Determining in Situ Periphyton Community Responses to Nutrient and Atrazine Gradients Via Pigment Analysis

Science of the Total Environment 515–516 (2015) 70–82

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Science of the Total Environment

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Determining in situ periphyton community responses to nutrient and atrazine gradients via pigment analysis

Rebecca L. Dalton a,⁎,CélineBoutina,b, Frances R. Pick a a Ottawa-Carleton Institute of Biology, 30 Marie Curie, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada b Science and Technology Branch, Environment Canada, 1125 Colonel By Drive, Raven Road, Carleton University, Ottawa, Ontario K1A 0H3, Canada

HIGHLIGHTS GRAPHICAL ABSTRACT

• Agrochemicals (herbicides and fertilizers) may impair surface waters and periphyton • In situ effects (experimental, existing gradients) assessed via pigment analyses • Few effects of atrazine and nutrients were observed in periphytometer experiments • Nitrate enrichment was related to increased biomass, particularly of green algae • Effects of nitrogen enrichment superseded those of phosphorus and atrazine

article info abstract

Article history: Agrochemicals, including fertilizers and herbicides, are significant contributors of non-point source pollution to Received 27 October 2014 surface waters and have the potential to negatively affect periphyton. We characterized periphyton communities Received in revised form 31 December 2014 using pigment markers to assess the effects of nutrient enrichment and the herbicide atrazine with in situ exper- Accepted 11 January 2015 imental manipulations and by examining changes in community structure along existing agrochemical gradients. Available online xxxx In 2008, the addition of nutrients (20 mg/L nitrate and 1.25 mg/L reactive phosphate), atrazine (20 μg/L) and a fi Editor: Mark Hanson combination of both nutrients and atrazine had no signi cant effect on periphyton biomass or community structure in a stream periphytometer experiment. In 2009, similar experiments with higher concentrations of atrazine Keywords: (200 μg/L) at two stream sites led to some minor effects. In contrast, at the watershed scale (2010) periphyton Periphyton biomass (mg/m2 chlorophyll a) increased significantly along correlated gradients of nitrate and atrazine but no Nutrients direct effects of reactive phosphate were observed. Across the watershed, the average periphyton community Atrazine was composed of Bacillariophyceae (60.9%), Chlorophyceae (28.1%), Cryptophyceae (6.9%) and Euglenophyceae Periphytometer (4.1%), with the Bacillariophyceae associated with high turbidity and the Chlorophyceae with nitrate enrichment. CHEMTAX Overall, effects of nitrate on periphyton biomass and community structure superseded effects of reactive phos- Flowing waters phate and atrazine. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

1. Introduction

In agricultural watersheds, aquatic primary producer communities, ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (R.L. Dalton), including periphyton, may be altered by exposure to agrochemicals [email protected] (C. Boutin), [email protected] (F.R. Pick). such as herbicides and nutrients from fertilizers. Since periphyton is a

http://dx.doi.org/10.1016/j.scitotenv.2015.01.023 0048-9697/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). R.L. Dalton et al. / Science of the Total Environment 515–516 (2015) 70–82 71 dominant group of primary producers in mid-sized streams and rivers 2. Materials and methods (Strahler stream order 4–6) (Vannote et al., 1980), agrochemicals have the potential to impair the environmental quality and ecological 2.1. Study area health of these systems. Nutrient enrichment can result in broad shifts in periphyton communities towards increased relative dominance of The South Nation River watershed, eastern Ontario, Canada the Chlorophyceae (green algae), particularly filamentous taxa, in a va- (3915 km2) is predominately agricultural with crops of corn and riety of surface waters (e.g. Cattaneo, 1987; Carrick and Lowe, 1988; soybean (Glycine max L. (Merr.)) typically planted in tile-drained Dodds, 1991; Chételat et al., 1999; Rober et al., 2011; Ferragut and de fields. The headwaters of this 177 km long river commence near the Campos Bicudo, 2012). Changes may also occur at a finer taxonomic St. Lawrence River (44°40′41″N, 75°41′58″W) and the river flows scale. For example, the Bacillariophyceae (diatoms) range in their sensi- north-easterly across a flat, poorly drained landscape until its conflu- tivity to eutrophication and organic pollution and are frequently used to ence with the Ottawa River (45°34′24″N, 75°06′00″W) with a historical assess water quality (reviewed in Kelly, 2013). (1915–2011) average annual discharge of 44.3 m3/s (Environment Direct effects of herbicides on periphyton are less clear because Canada, 2013). Twenty-four field sites located throughout the South herbicides occur in surface waters at variable concentrations several Nation River watershed were selected for study (Fig. 1). Field sites orders of magnitude lower than nutrients with pulses occurring following were paired by matching 12 sites surrounded by low agriculture with rain events. Of the herbicides, atrazine is of particular interest due to 12 sites surrounded by high agriculture. Each pair was located along its widespread usage on North American corn (maize) (Zea mays L.) the same tributary with the low agriculture site located upstream of crops, frequent detection in surface waters and concerns over its the high agriculture site (Fig. 1). The average distance between 10 of toxicity, mobility and persistence (Solomon et al., 1996; Gilliom et al., the 12 pairs was 8.8 ± 8.9 km (ranging from 1.5 to 33.7 km). Two 2006; Stone et al., 2014). Laboratory and micro/mesocosm studies have pairs of sites were located along different tributaries due to a lack of ac- shown that periphyton functional endpoints (e.g. primary production) cessible and suitable sites. Tributaries were generally hydrologically un- may recover while community changes (e.g. species composition) persist connected and varied in the intensity of surrounding agriculture. All following experimental exposure to herbicides in general (reviewed in sites (20 m stream length) were matched as closely as possible in Brock et al., 2000) and atrazine in particular (Hamala and Kollig, 1985). terms of visible features such as steam width, bank slope and canopy Early lentic enclosure studies noted a shift towards Bacillariophyceae cover. dominated periphyton communities following exposure to atrazine (100 μg/L) (Herman et al., 1986; Hamilton et al., 1987). 2.2. Physical and chemical characteristics of field sites Several studies have attributed atrazine exposure in the field to a shift towards atrazine tolerant Bacillariophyceae dominated periphyton Strahler stream order for all 24 field sites was determined from data communities but it is difficult to separate out effects of other stressors provided by the South Nation River Conservation Authority and Ontario (e.g. Guasch et al., 1998; Dorigo et al., 2004). A number of laboratory Ministry of Natural Resources (© Queen's Printer, 2013). Stream width and micro/mesocosm studies have aimed to tease apart the interaction was measured in triplicate at each site. Stream depth was measured in between atrazine and correlated stressors such as nutrient enrichment triplicate during base flow in August 2010. Maximum depth was mea- on periphyton communities. The effects of atrazine and nutrients on sured mid-channel, while average depth was measured mid-channel periphyton are expected to be antagonistic but results have been as well as halfway between the mid-channel and each bank. Surface mixed. For example, nutrients have been shown to have no effect on velocity was estimated by measuring the time for an orange wiffleball atrazine toxicity (Guasch et al., 2007). Others have found that both atra- to travel 1 m in triplicate in June and July 2010. Dissolved oxygen, pH, zine and nutrients can increase periphyton biomass through indirect temperature and conductivity were measured with a HydroLab 4a effects of atrazine on phytoplankton (Rohr et al., 2008; Halstead et al., Sonde (Hach Hydromet, Loveland, USA) once in May, June and July 2014). In contrast, Murdock et al. (2013) found conflicting trends be- 2010 at all sites. Duplicate mid-channel, integrated water samples tween field and laboratory studies. A key challenge in risk assessment (1 L) were taken in polypropylene bottles in May, June and July 2010 is finding the right balance between realistic field studies that may be to measure turbidity (LaMotte, Chestertown, USA) and planktonic variable and difficult to interpret and simplified experiments that may chlorophyll a. Water samples (500 mL) for planktonic chlorophyll a not be realistic of actual exposures and communities. In the present analysis were filtered through 1.5 μm glass fiber filters (type 934-AH, study, we aimed to find this balance by examining the effects of nutri- Whatman, Mississauga, Canada) and frozen at −30 °C until extraction ents and atrazine on natural periphyton communities in situ using of algal pigments. both experimental and field exposures. Agricultural intensity was calculated as the percentage of annual The main objective of the present study was to assess the effects of cropland in a 500 m radius surrounding each site (ArcMap v.10, ESRI, high nutrients, exposure to the herbicide atrazine and their relative Canada Ltd, Toronto, Canada) from Landsat satellite imagery (30 m res- effects on periphyton community structure in situ using two ap- olution) data provided by Agriculture and Agri-Food Canada (2008). proaches. First, natural communities of periphyton were exposed to Data used in Fig. 1 were from Natural Resources Canada (2009). Field experimental treatments of nutrients and atrazine at two stream/river sites were characterized by in-stream nitrogen (N) and phosphorus sites using periphytometers. Second, periphyton communities were (P), with elevated concentrations representing enrichment from syn- compared between paired sites surrounded by low or high agriculture thetic fertilizers and manure (Dubrovsky et al., 2010). Mid-channel, in- and across existing gradients of nutrients and atrazine in a large agricul- tegrated water samples (300 mL) were collected in polyethylene tural watershed. It was hypothesized that nutrient enrichment would terephthalate bottles for nutrient analysis in May, June and July 2010 result in Chlorophyceae dominated communities and atrazine exposure with duplicate samples collected for approximately 10% of all samples. would result in Bacillariophyceae dominated communities. It was Time-weighted-average atrazine concentrations (56 days between 18 predicted that nutrients and atrazine would have antagonistic effects May and 22 July 2010) were determined by passive sampling with and that the relative strength of each stressor would be concentration polar organic chemical integrative samplers (POCIS) deployed in trip- dependent. A secondary objective was to apply a chemotaxonomic licate as described in Dalton et al. (2014). In-stream and pigment analysis method developed for classifying Southern Ocean periphytometer reservoir nutrient and atrazine concentrations phytoplankton communities to freshwater periphyton communi- were also measured from duplicate samples on days 0, 7 and 14 of ties. Compared to microscopy, this approach reduces the time and each periphytometer experiment. For atrazine analysis, sub- taxonomic expertise required to determine changes in algal com- samples (20 mL) were frozen and stored at −30 °C in pre-cleaned munity structure. amber borosilicate vials until analysis. Nutrients, including reactive 72 R.L. Dalton et al. / Science of the Total Environment 515–516 (2015) 70–82

