was then evaporated in a Speed-Vac, and the dried samples were re-dissolved in 500lL methanol : water (4 : 1, v:v) and analysed on an Agilent HPLC (model 1100, Waldbronn, Germany) equipped with a reversed-phase column (Zorbax Eclipse XDB-C18, 3.5lm, 75 9 4.6 mm) maintained at 25°C. The mobile phase was 0.01% H3PO4 : CH3CN (50 : 50, v:v) at a flow
rate of 1 mL min 1, resulting in a retention time of TBZ of 4.3 min. The injection volume was 100lL. TBZ was detected at 225 nm using an 1100 photodiode array detector (DAD lamp, model Agilent 1100 series, Wald-bronn, Germany). TBZ recovery throughout the proce-dure was 95% (3 SE). TBZ dissipation (including losses by adsorption, photo- or biodegradation) was calculated as the per cent difference between the average TBZ con-centrations between water replacements.
Water temperature, conductivity, dissolved oxygen concentration and pH were measured at each sampling time (i.e. weekly) using portable metres (model 340i; WTW GmbH, Weilheim, Germany). Concentrations of soluble reactive phosphorus (Murphy & Riley, 1962) and nitrate (Nitrate test kit, Merck, Germany) were measured spectrophotometrically. Dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) were measured with a total organic carbon analyser (TOC-VCPN Shimadzu,
Tokyo, Japan).
Microbial biomass
Fungal biomass was estimated as ergosterol in leaves and sand following the protocol of Gessner & Schmitt
(1996) with some modifications. Fifteen leaf discs (diam-eter= 1 cm) or 5 mL of sand was lyophilised and extracted with 0.14 M KOH methanol (80 °C, 30 min
with agitation). The lipid extracts were purified by solid-phase extraction (tC18 cartridges, Sep-Pakâ Vac RC, 500 mg, Waters), and ergosterol was eluted with iso-propanol and concentrated fivefold under a stream of nitrogen. The concentrated eluate was then analysed by HPLC (Lachrom L-7400, Merck-Hitachi, Tokyo, Japan) equipped with an ODS-2 Hypersil column (250 9 4.6 mm, 5 lm particle diameter; Thermo Scien-tific, San Jose, CA, U.S.A.) using methanol as the mobile phase at a flow rate of 1.4 mL min 1. This resulted in a retention time of 6.5 min. Ergosterol was detected at 282 nm. Ergosterol quantification was based on stan-dards (≥95% purity; Sigma Aldrich, Steinheim, Ger-many) in the range of 0–200 lg mL 1. Fungal biomass
in terms of carbon was estimated based on an ergosterol content of 5.5 mg dry mass g 1 fungal biomass (Gessner
& Chauvet, 1993) and 43% carbon in fungal dry mass (Baldy & Gessner, 1997).
Bacterial biomass was estimated based on flow cytometric counts of bacterial cells (Borrel et al., 2012). One leaf disc and 1 mL of sand were placed in TE buf-fer (10 mM Tris, 1 mM EDTA) and fixed with
paraformaldehyde (2% final concentration). Samples were incubated for 1 h at room temperature and then treated with sodium pyrophosphate (10 mM final
con-centration) and sonicated (40 W, 40 kHz, 30 s9 2; Arti-gas, Romanı & Sabater, 2008) using a sonication bath (model FB 15048, Fisher Scientific, Leicestershire, U.K.). The bacterial suspensions were centrifuged at 800 g for 60 s and the supernatants diluted 10-fold and stained with SYBR Green I (Molecular Probes) before counting bacterial cells with a BD FACSCalibur flow cytometer (15 mW at 488 nm, Becton Dickinson, U.S.A.). Bacterial biomass was calculated from bacterial densities using a conversion factor of 2.2 9 10 13g C lm 3 (Bratbak &
Dundas, 1984) and considering a mean bacterial biovol-ume of 0.1lm3 (Theil-Nielsen & Søndergaard, 1998).
Functional indicators
The breakdown of Fagus sylvatica leaves was determined as leaf mass loss between days 0 and 35. At each sam-pling time, 12 leaf discs (1 cm diameter) from each stream channel were cut, dried (70°C for 48 h), weighed, ashed (450 °C for 5 h) and reweighed to deter-mine the ash-free dry mass (AFDM) remaining. Break-down rates were determined using an exponential decay
model with k as the breakdown coefficient (Petersen & Cummins, 1974).
