Phytoplankton-specific response to enrichment of phosphorus-rich surface waters with ammonium, nitrate, and urea.

Supply of anthropogenic nitrogen (N) to the biosphere has tripled since 1960; however, little is known of how in situ response to N fertilisation differs among phytoplankton, whether species response varies with the chemical form of N, or how interpretation of N effects is influenced by the method of analysis (microscopy, pigment biomarkers). To address these issues, we conducted two 21-day in situ mesocosm (3140 L) experiments to quantify the species- and genus-specific responses of phytoplankton to fertilisation of P-rich lake waters with ammonium (NH(4)(+)), nitrate (NO(3)(-)), and urea ([NH(2)](2)CO). Phytoplankton abundance was estimated using both microscopic enumeration of cell densities and high performance liquid chromatographic (HPLC) analysis of algal pigments. We found that total algal biomass increased 200% and 350% following fertilisation with NO(3)(-) and chemically-reduced N (NH(4)(+), urea), respectively, although 144 individual taxa exhibited distinctive responses to N, including compound-specific stimulation (Planktothrix agardhii and NH(4)(+)), increased biomass with chemically-reduced N alone (Scenedesmus spp., Coelastrum astroideum) and no response (Aphanizomenon flos-aquae, Ceratium hirundinella). Principle components analyses (PCA) captured 53.2-69.9% of variation in experimental assemblages irrespective of the degree of taxonomic resolution of analysis. PCA of species-level data revealed that congeneric taxa exhibited common responses to fertilisation regimes (e.g., Microcystis aeruginosa, M. flos-aquae, M. botrys), whereas genera within the same division had widely divergent responses to added N (e.g., Anabaena, Planktothrix, Microcystis). Least-squares regression analysis demonstrated that changes in phytoplankton biomass determined by microscopy were correlated significantly (p<0.005) with variations in HPLC-derived concentrations of biomarker pigments (r(2) = 0.13-0.64) from all major algal groups, although HPLC tended to underestimate the relative abundance of cyanobacteria. Together, these findings show that while fertilisation of P-rich lakes with N can increase algal biomass, there is substantial variation in responses of genera and divisions to specific chemical forms of added N.

Introduction

Human activities such as farming and industrial fixation of atmospheric nitrogen (N) have tripled the supply of N to the biosphere since 1960 and are expected to double present levels of N influx by 2050 to meet future demands for food production [1], [2]. In particular, application of N fertilisers will exceed 275 Tg N year−1 and will be concentrated in regions where centuries of farming may have saturated soils with phosphorus (P) [3], [4], increased P export to lakes [5], and overloaded surface waters with P [6]. In these regions, lakes already exhibit poor predictive relationships between P influx and algal abundance [7], continuously high concentrations of dissolved P despite abundant phytoplankton [3], [8], insufficient biological fixation of N to support primary production [9], [10], and strong positive correlations between N influx and total algal or cyanobacterial abundance [6], [11]. Taken together, these findings suggest that persistent fertiliser application has weakened the regulatory role of P [12], [13], and that pollution with N may further degrade water quality in P-rich lakes [11].

At present, most evidence shows that freshwater eutrophication ultimately arises from persistent increases in P influx from urban and other anthropogenic sources [14]–[16]. However, synthesis of laboratory studies [17], [18], in situ mesocosm experiments [19], [20], whole-ecosystem manipulations [21] (but see) [22], catchment-scale mass balances [11], [23], regional surveys [24] and palaeolimnological reconstructions [6], [11] also demonstrates that rises in N influx can independently increase algal biomass, alter the proportion of diazotrophic cyanobacteria, and increase toxicity of some algae, particularly in lakes with total P (TP) concentrations over 100 µg P L−1 and N : P mass ratios below ∼20∶ 1 [25]. Lack of reconciliation between these two robust data sets has resulted in vigorous and occasionally acrimonious debate over the unique and interactive roles of N in regulating baseline lake productivity [26], [27] and cultural eutrophication [13], [28], [29].