11 Ottawa River 11 12 10 10 12 1

Ottawa, 8 Canada 8 9 7 9 6 7 4 SC 6

eRiver 5 5

NB St. Lawrenc 3 4 New York State, USA 2 3 2 Land Cover Annual Crop Land 1 Perennial Crop Land and Pasture Natural Habitat Developed Land Water

010205Km

Fig. 1. Twelve paired ﬁeld sites (24 in total) in the South Nation River watershed (3915 km2), Canada. Sites were surrounded by low or high levels of agriculture. NB — North Branch South Nation River. SC — South Castor River.

− phosphate (RP), total phosphorus (TP), nitrate (NO3 ), total nitrogen and a screen to protect the filters (modified from Matlock et al. (1998) (TN) and dissolved inorganic nitrogen (DIN), were analyzed following and Kish (2006) and described in detail in Appendix B). Five experi- established methods of the Ontario Ministry of the Environment ments were conducted and all experiments included four treatments (2007a,b). Atrazine was measured using LC–MS/MS analyses per- prepared in distilled water: 1) control, 2) nutrient, 3) atrazine and 4) nu- formed on a high performance liquid chromatograph coupled with a trient + atrazine (Table 1). The 2008 experiment included five tandem mass spectrometer in multiple reaction monitoring mode replicates per treatment (N = 20) and the 2009 experiments included with positive electrospray ionization. Details of nutrient and atrazine six replicates per treatment (N = 24). chemical analysis are given in Appendix A of the Supplementary The exposure profile of the agrochemicals to periphyton via passive- material. diffusion from the periphytometers represented a “worst-case” scenario by maintaining concentrations higher than expected in-stream concen- 2.3. In-situ periphytometer experiments trations throughout the experiments. Similar to Kish (2006) and Ludwig et al. (2008), the nutrient treatment included an initial concentration − Passive-diffusion periphytometers were deployed in the South of 20 mg/L NO3 −N. An initial concentration of 1.25 mg/L RP–P was se- Castor River in 2008 and in both the South Castor River and North lected to give the Redfield ratio of N:P (16:1) (Redfield, 1958). In 2008, Branch of the South Nation River in 2009 (Fig. 1; Table 1). These two the atrazine treatment consisted of 20 μg/L because atrazine concentra- low agriculture sites were chosen because they were expected to be nu- tions in streams and rivers rarely exceed this amount (Solomon et al., trient limited and have limited exposure to atrazine. Periphytometers 1996). In 2009, atrazine was increased by an order of magnitude consisted of a solution reservoir, a membrane to reduce microbial con- (200 μg/L) to maintain experimental concentrations of atrazine tamination, a glass fiber filter substrate for periphyton colonization well above likely environmental concentrations throughout the R.L. Dalton et al. / Science of the Total Environment 515–516 (2015) 70–82 73

experiments. Atrazine was injected into periphytometers on day 7 to reduce existing biomass or day 0 to inhibit colonization (Table 1). The nu-

trient treatment was prepared from anhydrous sodium nitrate (NaNO3) and disodium hydrogen phosphate heptahydrate (Na2HPO4–7H2O) (Sigma-Aldrich, Oakville, Canada). The atrazine treatment was prepared using the commercial herbicide formulation Atrazine 480 (United Agri

N + 1.25 mg/L Reactive Products Canada Inc., Dorchester, Canada), containing 451 g/L of the – active ingredient atrazine (6-chloro-N-ethyl-N′-1-methylethyl-1,3,5- P + atrazine (same

– triazine-2,4-diamine) and 29 g/L of related triazines (for example, sima- zine and propazine). Periphytometers were deployed for 14 days. Preliminary experi-

phosphate concentration and timing as atrazine treatment) ments demonstrated that significant periphyton colonization occurred after a 14 day experimental period and this timeframe was also used in several earlier studies (Matlock et al., 1998; Kish, 2006; Ludwig et al., 2008). Periphytometers were deployed in July and August (Table 1), after expected peak in-stream atrazine concentrations (June) following the application of atrazine to corn crops (late April through to July). Periphytometers were oriented parallel to the stream g/L spiked on day 0 g/L spiked on day 7 g/L spiked on day 0 g/L spiked on day 7 μ μ μ μ g/L spiked on day 7 20 mg/L Nitrate current with the glass fiber filters perpendicular to the water's surface μ 200 200 200 200 (Matlock et al., 1998). Periphytometers were deployed subsurface at a uniform depth (10 cm) (Appendix B). Following each experimental P20

– period, periphytometer glass ﬁber ﬁlters were frozen at −30 °C until pigment extraction. The periphytometer and experimental set-up are describedindetailinAppendixB.