Sporulation rates of aquatic hyphomycetes were determined according to Artigas et al. (2008). Ten leaf discs or 5 mL of sand was placed in sterile 100-mL flasks containing 40 mL of 0.2-lm filtered water from the corresponding channels. The incubation conditions were the same as those used to prepare the inoculum for leaves. After 48 h, 20 mL of the spore suspensions was mixed with 100lL Triton-100 and shaken for 10 min at 80 rpm to detach fungal spores from parti-cles. The resulting suspensions were filtered through 5-lm nitrocellulose filters (Whatman, U.K.) and stained with 0.1% trypan blue in 60% lactic acid. Volumes limited to 5 mL were used for sand samples to avoid clogging of the filters and facilitate counting of the spores. For this purpose, the filters were mounted on microscope slides and viewed at 400-10009 magnifica-tion (Leica DM R, Wetzlar, Germany). Twenty fields were counted per leaf sample (≥500 spore counts per filter), whereas the whole filter was scanned for sand samples. Conidia were counted and identified based on Ingold (1975).
Potential extracellular enzyme activities were mea-sured in leaf and sand samples with commercial sub-strate analogues (Artigas et al., 2012). Cellobiohydrolase (EC 3.2.1.91) and b-glucosidase (EC 3.2.1.21) activities were determined using methylumbelliferyl substrates (MUF, Sigma), whereas phenol oxidase (EC 1.14.18.1) activity was measured using 3,4-dihydroxy-L
-phenylala-nine (L-DOPA, Sigma). Two leaf discs or 1 mL of sand was placed in 3 mL of water from the stream channels corresponding to the samples, and the MUF or L-DOPA substrates were added at saturating concentrations (0.3 mM forb-glucosidase and 1.5 mM for
cellobiohydro-lase and phenol oxidase). The samples were incubated for 1 h at 19°C in the dark with stirring. Blanks for determining enzyme activity in water (without leaves and/or sand) were simultaneously incubated. The enzy-matic activity of cellobiohydrolase and b-glucosidase was stopped by adding (1 : 1, V:V) glycine buffer (pH 10.4), and fluorescence was measured (365 nm excitation wavelength, 455 nm emission wavelength) with a fluo-rometer (SFM 25, Kontron Instruments, Milano, Italy). For phenol oxidase, fluorescence was measured at 460 nm without buffer being added. Potential activities are expressed aslmol MUF g1AFDM h 1 for b-glucosi-dase and cellobiohydrolase and as nmol 2,3-dihydroin-dole-5,6-quinone-2-carboxylate (DIQC) g 1 AFDM h 1 for phenol oxidase.
Statistical analyses
A three-way repeated-measures ANOVA was used to determine effects of TBZ (Factor 1: control versus con-taminated), substratum (Factor 2: leaves versus sand) and mixture (Factor 3: single versus mixed substratum) factors on microbial biomass and functional indicators of microbial communities. All variables were log-trans-formed to meet the assumptions of normality and homo-geneity of variance. The probability values within groups (time, factors and their interactions) were adjusted for sphericity using the Greenhouse–Geisser correction. The proportional change of the responses of biological indicators to TBZ over 5 weeks (%R 1 SE) was calculated using the equation %R= ((contaminated – control)/control) 9 100. Finally, differences in water chemistry (TBZ, DOC, nutrient concentrations) between TBZ-contaminated and control channels and among channels stocked with leaves, sand or leaves+sand were tested by two-way repeated-measures ANOVA. Separate post hoc comparisons (Tukey’s test, P< 0.05) were applied to determine differences in water chemistry (channels containing leaves, sand or leaves+sand) and characteristics of microbial communities associated with leaves and sand (alone versus mixed substratum in con-trol versus contaminated conditions).