Continuing uncertainty over ecosystem consequences of N pollution may arise in part because of comparatively limited understanding of how effects of N may vary with phytoplankton identity, nutritional capability and status, and the chemical form of added N [18], [30], [31]. At a coarse taxonomic level, preliminary evidence suggests that fertilisation of eutrophic waters with ammonium (NH4 +) or urea ([NH2]2CO) increases the in situ abundance of non-N-fixing cyanobacteria [20], [25] which exhibit efficient light use [32] and superior NH4 + uptake kinetics [33], as well as chlorophytes which can sustain rapid growth using diverse N sources if sufficient light is present [34], [35]. In contrast, the competitive advantage of diazotrophic cyanobacteria can be lost following N fertilisation because N uptake suppresses formation of heterocysts and nitrogenase enzyme complexes needed for biological fixation of N2 [20], [36]. Finally, addition of nitrate (NO3 −) to P-rich waters can favour production of diatoms if silica (Si) is available [37], [38], possibly due to non-saturating uptake kinetics for this compound [39]. However, despite these broad generalizations, substantial uncertainty surrounds the in situ response of individual species or genera of phytoplankton to fertilisation with N [40]–[42] due to low taxonomic resolution of prior N studies (division-level), substantial overlap among algae in in vitro nutrient-uptake capabilities (N half-saturation constant, Ks, = 1–14 µg N L−1) and maximum growth rates (N-sufficient Vmax = 0.2–8.0 ln units day−1) [30], [31], [43], and the high degree of environmental simplification in laboratory and microcosm studies [44].

In this study, we conducted 21-day long mesocosm (3140 L) experiments in summer and autumn to quantify the in situ response of over 140 individual phytoplankton taxa to enrichment of P-rich freshwaters with NH4 +, NO3 − and urea. Effects of N addition on total algal abundance and gross community composition in this experiment have been analysed previously using high performance liquid chromatography (HPLC) and reported in [25]. Instead the unique objectives of the present study are four-fold: 1) to use microscopic analysis to quantify interspecific variation among algae in the response to N amendments; 2) to determine how phytoplankton-specific responses vary with the chemical form of added N; 3) to evaluate the influence of the taxonomic resolution of microscopic analysis (division, genus, species) on interpretation of N effects on phytoplankton, and; 4) to compare changes in phytoplankton assemblages derived from microscopic enumeration of cell densities and chromatographic analysis of algal pigments. As suggested elsewhere, biomarker-based analyses might be biased by limited taxonomic resolution, phylogenetic variation in cellular pigment content, or changes in ambient environmental conditions (light, temperature, nutrient availability) which uniquely influence the cellular quota of pigments [45]–[47]. Improved understanding of the nature of taxon-specific responses to N influx may help protect aquatic ecosystems against future pollution with agricultural N [2], optimise wastewater treatment procedures [28], and resolve on-going debate concerning the respective roles of N and P in regulating cultural eutrophication [48].

Methods

Study Site

The experiments were conducted in Wascana Lake (Fig. 1), an unstratified, 0.5 km2, 7-m deep basin located within the central urban park of the City of Regina, Saskatchewan, Canada (50°26.17′N, 104°36.91′W). Regional evaporation (∼60 cm year−1) exceeds precipitation by two-fold, the climate is classified as sub-humid continental, and mean monthly air temperatures vary by up to 35°C (19°C in July, −16°C in January) [49]. Wascana Lake is fed by Wascana Creek, a permanent stream which drains a 1400 km2 agricultural basin [49]. Snow melt during March–April typically accounts for 80% of annual runoff in the region [50] resulting in seasonally variable, but generally low, water residence in the lake (decadal mean ∼0.07 yr) [49].

Figure 1

Map of Wascana Lake, Saskatchewan, Canada.

Map shows a) the continental location, b) the gross drainage area (1400 km2) and c) depth contour map with the location of the mesocosm experiment (shaded box).

Wascana Lake has been monitored biweekly (May–Aug) since 1996 and exhibits elevated but annually-variable mean (±SD) summer concentrations of Chl a (39±48 µg L−1), soluble reactive P (SRP) (192±161 µg P L−1), total dissolved P (TDP) (299±208 µg P L−1), NO3 − (119±217 µg N L−1), NH4 + (94±203 µg N L−1), total dissolved N (TDN) (1327±726 µg N L−1), and dissolved organic carbon (DOC) (16.2±4.0 mg C L−1). Consequently, mean mass ratios of TDN : SRP are low (6.9±6.6). Typical plankton phenology includes a progression from a spring community composed of diatoms, cryptophytes, and copepods (Diacyclops thomasi, Leptodiaptomus siciloides), through a pronounced June clearwater phase with high densities of large-bodied Cladocera (D. magna, D. pulicaria, D. galeata mendotae) [51], to a summer and autumn community composed of small-bodied zooplankton (Eubosmina coregoni, Bosmina longirostris, Diaphanosoma birgei, Daphnia retrocurva, Ceriodaphnia, Chydorus, rotifers) and abundant cyanobacteria [8], [49]. Overall, cryptophytes, chrysophytes and chlorophytes comprise <20% of summer algal biomass. Instead, N-fixing Aphanizomenon flos-aquae and Anabaena spp. are abundant immediately after the clearwater phase, Microcystis spp. are common in the warm (>25°C) surface waters during August, Planktothrix agardhii is dominant during late-August through September, and Phormidium (and Cyclotella) spp. increase thereafter. Consequently, late-summer concentrations of the cyanobacterial toxin microcystin (MC) can be 10-fold greater than the upper limits recommended by the World Health Organization for drinking water (1 µg MC L−1).