2.4. Periphyton between paired sites and across watershed scale agrochemical gradients

In 2010, periphyton was collected from 24 sites located throughout the South Nation River watershed (Fig. 1). Periphyton was colonized on white high density polyethylene shields used to protect POCIS

N + 1.25 mg/L Reactive phosphate 2

– (Dalton et al., 2014). Each shield had a total surface area of 372 cm . − Accumulated periphyton was removed from the shields after 28 days and then allowed to re-colonize the shields for another 28 days. At each site, one upstream and one downstream facing shield was collected. Upon retrieval, the shields were stored at −30 °C in darkness until

20 mg/L Nitrate processing. Periphyton was removed from the shields with a plastic putty knife and nylon brush and each sample (350 mL final volume in tap water) homogenized in a blender. A sub-sample (5–20 mL) was filtered through a 1.5 μm Whatman glass fiber filter (type 934-AH) and frozen at −30 °C until pigment extraction. Triplicate sub-samples fi Control Nutrient Atrazine Nutrient + atrazine Distilled water were processed for six shields and yielded an average coef cient of variation of 8.2% (range 2.6 to 14.0%).

2.5. Extraction and quantitation of algal pigments 7 Aug. 2008 – In-stream planktonic chlorophyll a was measured by extracting algal 28 July 2009 19 Aug. 2009 23 July 2009 20 Aug. 2009 – – – – pigments in acetone and dimethyl sulfoxide (Burnison, 1980)and calculating chlorophyll a with spectrophotometry (Pye Unicam SP8- eld sites in the South Nation River watershed, Canada (2008 and 2009). fi 100 UV–Visible spectrophotometer, Thermo Fisher ScientificInc., Waltham, USA or Varian Cary100 UV–Visible spectrophotometer, Agilent Technologies, Mississauga, Canada) using a trichromatic equa- tion (Jeffrey and Humphrey, 1975). HPLC was used to identify algal pigments for chemotaxonomic determination of the contribution of major algal groups to chlorophyll a biomass for both the periphytometer experiments and the cross-watershed field study (Table C.1). Pigment extraction was modified from Buffan-Dubau and Carman (2000) and conducted in darkness or low light. Glass fiber filters were freeze dried (48 to 72 h) (Super Modulyo freeze dryer, Fisher Scientific, Ottawa, Canada). Samples were sonicated in 5 mL (periphytometer samples) or 10 mL (cross-watershed periphyton samples) of HPLC grade acetone (Fisher Scientific) for 30 s and algal pigments were extracted for 24 h at −30 °C. Samples were centrifuged at 4000 rpm at 4 °C for 15 m and filtered through 0.2 μm 13 mm diameter PTFE syringe filters (Fisher Scientific). Cross-watershed periphyton samples (2010) were diluted in 5 2009 SC 0 South Castor River R. 5 4 2009 SC 7 South Castor River R. 9 1 2008 SC 7 South Castor R. 24 July Experiment Code Site Dates Treatments 3 2009 NB 0 North Branch South Nation R. 6 2 2009 NB 7 North Branch South Nation R. 14

Table 1 Overview of periphytometer experiments conducted at two acetone to achieve approximately 40 ng chlorophyll a on column, as 74 R.L. Dalton et al. / Science of the Total Environment 515–516 (2015) 70–82

Table 2 Periphyton pigment biomass (mg/m2) from in situ studies. Average values (±standard deviation) are shown with minimum and maximum values in brackets. Data from periphytometer experiments include all experimental treatments.

Pigment 2008 periphytometer experiment 2009 periphytometer experiments (North Branch 2010 periphyton at 24 South Nation R. (South Castor R.) (N = 20) South Nation and South Castor R.) (N = 96) watershed sites (N = 60)

Chlorophylls Chlorophyll a 24.74 ± 7.22 (10.96–38.66) 1.82 ± 1.09 (0.35–5.07) 38.00 ± 38.93 (1.07–152.21) Chlorophyll b 0.90 ± 0.26 (0.37–1.51) 0.04 ± 0.02 (0.01–0.11) 4.53 ± 7.38 (0.11–51.25)

Chlorophyll c2 0.14 ± 0.03 (0.08–0.21) 0.004 ± 0.004 (0.000–0.026) 0.25 ± 0.26 (0.01–1.05)

Carotenes α-Carotene 0 0 0 β-Carotene 2.23 ± 0.18 (1.94–2.59) 0.08 ± 0.11 (0.00–0.41) 1.34 ± 1.98 (0.00–10.24)

Xanthophylls Alloxanthin 0.31 ± 0.11 (0.17–0.62) 0.08 ± 0.11 (0.00–0.42) 0.87 ± 1.21 (0.00–5.62) Diadinoxanthin 1.23 ± 0.34 (0.73–2.04) 0.04 ± 0.10 (0.00–0.47) 1.46 ± 2.50 (0.00–11.85) Diatoxanthin 0 0.04 ± 0.10 (0.00–0.38) 0.10 ± 0.37 (0.00–2.13) Echinenone 0 0 0 Fucoxanthin 10.21 ± 3.15 (5.02–17.55) 0.97 ± 0.60 (0.10–2.67) 11.14 ± 12.98 (0.22–53.28) Lutein 0.39 ± 0.12 (0.17–0.67) 0.07 ± 0.11 (0.00–0.45) 1.30 ± 1.78 (0.00–10.93) Peridinin 0 0 0 Zeaxanthin 0 0.02 ± 0.06 (0.00–0.22) 0.09 ± 0.34 (0.00–1.91)

determined from test samples. Pigments were separated following land use, atrazine concentrations, major nutrient fractions and chloro- Zapata et al. (2000) using a Waters (Mississauga, Canada) HPLC (626 phyll a concentrations between low and high agriculture sites were pump, 717 plus autosampler and 600 s controller) with a 2996 photodi- compared using paired t-tests. The assumption of normality of differ- ode array and a 2475 multi-wavelength ﬂuorescence detector with a ences between pairs was evaluated with Shapiro–Wilk's tests. Non-