Results
Tebuconazole concentration and water quality
Analyses of water samples taken after each water exchange suggest that the channels were contaminated with TBZ at an average concentration (1 SE) of 10.7 0.3 lg L 1 (Fig. 1). By the end of each week, TBZ concentrations had dropped to an average of 6.6 0.1 lg L 1. Weekly TBZ dissipation varied between substrata (P< 0.001) and over time (P < 0.001). The great-est dissipation was generally observed in channels containing leaves+sand (43.8 1.8%) followed by chan-nels with leaves (36.3 4.0%) and sand (34.7 2.0%) (Tukey’s test, P< 0.05).
Water temperature, conductivity, dissolved oxygen concentration and pH were similar in all channels dur-ing the 5 weeks of the experiment (Table 1). Differences were observed in DOC and TDN concentrations among channels containing different substrata (P< 0.002), being greater in channels with leaves than in channels with sand only or leaves+sand (Tukey’s test, P < 0.05). These differences were more marked at the beginning of the experiment (weeks 1–3) and tended to decrease during
the last week (data not shown). Conversely, no signifi-cant differences were observed in PO43 and NO3
concentrations (P= 0.67 and P = 0.085, respectively). Overall, DOC concentration tended to decrease and nitrogen concentration (including TDN and NO3 ) to
increase over time (P< 0.0001).
TBZ-contaminated channels showed lower TDN con-centrations than control channels irrespective of the sub-stratum (P< 0.005). The differences were first observed in channels containing sand and leaves+sand (1.2–1.5 times lower concentrations in contaminated channels in weeks 1 and 2) and later in channels containing leaves (1.2 times lower, weeks 3 and 4).
Microbial biomass
TBZ had significant effects on microbial biomass associ-ated with leaves and sand, although effects on fungi and bacteria differed (Table 2). TBZ decreased fungal biomass in channels with leaves (%R 1 SE = 17.2 4.5%) and sand ( 20.5 3.6%) and to a lesser extent also in chan-nels with mixed substratum ( 9.5 6.8%) (Tukey’s test, P< 0.05). These effects immediately appeared in the channels containing sand (week 1) and later in the channels with leaves (weeks 2–3; Fig. 2). Fungal biomass was higher in leaves (4.6 0.3 mg fungal C g 1 AFDM, 5-week average in control channels without TBZ) than in sand (0.040 0.002 mg fungal C g 1 AFDM) with clear
Fig. 1 Tebuconazole concentrations in water samples from experi-mental stream channels containing leaves, sand and leaves+sand. Values are means 1 SE (n = 3) of weekly measurements made during the 5-week experiment. The weekly dissipation of TBZ in water is represented by dashed lines.
peaks occurring in leaves in week 2 (Fig. 2a,b). TBZ increased the average bacterial biomass in channels with leaves (+50.0 21.1%) or sand (+34.5 15.6%) (Tukey’s test, P< 0.05), but never in channels with mixed substra-tum (Table 2, Fig. 2). Bacterial biomass tended to be higher on sand (0.17 0.01 mg bacterial C g 1 AFDM) than on leaves (0.04 0.01 mg bacterial C g 1AFDM).
Functional indicators
Leaf mass loss was not significantly affected by TBZ, but differed among channels containing leaves and leaves mixed with sand. Breakdown rates in channels contain-ing only leaves were 0.0018 and 0.0016 day 1 (control and TBZ-contaminated, respectively), whereas rates for leaves in channel with mixed substratum were 0.0043 and 0.0038 day 1 (control and TBZ-contaminated, respectively).
Sporulation rates of aquatic hyphomycetes differed between substrata (Table 2, Fig. 3). TBZ effects were only observed in mixed substratum with leaves and sand where sporulation rates increased (%R 1
SE= +82.3 27%), especially during weeks 3 and 4 (Table 2). TBZ had no clear effects on the sporulation of aquatic hyphomycetes. A sporulation peak in control channels with leaves in week 1 shifted to weeks 4–5 in TBZ-contaminated channels with leaves. A total of 14 aquatic hyphomycete species were identified on leaves and sand at the end of the experiment on day 35. The presence of TBZ significantly decreased the number of spores of Lunulospora curvula, Lemonniera aquatica and Clavariopsis aquatica and increased that of Diplocladiella scalaroides and Margaritispora aquatica.
With one exception, potential extracellular enzyme activities were highest in the second week of the experi-ment (Fig. 4). After 2–3 weeks of exposure, TBZ contami-nation reduced the potential phenol oxidase activity in channels containing sand (%R 1 SE = 36.5 6.2, Table 2, Fig. 4c), at least temporarily. Conversely, the potential phenol oxidase activity associated with leaves was not affected by TBZ. Potentialb-glucosidase and cel-lobiohydrolase activities were greater in leaves (2.3 0.2 and 1.9 0.1 lmol MUF g 1 AFDM h 1, respectively) than in sand (0.60 0.08 and 0.40 0.03 lmol MUF g 1
Table 1 Water physical and chemical characteris-tics measured in experimental stream channels containing leaves (L), sand (S) or leaves+sand (L+S) and exposed or not (controls) to TBZ. Values are means with standard errors in parentheses of measurements taken in each of three replicate stream channels on five sampling dates (n= 15)
Variable L Control L TBZ S Control S TBZ L+S Control L+S TBZ Temperature (°C) 18.8 (0.1) 18.7 (0.1) 19.1 (0.1) 19.0 (0.1) 18.4 (0.1) 18.8 (0.1) Conductivity (lS cm 1 ) 290 (4) 288 (4) 299 (5) 291 (5) 287 (5) 287 (4.8) Oxygen (%) 95.4 (0.6) 95.4 (0.6) 95.4 (0.6) 95.4 (0.6) 95.4 (0.6) 95.4 (0.6) pH 7.2 (0.1) 7.3 (0.1) 7.1 (0.1) 7.4 (0.1) 7.4 (0.1) 7.3 (0.1) DOC (mg L 1) 9.5 (1.1) 9.4 (1.1) 7.5 (0.9) 7.4 (1) 7 (0.9) 7.3 (0.9) N-NO3 (lg L 1) 474 (30) 493 (32) 498 (39) 479 (37) 483 (42) 450 (39) P-PO43 (lg L 1) 11.2 (2.6) 11.9 (2.6) 12.3 (2.6) 12.8 (3.1) 7.4 (1) 10.9 (1.8) TDN (mg L 1) 1.5 (0.1) 1.4 (0.1) 1.2 (0.1) 1.1 (0.1) 1.1 (0.2) 1.0 (0.2)
Table 2 Results of three-way repeated-measures ANOVA applied to biological indicators including fungal and bacterial biomass, sporulation of aquatic hyphomycetes and potential extracellular activities of three enzymes:b-glucosidase (BG), cellobiohydrolase (CBH) and phenol oxidase (PO). Probability values based on the Greenhouse– Geisser statistic are listed for each of the sources of variation tested including time (T), tebucona-zole (TBZ), substratum (S) and mixture (M) as well as their interactions. Interactions between T, S and M are not represented. Statistically signifi-cant P-values are highlighted in bold.
Source of
variation Fungi Bacteria Sporulation BG CBH PO
T <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 T9 TBZ 0.09 0.58 0.09 <0.05 <0.001 0.65 S <0.001 <0.001 <0.001 <0.001 <0.001 0.19 M 0.15 0.19 <0.001 <0.005 <0.05 <0.001 TBZ <0.05 <0.05 0.63 0.10 0.42 0.24 S9 M 0.14 0.57 <0.05 <0.05 0.16 <0.001 S9 TBZ <0.05 0.365 0.44 0.08 0.49 <0.005 M9 TBZ 0.44 <0.05 0.26 0.37 0.23 0.14 S9 M 9 TBZ 0.62 0.35 0.45 <0.05 0.19 0.09
AFDM h 1, respectively) (Table 2). TBZ effects on these activities were time dependent (Table 2). For instance, an increase in potentialb-glucosidase and cellobiohydrolase activities was observed in week 4 in channels with leaves only (+78 and +93%, respectively) and in leaves (+17 and +34%) and sand (+17 and +59%) in the channels with mixed substratum.
Discussion
The TBZ concentrations tested in our experiment of 10.7 0.3 lg L 1were environmentally relevant (Beren-zen et al., 2005; Rabiet et al., 2010). Although concentra-tions decreased by c. 40% within a week of contact with submerged leaves and sand, they affected both leaf- and
Fig. 2 Fungal (a–d) and bacterial (e–h) biomass associated with leaves and sand in control and TBZ-contaminated experi-mental stream channels containing leaves, sand or leaves+sand. Values are means 1 SE (n = 3). Differences between treatments are marked by letters (a> b; Tukey’s test, P < 0.05).
sand-associated microbial communities and both target (fungi) and nontarget (bacteria) organisms. However, toxicity varied between microbial communities associ-ated with leaves and sand, and mixing of the two sub-strata tended to decrease the TBZ effects.