Symmetry C8 analytical column (3.6 × 150 mm, average particle size parametric Wilcoxon signed rank tests were conducted when transfor- 4 μm, mobile phase A: methanol:acetonitrile:aqueous pyridine solution mations did not improve normality. In-stream concentrations of atra- (50:25:25), B: methanol:acetonitrile:acetone (20:60:20), 25 μL injec- zine, the major nutrient fractions and biomass of the controls were tion). Pigments were quantified using Waters Empower-2 Software. compared between periphytometer experiments with one-way analy- To date the program CHEMTAX (Chemical Taxonomy) (Mackey sis of variance (ANOVA). Two-way ANOVAs were used to examine the et al., 1996) has been used primarily for classification of marine phyto- effects of periphytometer treatment (control, nutrient, atrazine, nutri- plankton (Higgins et al., 2011 and references therein), less commonly ent + atrazine) and algal class (Bacillariophyceae and Chlorophyceae, for freshwater phytoplankton (Descy et al., 2009 and references there- as well as Cryptophyceae and Euglenophyceae for the 2008 experi- in) and rarely for freshwater periphyton (Lauridsen et al., 2011). In ment) on chlorophyll a biomass. Algal class and treatment were consid- total, 13 pigments were measured (Table 2) and were associated with ered fixed factors in factorial two-way ANOVAs for each periphytometer eight algal classes found in freshwater (Table C.1; Jeffrey et al., 2011). experiment. Differences between treatment levels were assessed using The number of algal classes analyzed was reduced by removing classes Sidak post-hoc tests. − with unique pigment markers that were not detected and those with Linear regression was used to assess the effects of atrazine, NO3 ,RP low concentrations of shared pigment markers that could not be distin- and DIN:RP (separately) on periphyton biomass across the watershed. guished between multiple classes. Initial CHEMTAX (v.1.95) analyses Pearson's correlations were subsequently used to assess the correlation − were conducted using chlorophyll a, chlorophyll b, β-carotene, between atrazine, NO3 , RP and DIN:RP and between these agrochemi- alloxanthin, diadinoxanthin, fucoxanthin and lutein to identify the con- cals and land use. A paired t-test was conducted to assess differences tribution of the Bacillariophyceae, Chlorophyceae, Cryptophyceae and in overall periphyton biomass between paired sites. A factorial 2-way Euglenophyceae to total periphyton biomass. Pigment concentrations ANOVA was used to examine effects of algal class (Bacillariophyceae, were very low in the 2009 periphytometer experiments (Table 2), lim- Chlorophyceae, Cryptophyceae and Euglenophyceae) and agriculture iting the ability of CHEMTAX to reliably distinguish between multiple impact (low or high) on chlorophyll a biomass. A GLM was used to as- − algal classes. The two dominant algal classes, the Bacillariophyceae sess effects of algal class (fixed factor) and atrazine, NO3 and RP (covar- and Chlorophyceae, were classified for these data using chlorophyll a, iates) on chlorophyll a biomass. Interactions terms between covariates the Bacillariophyceae marker fucoxanthin and both chlorophyll b and were included. Preliminary analysis included tributary as a random fac- lutein for Chlorophyceae markers. An initial pigment to chlorophyll a tor in mixed linear models but in the case of the GLM, F-statistics for ratio matrix was created by averaging pigment ratios from culture/ some interactions could not be calculated. Since mixed linear model re- field experiments using data reviewed in Higgins et al. (2011). Pigment sults were generally similar to those of the 2-way ANOVA and GLM, and ratios were subsequently optimized by running an additional 60 ran- Wald statistics (Wald Z = 1.695; p = 0.090 and Wald Z = 1.854; p = domized ratios for each dataset (cross-watershed study and five 0.064 respectively) provided evidence that the random variable was periphytometer experiments). The best solution, having the lowest not significant, the simpler models were used. General linear model as- root mean square residual, was used to calculate algal class abundances sumptions of normality of residuals and homogeneity of variance were and the best six solutions used to calculate average pigment ratios and assessed using Shapiro–Wilk's and Levene's tests respectively where their standard deviations for the final pigment ratio matrices. Details appropriate. Data were transformed if necessary using square root or of the optimization procedure and selection of algal classes and pig- logarithmic transformations to best meet these assumptions. ments are given in Appendix C. Partial canonical correspondence analysis (partial CCA) was used to examine the relationship between periphyton community structure 2.6. Data analysis and agrochemicals, using environmental covariables to account for physico-chemical differences between field sites. Variables were nor- Univariate statistical analyses were performed using SPSS v21 (IBM malized if necessary following an assessment of normality using Corp., Armonk, USA). Differences in physical and chemical variables, Shapiro–Wilk's tests and then standardized to a mean of 0 and standard R.L. Dalton et al. / Science of the Total Environment 515–516 (2015) 70–82 75

Table 3 Physical and chemical characteristics of 12 paired (24 in total) stream/river sites in the South Nation River watershed, Canada. Signiﬁcant (p b 0.05) paired t-tests comparing average values between paired sites are in bold.

Variable Units Average ± standard deviation Average ± standard deviation N Paired t-test comparing average (range in brackets) (range in brackets) values (df = 11)

Low agriculture sites High agriculture sites

Physical stream characteristics Strahler stream order n/a 4.5 ± 0.90 (3–6) 4.7 ± 0.89 (4–6) 24 Wilcoxon p = 0.157a Stream width m 14.2 ± 7.6 (4.7–32.6) 16.5 ± 11.2 (5.6–48.5) 72 t = −0.919; p = 0.378 Average baseﬂow depth cm 66 ± 30 (33–115) 66 ± 45 (18–182) 216 t = 0.063; p = 0.951 Maximum baseﬂow depth cm 89 ± 44 (40–190) 90 ± 67 (22–258) 72 t = −0.036; p = 0.972 Surface velocity m/s 0.091 ± 0.086 (0.024–0.321) 0.094 ± 0.055 (0.009–0.202) 144 t = −0.096; p = 0.926

Water chemistry pH n/a 7.85 ± 0.24 (7.42–8.08) 8.06 ± 0.24 (7.75–8.63) 72 t=−3.862; p = 0.003 Dissolved oxygen mg/L 5.93 ± 1.80 (2.21–8.25) 7.74 ± 2.20 (4.96–12.55) 72 t=−3.220; p = 0.008 Temperature °C 20.95 ± 1.90 (18.42–24.08) 21.90 ± 1.62 (18.46–23.94) 72 t = −2.021; p = 0.068 Conductivity μS/cm 521.4 ± 136.4 (343.9–788.7) 532.2 ± 100.2 (366.5–690.0) 72 t = −0.748; p = 0.470 Turbidity NTU 9.3 ± 5.5 (2.3–18.1) 17.2 ± 13.6 (2.4–50.9) 144 t=−2.294; p = 0.043 Planktonic chlorophyll a μg/L 7.2 ± 4.1 (2.6–15.1) 8.5 ± 4.1 (3.8–16.2) 144 t = −1.052; p = 0.315

a Non-parametric Wilcoxon signed rank tests were conducted when paired t-test assumptions of normality of differences between pairs were not met after data transformations. deviation of 1 (z-score transformation). Of the potential covariables, paired sites (Table 3). Strahler stream order ranged from 3 near the stream order, stream width, average depth, maximum depth, surface headwaters to 6 along the main branch of the South Nation River. Across velocity, conductivity and turbidity were included as environmental the watershed, sites differed in terms of stream width, baseflow depth variables in an initial full CCA. Temperature was not included because (both average and maximum) and surface velocity. However, paired it was reflective of ambient temperatures and expected to vary more be- sites were similar in terms of these physical characteristics, as well as tween sampling dates than between field sites. Dissolved oxygen and other characteristics including temperature, conductivity and plankton- pH were excluded because although they may influence periphyton ic chlorophyll a. High agriculture sites were more turbid, more alkaline communities, they may also be affected by periphyton. Symmetric and had a higher concentration of dissolved oxygen compared to low biplot scaling was used (ter Braak and Šmilauer, 2002). Forward step- agriculture sites (Table 3). wise regression and partial Monte Carlo permutation tests were used The percentage of surrounding annual crop land was higher at high to calculate F-ratios and test the significance of the environmental var- agriculture sites compared to low agriculture sites (Table 4). Time- iables at p b 0.05 (ter Braak and Šmilauer, 2002). Significant environ- weighted-average concentrations of atrazine (0.004 to 0.412 μg/L) mental variables were then used as covariables in a subsequent partial were also significantly higher at high agriculture sites (Table 4). Con- CCA where the ordination was constrained to variation in periphyton centrations of RP and TP were similar between paired sites between − class data explained by atrazine and June concentrations of NO3 ,RP May and July 2010. Nitrate ranged in concentration by three orders of − and the ratio DIN:RP. Planktonic chlorophyll a was also included as magnitude across the watershed. Although NO3 tended to be higher an environmental variable because phytoplankton and periphyton at high agriculture sites compared to low agriculture sites, the trend can be negatively correlated. Multivariate analyses were conducted was statistically significant only for data averaged from May, June and using CANOCO v.4.5 (Plant Research International, Wageningen, The July samples. Total nitrogen followed similar trends, driven by changes − Netherlands). in NO3 . Ratios of N:P also varied and the ratio of DIN:RP was significantly higher at high agriculture sites for data averaged from May, June and 3. Results July samples (Table 4). In-stream concentrations of agrochemicals were also measured 3.1. Characteristics of field sites at two field sites during the five periphytometer experiments (July–August 2008 and 2009). Atrazine concentrations were low at Physical and chemical characteristics varied across 24 field sites in both of these low agriculture field sites (Table 5). In-stream concen- − the South Nation River watershed but were typically similar between trations of RP, TP and NO3 and ratios of DIN:RP and TN:TP differed

Table 4 Agriculture land use (percentage of surrounding annual crop land) and in-stream agrochemicals at 12 paired (24 in total) stream/river sites in the South Nation River watershed, Canada. Concentrations of atrazine, major nutrient forms (μg/L) and their ratios were averaged from samples taken in May, June and July 2010. Statistics in bold are signiﬁcant at p b 0.05.

Chemical Abbreviation Time Low agriculture sites High agriculture sites Paired t-test (df = 11)

Annual crop land 30.2 ± 22.7 (6.7–83.9) 67.0 ± 15.8 (44.3–97.4) t=−4.865; p b 0.001 Atrazine ATR May–July 0.098 ± 0.102 (0.004–0.364) 0.178 ± 0.128 (0.014–0.412) t=−2.480; p = 0.031 Reactive phosphate RP June 35 ± 20 (10–69) 40 ± 27 (4–102) t = −0.781; p = 0.451 May–July 54 ± 29 (20–110) 59 ± 25 (22–103) t = −0.872; p = 0.402 Total phosphorus TP June 55 ± 20 (30–85) 57 ± 20 (24–102) t = −0.305; p = 0.766 May–July 81 ± 32 (39–131) 86 ± 28 (48–139) t = −0.574; p = 0.577 − Nitrate NO3 June 1356 ± 1476 (3–4235) 2379 ± 1829 (117–5404) t = −2.087; p = 0.061 May–July 643 ± 689 (8–1784) 1271 ± 836 (70–2503) Wilcoxon p = 0.004a Total nitrogen TN June 2286 ± 1413 (851–5019) 3204 ± 1740 (1027–6144) t = −1.955; p = 0.076 May–July 1649 ± 611 (895–2704) 2194 ± 772 (1155–3447) Wilcoxon p = 0.012a Dissolved inorganic nitrogen: reactive phosphate DIN:RP June 41 ± 47 (2–157) 113 ± 157 (3–564) t = −2.181; p = 0.052 May–July 20 ± 18 (2–55) 51 ± 54 (2–190) t=−2.598; p = 0.025 Total nitrogen: phosphorus TN:TP June 42 ± 23 (15–85) 64 ± 43 (16–150) t = −1.901; p = 0.084 May–July 27 ± 9 (13–41) 35 ± 18 (12–65) t = −1.720; p = 0.113

a Non-parametric Wilcoxon signed rank tests were conducted when paired t-test assumptions of normality of differences between pairs were not met after data transformations. 76 R.L. Dalton et al. / Science of the Total Environment 515–516 (2015) 70–82

between experiments. Total nitrogen was similar between experiments (Table 5). 0.503 = 0.655 =0.716 = 0.624 = 0.498 = = 0.237 2 2 2 2 2 2 3.2. In-situ periphytometer experiments 0.001; R 0.001; R 0.001; R b b b Biomass of the controls was an order of magnitude higher in 2008 (experiment 1) compared to 2009 (experiments 2–5) (Fig. 2)(pb 0.001). Total rainfall in 2009 was substantially higher than that in 2008(334mmversus139mmrespectivelyduringJulyandAugust nd 2009). Minimum and maximum F = 6.704; p = 0.001; R F = 12.833; p F = 6.838; p = 0.001; R F = 16.980; p F = 11.224; p at the Ottawa Macdonald-Cartier International Airport, Canada) (Environment Canada, 2014). In 2009, biomass of the controls was similar between the two experiments conducted at each site (p ≥

2937) a F = 2.097; p = 0.109; R 0.099). In 2008, the Cryptophyceae and Euglenophyceae represented 2207) ab – – 0.027) n/a only 4.0 ± 1.4 and 1.5 ± 1.0% of total chlorophyll a biomass respec- – 88) ab 68) ab 26) abc 44) ab – tively. Subsequent analyses examined the contributions of only the – – 0.020

b Bacillariophyceae and Chlorophyceae to biomass. Biomass of the Bacillariophyceae was signiﬁcantly higher than biomass of the ≥ ≤

0.020 ( Chlorophyceae for all experiments (Fig. 2;F 205.152; p 0.001). b 46 ± 17 (32 Overall, periphytometer biomass consisted of an average of 92.8 ± 6.3% (69.9 to 99.4%) Bacillariophyceae. Conversely, periphytometer

0.034) biomass was on average 7.2 ± 6.3% (0.6 to 30.1%) Chlorophyceae. – 1717) a 1114 ± 860 (386 2363) a 1820 ± 880 (1057 –

– No interaction between algal class and periphytometer treatment was observed for any experiment (F ≤ 0.303; p ≥ 0.825). Biomass 141) a 49 ± 30 (22 – 72) a 24) bc 21 ± 4 (18 37) b 39 ± 5 (32 was similar across treatments for experiments 1 (2008 SC 7), 2 (2009 – – NB 7) and 3 (2009 NB 0) (F ≤ 0.490; p ≥ 0.656). Significant differences between treatments were observed for experiment 4 (2009 SC 7) (F = 2.875; df = 7,40; p = 0.048; R2 = 0.869) and experiment Exp. 4 (2009 SC 7) Exp. 5 (2009 SC 0) 1-Way-ANOVA (df = 4,27) 5 (2009 SC 0) (F = 3.163; df = 7,40; p = 0.035; R2 = 0.844). Biomass of the control was significantly higher than the atrazine treatment in . experiment 4 (2009 SC 7) (p = 0.042) and significantly higher than 1597) a 2064 ± 258 (1753 – all experimental treatments in experiment 5 (2009 SC 0) (p ≤ 0.021) 401) c 1470 ± 236 (1183 Table 1 – (Fig. 2). However, overall total biomass was very low and variability 40) ab 19 ± 6 (11 66) a 34 ± 3 (29 33) c 62 ± 11 (48 – – – 21) c 88 ± 38 (51 – –

– high, limiting the biological signiﬁcance of this result. Measured

2009 NB 0) − concentrations of NO3 , RP and atrazine in periphytometer solution reservoirs from samples taken on days 0, 7 and 14 are described in 0.020 0.032 ± 0.002 (0.029 b Appendix B.

3.3. Periphyton between paired sites and across watershed scale 1781) a 1435 ± 147 (1275 640) bc 304 ± 80 (223 – agrochemical gradients – 44) b 50 ± 9 (42 21) c 28 ± 9 (20 – 42) b 14 ± 6 (9 54) ab 29 ± 3 (24 – Across the watershed, average periphyton biomass was 37.2 ± 38.7

2009 NB 7) Exp. 3 ( 2 0.05). Experiment codes correspond to (1.6 to 138.5) mg/m chlorophyll a (Fig. 3). On average, the periphyton b community was composed of 60.9 ± 12.9% (26.1 to 80.8%) 0.020 b Bacillariophyceae, 28.1 ± 15.0% (2.3 to 64.8%) Chlorophyceae, 6.9 ± 5.4% (0.0 to 15.7%) Cryptophyceae and 4.1 ± 3.1% (0.0 to 11.9%) Euglenophyceae. Total biomass varied across the watershed but was sim- 2759) a 1370 ± 383 (811 1961) a 507 ± 150 (317 – ilar between paired low and high agriculture sites (Fig. 3; t = 0.728, df = – cantly different (p