Dissipation of TBZ was greater in channels containing both leaves and sand than in channels containing either
substratum alone. The average TBZ dissipation of 44% in 7 days is lower than values reported for stream peri-phyton (84–93% in 24 h in Tlili et al., 2011; 60-75; % in
Fig. 3 Sporulation rates of aquatic hyphomycetes on leaves (a,b) and sand (c,d) in control and TBZ-contaminated experimental stream channels. Values are means 1 SE (n = 3).
Fig. 4 Potential phenol oxidase activity associated with leaves (a,b) and sand (c,d) in control and TBZ-contaminated experimental stream channels. Values are means 1 SE (n = 3). Differences between treatments are marked by letters (a> b; Tukey’s test, P< 0.05).
22 days in Artigas et al., 2014). In agreement with results by Dimitrov et al. (2014), a fraction of the added TBZ was probably adsorbed to leaves as well as to mineral particles and fine-particulate organic matter in sand, but a non-negligible fraction (56% in channels containing leaves+sand) remained dissolved in the water and was probably available to microbes. Preliminary experiments revealed that TBZ adsorption to surfaces of the experi-mental channel (including pipes and pumps) accounts for only a small fraction (<10%) of the initial amounts applied (C. Margoum, unpubl. data). Greater TBZ dissi-pation in channels with leaves and sand suggests that the greater surface area in the experimental channels containing both substrata increased TBZ adsorption. However, TBZ dissipation was not additive in the chan-nels containing leaves and sand compared to chanchan-nels with either substratum alone, suggesting that part of the observed TBZ dissipation could have been due to processes other than adsorption, such as photo- or biodegradation.
Channels containing either leaves or sand dissipated similar amounts of TBZ during the experiment. This out-come was unexpected given that TBZ has a greater affin-ity to substrata rich in organic carbon, such as leaves, than to the mineral surface of sand (Pereira et al., 1996). However, the sand used in our experimental channels, as well as in natural stream beds, contained substantial amounts of organic matter as well. Therefore, the similar dissipation of TBZ in both types of channels might still be in line with the importance of organic matter as primary adsorption site for TBZ, which would suggest a key role of sediment organic matter for dissipating TBZ in streams.
With the exception of phenol oxidase, similar responses to TBZ contamination were observed between microbial communities associated with leaves and sand in channels containing either substratum alone. These common responses were characterised by a reduction in fungal biomass by 17–20%, in agreement with the mode of action of TBZ as an inhibitor of ergosterol biosynthe-sis (Copping & Hewitt, 1998), and a corresponding increase in bacterial biomass by 30–50%. This increase in bacterial biomass in the contaminated channels is at variance with results of earlier experiments with leaf lit-ter (Bundschuh et al., 2011; Artigas et al., 2012), but in accordance with data on soil and plankton microbial communities (Cycon et al., 2006; Artigas et al., 2014). The increase in bacteria could be explained by (i) reduced resource competition when fungi are stressed (Mille-Lindblom, Fischer & Tranvik, 2006), (ii) enhanced sup-ply of nutrients released from killed fungi (Cycon et al.,
2006) or (iii) reduced interference competition (e.g. low-ered production of fungal antibiotics) by stressed fungi (Drews, 2000). However, the second hypothesis was not supported by our data on TDN and phosphate concen-trations, which were lower (TDN) in the contaminated than in the control channels, or similar (phosphate). The gradual increases in dissolved nitrogen concentrations (TDN and NO3 ) observed during the experiment in
channels containing leaves were probably due to leach-ing of N from leaves and could have masked a nutrient-mediated stimulation of bacteria. Another possible explanation is an inhibition of protozoan grazers of bac-teria by the fungicide (Ekelund, 1999), as previously sug-gested for plankton (Artigas et al., 2014). It must be noted, however, that the stimulatory effect of TBZ on leaf-associated bacteria (biomass increase of 0.02– 0.04 mg C g 1 AFDM compared to control channels without TBZ) was small compared to the negative effect on fungi (biomass decrease by 1.0–1.2 mg C g 1AFDM).