ﬁ 11; p = 0.482). A factorial two-way ANOVA was used to examine effects 58) b 45 ± 7 (39 40) a 16 ± 4 (11 – 55) a 32 ± 12 (15 54) b 35 ± 5 (30 – – of algal class and agricultural impact (low versus high) on biomass. Bio- mass differed between algal classes (F = 18.936; df = 3,88; p b 0.001; R2 = 0.393), with higher biomass observed for the Bacillariophyceae g/L) averaged (±standard deviation) from days 0, 7 and 14 of periphytometer experiments conducted in the South Nation River watershed, Canada (2008 a 0.020 μ b 1147 ± 521 (745 and Chlorophyceae compared to the Cryptophyceae and Euglenophyceae (Fig. 3). Biomass did not differ between low and high agriculture sites (F = 0.158; df = 1,88; p = 0.692; R2 = 0.393) and no interaction was observed between algal class and level of agricultural impact (F = − 3 0.038; df = 3,88; p = 0.990; R2 = 0.393) (Fig. 3). DIN:RP 37 ± 9 (30 – Linear regression was used to examine the relationship between periphyton biomass and agrochemical gradients, as measured by − concentrations of atrazine and June NO3 ,RPandDIN:RP.Juneconcen- trations were selected because in-stream concentrations of nutrients from fertilizers were expected to peak in June. The percentage of surrounding annual crop land was correlated with atrazine (Pearson − correlation coefﬁcient (PCC) = 0.491; p = 0.015), June NO3 (PCC = 0.500; p = 0.013), DIN:RP (PCC = 0.563; p = 0.004) but not RP

reactive phosphate (PCC = −0.002; p = 0.992). Subsequent analysis focused on direct ChemicalAtrazine Abbreviation Exp. 1 (2008 SC 7) ATR Exp. 2 ( Reactive phosphate RP 31 ± 7 (22 – Nitrate NO Total phosphorus TP 47 ± 6 (37 – Dissolved inorganic nitrogen: Total nitrogenTotal nitrogen: phosphorus TN:TP TN 41 ± 10 (34 1899 ± 559 (1396 Table 5 In-stream concentrations of atrazine and nutrients ( values are in brackets. Values followed by different letters are signi measures of agrochemicals. All four measures showed a general trend R.L. Dalton et al. / Science of the Total Environment 515–516 (2015) 70–82 77

A) Exp. 1 (2008 SC 7) B) Exp. 1 (2008 SC 7) 40 a 40 a ) ) 2 2 aaa aaa 32 32 (mg/m (mg/m 24 24 a a 16 16

8 8 Chlorophyll Chlorophyll Chlorophyll Chlorophyll 0 0 C NUT ATR N+A C NUT AT N+A Treatment Treatment

C) Exp. 2 (2009 NB 7) D) Exp. 3 (2009 NB 0) 5 5 ) ) 2 2 4 4 a 3 a aa 3 2 2 a a aa 1 1 Chlorophyll (mg/m a 0 Chlorophyll (mg/m a 0 C NUT AT N+A C NUT AT N+A Treatment Treatment

E) Exp. 4 (2009 SC 7) F) Exp. 5 (2009 SC 0) 5 a 5

) ab ) 2 2 4 ab 4 b ba 3 3

2 2 b b

1 1 Chlorophyll (mg/m a Chlorophyll a (mg/m a Chlorophyll 0 0 C NUT AT N+A C NUT AT N+A Treatment Treatment

Fig. 2. Average contribution of the Bacillariophyceae and Chlorophyceae to chlorophyll a biomass (mg/m2) in periphyton colonized on periphytometers for 14 days in the South Castor R (SC) or North Branch South Nation R (NB). The Cryptophyceae and Euglenophyceae are also shown in A). Standard deviation is shown for average total biomass. Treatments consisted of a distilled water control (C), nitrate and reactive phosphate nutrients (NUT), atrazine (ATR) or both nutrients and atrazine (N + A). Atrazine was spiked on day 0 or day 7. Treatments followed by different letters are statistically different (p b 0.05), N = 20 (A, B) or N = 24 (C–F). Experiment numbers correspond to Table 1. of increasing periphyton biomass as agrochemicals increased (Fig. 4). 0.213; R2 = 0.538) and atrazine had no effect (F = 0.691; df = 1,85; − 2 Both atrazine and NO3 concentrations had a significant positive effect p=0.408;R = 0.538). on periphyton biomass, whereas RP concentrations and DIN:RP had The response of the periphyton communities to agrochemicals no effect (Fig. 4). Changes in periphyton biomass along agrochemical was examined further using multivariate analysis. An initial CCA in- gradients were assessed using a GLM examining effects of algal class, dicated that of the environmental variables, turbidity, surface veloc- − atrazine and June concentrations of NO3 and RP. The ratio DIN:RP was ity and average depth explained a significant amount of variation in − not included because it was highly correlated with NO3 (PCC = periphyton community structure between field sites (Fig. 5). Turbid 0.823; p b 0.001). Interactions between measures of agrochemicals sites were associated with the Bacillariophyceae, high velocity with the were included in the GLM due to the observed correlation between var- Euglenophyceae and deep sites with the Cryptophyceae (Fig. 5). Turbid- − iables. Atrazine was correlated with NO3 (PCC = 0.561; p = 0.004) and ity, surface velocity and average depth were included as covariables in − NO3 with RP (PCC = 0.407; p = 0.048); these relationships limit the a subsequent partial CCA to examine the influence of agrochemicals model's strength in detecting the influence of individual agrochemicals. on periphyton communities, after accounting for physical and chemical No significant interactions were observed in the model (F ≤ 2.618; df = differences between field sites. Maximum depth was also included as 1,85; p ≥ 0.109; R2 = 0.538). The contribution of algal classes to periph- a covariable because it contributed to the significance of average yton biomass differed significantly (F = 24.016; df = 3,85; p b 0.001, depth (i.e. average depth no longer explained a significant amount of R2 = 0.538) between all classes (p ≤ 0.32), except between the variation once maximum depth was excluded). The partial CCA was Cryptophyceae and Euglenophyceae which had a similar biomass constrained to variation in algal class biomass explained by linear (p = 0.956). After controlling for the other agrochemicals, nitrate had combinations of planktonic chlorophyll a, atrazine and June concentra- − a significant positive effect on overall biomass (F = 6.205; df = 1,85; tions of NO3 , RP and DIN:RP. The model was only able to retain four p = 0.015; R2 = 0.538), whereas RP (F = 1.572; df = 1,85; p = environmental variables (since only four algal classes were examined) 78 R.L. Dalton et al. / Science of the Total Environment 515–516 (2015) 70–82

150 relative to P were associated with higher Bacillariophyceae biomass 125 (Fig. 6). ) 2

100 4. Discussion (mg/m a 75 4.1. Experimental effects of nutrients and atrazine on periphyton

50 In the present study, the experimental addition of nutrients and

Chlorophyll 25 atrazine generally had no effect on periphyton biomass or community composition. The addition of nutrients did not result in an increase in 0 periphyton biomass in either the South Castor River or North Branch 123456789101112 of the South Nation River, suggesting that these field sites were not Tributary strongly nutrient limited. The addition of 20 μg/L atrazine clearly had no effect on periphyton biomass or community composition. In con- Fig. 3. Biomass (mg/m2 chlorophyll a)ofthe Bacillariophyceae, Chlorophyceae, trast, an increase in chlorophyll c relative to chlorophyll a at 12 μg/L Cryptophyceae and Euglenophyceae in periphyton communities at 12 paired sites (24 atrazine was observed in an earlier periphytometer experiment, sug- in total) in the South Nation River watershed, Canada. Sites were paired along tributaries. Low agriculture sites (left column) were located upstream of high agriculture sites (right gesting that chlorophyll c containing Bacillariophyceae were more atra- column). Tributary numbers correspond to Fig. 1. zine tolerant compared to the Chlorophyceae (Kish, 2006). Detenbeck et al. (1996) observed reductions in gross primary productivity at 15 μg/L atrazine but a similar reduction was not observed at higher concentrations. However, they also provided evidence of a shift in and excluded the variable with the lowest conditional effects. Nitrate species dominance or selection for tolerant species at ≥50 μg/L atrazine. was dropped from the model because DIN:RP explained more variation In the present study, periphyton communities in these two temper- and these two variables had very similar effects on periphyton commu- ate streams were generally not inhibited by higher concentrations of nities. Atrazine, DIN:RP and phytoplankton were closely related and atrazine (200 μg/L). Effects of atrazine were observed for two experi- were explained by Axis 1, whereas RP was associated with Axis 2 ments but periphyton biomass in atrazine and nutrient treatments (Fig. 6). Of the variables, only DIN:RP explained a significant amount were not significantly different, suggesting that there was no biological- of variation. Sites enriched with N relative to P were associated with ly significant effect of atrazine. Very low biomass in these experiments, higher Chlorophyceae biomass, whereas sites less enriched with N likely due to high rainfall limiting colonization and increasing scouring,

A) B) 150 150 ) ) 2 2 2 F=5.081; p=0.034; R =0.188 F=7.493; p=0.012; R2=0.254 100 100 (mg/m (mg/m a a

50 50 Chlorophyll Chlorophyll 0 0 0.000 0.150 0.300 0.450 0 1500300045006000 Atrazine (μg/L) June Nitrate (μg/L)

C) D) 150 150 ) ) 2 2 F=0.810; p=0.378; R2=0.036 F=3.603; p=0.071; R2=0.141 100 100 (mg/m (mg/m a a

50 50 Chlorophyll Chlorophyll Chlorophyll 0 0 0255075100125 0 150 300 450 600 June RP (μg/L) June DIN:RP

2 − Fig. 4. Relationship between periphyton biomass (mg/m chlorophyll a) at 12 paired sites (24 in total) in the South Nation River watershed, Canada and A) atrazine, B) June nitrate (NO3 ), C) June reactive phosphate (RP) and D) June ratio of dissolved inorganic nitrogen to reactive phosphate (DIN:RP). Regression statistics and signiﬁcant trend lines are shown. R.L. Dalton et al. / Science of the Total Environment 515–516 (2015) 70–82 79

1.5 reduced the ability of the periphytometers to detect treatment effects. There may also be several other explanations for the apparent lack of 2 sensitivity to atrazine. The sensitivity of algal species to atrazine varies. For example, Fairchild et al. (1998) observed 50% inhibition concentra- 7 12 tions (IC50s) ranging from 94 μg/L for Chlorella sp. (Chlorophyceae) to N μ 5 3000 g/L for Anabaena sp. (Cyanophyceae). A review of toxicity data found the lowest reported IC50 for the Chlorophyceae was b1 μg/L (chlo- Max. depth Turbidity p=0.002 Aver. depth p=0.012 rophyll production over 7 days for 34 spp.) compared to 19 μg/L for the 11 8 3 6 Width Bacillariophyceae (population growth over 48 h for 46 spp.) (US EPA, 9 1 Conductivity 5 2012). Increased sensitivity of the Chlorophyceae to atrazine compared 0 4 2 to the Bacillariophyceae has been associated with higher rates of 11 Order 10 8 6 10 atrazine accumulation in the Chlorophyceae (Tang et al., 1998). The ex- 1 9 perimental conditions here may have selected an atrazine tolerant 12 3 Bacillariophyceae dominated community. Some biofouling occurred on the periphytometer screens which could have lowered light expo- Velocity p=0.010 7 sure, resulting in conditions more favorable for the Bacillariophyceae 4 (Guasch and Sabater, 1998). Also, the Bacillariophyceae are early colo- nizers (Guasch et al., 1997) and may have been more likely to colonize Axis 2 (16.3% species, 22.3% species-environment) the periphytometers over the relatively short test period.

-1.5 4.2. Changes in periphyton across agrochemical gradients -1.5 0 1.5

Axis 1 (54.7% species, 74.4% species-environment) Changes in periphyton biomass were observed across agrochemical gradients with a general trend towards increased periphyton biomass − Fig. 5. Canonical correspondence analysis (CCA) ordinating field sites by biomass of the ■ with increasing agrochemical concentration. Both atrazine and NO3 had significant positive effects on periphyton biomass, whereas RP had no ef- Bacillariophyceae, ● Chlorophyceae, ▲ Cryptophyceae and ◆ Euglenophyceae, constrained fi to variation explained by stream order, stream width, maximum depth, average depth, fect. However, effects of atrazine were not signi cant when multiple, cor- surface velocity, conductivity and turbidity. Twelve paired sites (24 in total) in the South related agrochemicals were modeled simultaneously. Periphyton Nation River watershed, Canada are shown with paired sites represented by the same communities were generally dominated by the Bacillariophyceae and number and high agriculture sites underlined. Significant variables (p b 0.05) are shown in Chlorophyceae to a lesser degree, with minor contributions of the bold. Cryptophyceae and Euglenophyceae. Nitrate enrichment relative to reactive phosphate was associated with the Chlorophyceae. In contrast, Munn et al. (2002) found that U.S. agricultural streams were dominated by either Bacillariophyceae or Cyanophyceae with Chlorophyceae