The main difference in the responses of leaf- and sand-associated microbial communities to TBZ was in phenol oxidase activity. The potential activity of this enzyme, which is involved in the microbial breakdown of lignin (McLatchey & Reddy, 1998), tended to be reduced in channels containing only sand ( 36.5%) but not when leaves were present. This could be related to the observed decrease in fungal biomass in the contami-nated channels containing sand in the absence of leaves, although calculations of biomass-specific phenol oxidase activity at the end of the experiment still showed differ-ences between control (175 13 mmol DIQC h 1g 1 microbial C) and contaminated channels (95 1 mmol DIQC h 1 g 1 microbial C). Shifts in fungal community
composition towards tolerant phenol oxidase-producing species could have circumvented a negative effect of TBZ on this enzyme. The decreased sporulation of Lem-moniera aquatica and Clavariopsis aquatic in the presence of the fungicide, which had also been observed by Bundschuh et al. (2011), and increased sporulation by Diplocladiella scalaroides and Margaritispora aquatica indi-cate that community shifts did indeed occur. A similar effect might account for the weak TBZ impacts on potential b-glucosidase and cellobiohydrolase activities, although this result contrasts with a previous study on these two enzymes associated with microbial communi-ties on alder and poplar leaves (Artigas et al., 2012).
Mixing leaves and sand weakened the observed TBZ effects on fungal and bacterial biomass compared to the effects on channels with either substratum alone. This difference could be due to an increased surface area and higher organic matter content in the channels with
mixed substratum, resulting in decreased fungicide exposure of microbial biomass even though all contami-nated channels received TBZ at the same average con-centration of 10.7 lg L 1. However, the response to TBZ of the sand-associated communities not only decreased, but somewhat differed between channels containing either only one or both substrata. In particular, TBZ con-tamination increased fungal biomass in sand when leaves were present (14.0 4.8%). This might indicate a refuge function of sand for fungi during TBZ-contamina-tion episodes. However, the increase of fungal biomass in sand could also be due to ergosterol-containing frag-ments of stressed or killed fungi appearing in the fine-particulate organic matter pool of the sediments rather than reflecting an increased presence of active fungi. Mixing leaves and sand in the experimental stream channels also promoted the breakdown of Fagus sylvatica leaves (compared to channels containing only leaves), suggesting a favourable influence of sand such as enhanced litter breakdown by microorganisms from sand. However, TBZ did not affect leaf breakdown rate, in contrast to a previous experiment (Artigas et al., 2012), possibly because of the recalcitrant nature of Fa-gus sylvatica leaves, which is an exceedingly slow leaf species to decompose (Gessner & Chauvet, 1994).
In summary, this study shows that TBZ can reduce fun-gal biomass, increase bacterial biomass and, to some extent, modify the enzymatic potential of microbial com-munities associated with leaves and sand in streams. However, the observed effects were weak compared to those observed in other studies (Artigas et al., 2012), possibly because of differences in TBZ concentrations to which the microbial communities were effectively exposed. A notable observation in this context is that mix-ing leaves and sand, which promoted leaf breakdown, tended to weaken TBZ effects on microbial communities. Caution is needed, however, when extrapolating results from TBZ toxicity tests in simplified indoor stream chan-nels to real ecosystems. For example, fungal biomass in leaves in the present study was almost an order of magni-tude lower than that reported from other field investiga-tions (e.g. Gessner & Chauvet, 1994). The initial fungal inoculum and the absence of a constant replenishment from upstream in natural streams are likely to influence the microbial colonisation of, and succession on, substrata in laboratory settings and could also affect responses to fungicides. Therefore, complementary field tests of the fate and ecotoxicological effects of TBZ on microbial com-munities are required as a basis for improved risk assess-ment of fungicides contaminating streams (Fernandez et al., 2015).
Acknowledgments
We thank Fanny Perriere, Jonathan Colombet and Gene-vieve Bricheux for help in the laboratory, and two anonymous reviewers for their comments and sugges-tions.
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