2 representing only 1% of total periphyton relative abundance, while Nelson et al. (2013) found that the Bacillariophyceae, Chlorophyceae and Cyanophyceae showed no consistent response to nutrient augmen- 2 tation in 30 U.S. streams. Similarly to the present study, Chételat et al. (1999) found the percentage of both Chlorophyceae and Bacillariophyceae biomass increased with increasing nutrients. The influence of nutrients on periphyton biomass and community 1 structure was examined further by comparing differences in the distri- Atrazine 6 5 bution of P and N across the watershed. Sites within the South Nation p=0.686 6 DIN:RP 9 River watershed were characterized by both N and P enrichment with 4 7 0 p=0.016 9 311 11 10 5 4 concentrations generally exceeding environmental thresholds for 8 1 8 stream impairment due to eutrophication (TP N30 μg/L and TN Phytoplankton 7 10 p=0.176 12 12 RP N1100 μg/L for the Mixedwood Plains of Ontario) (Chambers et al., p=0.444 3 2012). Nitrate was particularly enriched at high agriculture sites. Ratios of N:P N16:1 indicate P limitation (Redfield, 1958) but nutrient limitation is also a function of absolute concentrations of N and P. Keck and Lepori (2012) observed a wide transition in the probability of N limitation in stream/rivers and found that nutrient limitation was difficult to Axis 2 (5.2% species, 15.8% species-environment) predict except at extreme ratios of N:P (e.g. b1:1 and N100:1). They pro- posed that while individual species may be limited by specificoptimal -2 N:P ratios, communities may be able to take advantage of additions of 1- 0 1 either N or P through increased nutrient acquisition, reduced loss of Ax is 1 (25.0% spe cie s, 76.8% spe cie s-e nvironme nt) the scarcest nutrient and shifts in community structure. In the present study, it appeared that the Chlorophyceae were best able to take advan- Fig. 6. Partial canonical correspondence analysis (partial CCA) ordinating field sites tage of N enriched conditions. by biomass of the ■ Bacillariophyceae, ● Chlorophyceae, ▲ Cryptophyceae and ◆ A gradient of atrazine was observed across the watershed but all Euglenophyceae, constrained to variation explained by June reactive phosphate (RP), June sites had time-weighted-average concentrations well below Canadian ratio of dissolved inorganic nitrogen to reactive phosphate (DIN:RP), atrazine and water quality guidelines for the protection of aquatic life (1.8 μg/L) planktonic chlorophyll a concentrations (phytoplankton) using surface velocity, turbidity, (Canadian Council of Ministers of the Environment, 1999). Atrazine is average depth and maximum depth as covariables. Twelve paired sites (24 in total) in the not expected to have direct toxic effects on periphyton at environmen- South Nation River watershed, Canada are shown with pairs represented by the same number and high agriculture sites underlined. Significant variables (p b 0.05) are shown tally relevant concentrations (Solomon et al., 1996), although inhibitory in bold. effects on algal communities have been observed at concentrations as 80 R.L. Dalton et al. / Science of the Total Environment 515–516 (2015) 70–82 low as 0.1 and 1 μg/L (Pannard et al., 2009). Nutrients and atrazine were uncertainty in the analysis and the variability observed in pigment ra- expected to have contrasting effects on periphyton with nutrients stim- tios between datasets. Several studies have compared pigment analysis ulating growth and atrazine inhibiting growth. However, atrazine can with microscopy (reviewed in Wright and Jeffrey, 2006), including for both stimulate (10 μg/L) (Murdock and Wetzel, 2012) and inhibit pe- freshwater periphyton (e.g. Lauridsen et al., 2011) and have found riphyton (100 μg/L) (Guasch et al., 2007). Indirect positive effects of at- that while the two methods are generally correlated, discrepancies razine (100 μg/L) may also occur through a reduction in phytoplankton occur. A major difference in the two techniques is that biomass is gener- and subsequent increase in light and nutrient availability (Rohr et al., ally determined from estimates of biovolume for microscopy and from 2008; Halstead et al., 2014). Clearly, the response of atrazine is direct measures of chlorophyll a in pigment analysis. Microscopy pro- dependent on both the concentration, exposure duration and species vides a finer taxonomic resolution but pigment analysis is a useful present, as demonstrated by conflicting trends in the response of high-throughput tool for examining broad changes in algal class compo- periphyton to interactions between nutrients and atrazine in field and sition. In the present study, estimates of algal class with pigment analy- laboratory studies (Murdock et al., 2013). sis provided a reasonable estimate of the major algal classes due to the Recovery of periphyton functional endpoints from short term atra- presence of unique pigment markers and illustrated broad changes in zine exposure often occurs (e.g. Prosser et al., 2013,reviewedin community structure with nitrate enrichment. Solomon et al., 1996 and Brock et al., 2000). However, prior exposure to atrazine may also complicate the response of algae to agrochemicals because it tends to increase community tolerance towards atrazine 5. Conclusions (Guasch et al., 1998, 2007) and favor the Bacillariophyceae compared to the Chlorophyceae (Fairchild et al., 1998; Lockert et al., 2006). Given that atrazine is a herbicide, we expected to observe negative Andrus et al. (2013) found a lack of response of periphyton communi- effects of atrazine on periphyton, at least under experimental condi- ties to in-stream atrazine concentrations and attributed the lack of re- tions. Atrazine did not have significant, observable effects on periphyton sponse to either insufficient atrazine concentrations/exposure biomass and community structure, although separating effects of durations or the presence of tolerant Bacillariophyceae dominated com- agrochemicals was challenging due to their correlation. Experimental munities. In the South Nation River watershed, exposure to atrazine nutrient enrichment did not alter periphyton communities, suggesting over a number of years may have led to the selection of tolerant taxa. At- that the two Bacillariophyceae dominated stream sites were not nutri- razine contamination is widespread throughout the watershed (Dalton ent limited. However, at the watershed scale nutrient enrichment had et al., 2014) and exposure to atrazine appears to have led to decreased a significant effect on periphyton biomass and community structure. − sensitivity in populations of duckweed (Lemna minor)inthiswatershed Specifically, biomass increased along a gradient of increasing NO3 and − (Dalton et al., 2013). NO3 enrichment was associated with increased Chlorophyceae. Chang- Interactions between nutrients and atrazine complicate and poten- es in periphyton biomass and community structure were observed even tially mask the relative strength of their effects on periphyton commu- though the watershed was generally enriched in both N and P. These nities. Antagonistic effects would dampen the response whereas results support the theory that, unlike individual species, communities synergistic or additive effects would enhance the response but make are able to respond to additions of N or P through shifts in community separating individual effects difficult. For example, variance partitioning structure towards species best able to utilize these nutrients (Keck determined that atrazine had few effects on Midwest (U.S.) agricultural and Lepori, 2012). The present study highlights that nutrient enrich- streams but that significant ambiguity existed in determining effects of ment has the potential to impair the environmental quality and ecolog- specific variables on periphyton communities due to the variation ical health of surface waters under a range of nutrient limiting scenarios shared between groups of variables (Andrus et al., 2015). In the South and provides further evidence for the need to regulate nitrogen. − Nation River watershed, NO3 and atrazine were highly correlated because of their widespread use on corn crops, a major annual crop in this watershed. Effects of atrazine could not be clearly distinguished Acknowledgments − − from those of NO3 and effects of NO3 on periphyton superseded observable effects of atrazine and RP. This research was funded by grants to F. R. Pick (036751) and C. Boutin (249771) from the Natural Sciences and Engineering Research 4.3. Chemotaxonomic characterization of freshwater periphyton Council of Canada and to C. Boutin from Environment Canada. We thank Dr. Irene Gregory-Eaves' lab (McGill University), particularly In the present study, CHEMTAX was used, for the first time to our Kyle Simpson, Leen Stephan and Zofia Taranu for HPLC analysis of pe- knowledge, to characterize freshwater periphyton communities at the riphyton pigments. Thank you to France Maisonneuve (Environment watershed scale. Across the watershed, the main taxonomic groups of Canada) for the analysis of atrazine in periphytometer samples and to periphyton based on pigment analysis were the Bacillariophyceae, Dr. Ammar Saleem (Laboratory for the Analysis of Natural and Synthetic Chlorophyceae, Cryptophyceae and Euglenophyceae. Pigment markers Environmental Toxins, University of Ottawa) who was a key collabora- were too low to include the Cyanophyceae, Rhodophyceae and tor in the development of the atrazine analytical method for POCIS sam- Chrysophyceae in the analysis and although these groups are less com- ples. Nutrients were analyzed at the Robert O. Pickard Environmental mon, they may occur in periphyton communities (Allan, 1995). One Centre Laboratory of the City of Ottawa (Canada). Thank you to Philippe limitation of chemotaxonomy is that interpretation of the absence of Thomas (Environment Canada) and the Geographic, Statistical and Gov- pigments is less reliable than the presence of pigments because minor ernment Information Centre (University of Ottawa) for assistance with pigments may be unidentified or unreported (Higgins et al., 2011). In GIS analysis. We also thank Ashley Alberto, Elias Collette, David the present study a number of pigments had low concentrations even Lamontagne, Christina Nussbaumer, Luba Reshitnyk and Philippe when chlorophyll a was high and it was unclear whether absent pig- Thomas for their assistance in the field and/or processing periphyton ments and their related algal classes were truly absent or simply samples. below the limit of detection. A further limitation is that pigment ratios must be constant across samples (Mackey et al., 1996). Pigment ratios are known to vary between species and even strains (Jeffrey and Supplementary material Wright, 1994), may be altered by light and nutrient regimes (Higgins et al., 2011) and are currently underdeveloped for freshwater algae Supplementary material for this article can be found online at http:// (Lauridsen et al., 2011). These issues may have contributed to dx.doi.org/10.1016/j.scitotenv.2015.01.023. R.L. Dalton et al. / Science of the Total Environment 515–516 (2015) 70–82 81

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