Mesocosm Experiments

Three-week long mesocosm experiments were conducted during both August and September of 2008, as described in [25]. Briefly, 2-m wide by 1-m deep, cylindrical, opaque white poly-weave enclosures (3140 L) were open to the atmosphere, closed to the sediments, and located in a sheltered embayment (Fig. 1). Enclosures were filled passively (drawn up from depth), attached to anchored floating frames, and assigned treatments at random. No attempt was made to circulate these well-mixed mesocosms or to modify zooplankton densities, although minnow traps were added to each enclosure and checked routinely to remove planktivorous fish. Advantages and limitations of this experimental design for eutrophication studies are detailed in [20] who studied division-level effects of urea on algal communities and [25] who also contrasted urea with nitrate and ammonium effects, but did not analyse species- and genus-level responses.

Each experimental treatment consisted of three replicates to which N was added on days 0, 7, and 14 as sodium nitrate (NaNO3), ammonium chloride (NH4Cl) or urea ((NH2)2CO). All trials received 6 mg N L−1 per week, whereas unamended enclosures served as controls. Nitrogen additions were intended to increase soluble N : P to >22∶ 1 by mass to suppress N-fixing cyanobacteria [52], were based on decadal mean concentrations of SRP and TDN, and were within the range of N content observed in regional lakes [50].

Sampling took place on days 0, 3, 7, 14 and 21 between 10 00 h and 14 00 h, immediately after N addition on day 0, and before N additions on days 7 and 14. Water was collected from mesocosms using a 6-L Van Dorn bottle deployed centrally at 0.5-m depth. All water samples were screened (247-µm mesh) in the field to remove invertebrates, filtered through 0.45-µm pore membrane filters within 2 hr, and stored in darkness at 4°C until analysed for chemical solutes. Samples of 100 mL were collected from Van Dorn bottles and preserved with Lugol’s iodine solution for microscopic analysis of phytoplankton species composition and abundance. In addition, particulate organic matter (POM) was concentrated on GF/C glass fibre filters (nominal pore 1.2-µm), and stored frozen (−10°C) in darkness until analysis of biomarker pigments using HPLC. Physical (Secchi disk transparency, temperature) and chemical parameters (conductivity, dissolved O2, pH) were also recorded in the field following standard protocols [11], [49] and are reported in [25].

Laboratory Analysis

Preserved phytoplankton were identified, enumerated and measured using a Leica model DM IRB inverted light microscope (Leica Microsystems, Concord, Canada) and the Utermöhl sedimentation technique [53]. Aliquots of 2-mL were settled for 24 h prior to enumeration and analysis. Counts of cells, filaments and colonies were conducted at 100×, 400× or 1000× magnification, depending on the size and abundance of algal units, and were identified following the taxonomic conventions of [54]–[56]. Enumeration at 100× was made on every second transect within 50% of the depositional area of slides, those at 400× on one horizontal and one vertical transect, and counts at 1000× on 30 random fields of view, such that over 300 algal units were recorded for each sample. Cell measurements were conducted for abundant taxa at 1000× magnification on 30 randomly-selected individuals from a composite sample representing all enclosures and sampling events. Biovolume calculations followed [57] and were converted to cellular biomass by assuming that 1 mm3 of volume was equivalent to 1 mg of wet-weight biomass.

Phytoplankton pigments were extracted from POM using a mixture of acetone : methanol (80∶ 15, by volume) and were quantified using standard methods including spectrophotometry [58] and HPLC analyses [59]. HPLC used an Agilent model 1100 system (Agilent Technologies Inc, Mississagua, Canada) fitted with photodiode-array and fluorescence detectors and was calibrated using authentic pigment standards. Chlorophyll a (Chl a) concentrations were used to estimate total algal abundance (µg L−1), while other taxonomically-diagnostic pigments (nmol L−1) were used to quantify changes in siliceous algae (mainly diatoms and chrysophytes) (fucoxanthin), cryptophytes (alloxanthin), dinoflagellates (peridinin), chlorophytes (Chl b) and colonial cyanobacteria (myxoxanthophyll). Pigments from N-fixing cyanobacteria (canthaxanthin, aphanizophyll) were near detection limits during the 2008 experiments and were not included in further analyses.

Concentrations of dissolved nutrients were conducted at the University of Alberta Water Chemistry Laboratory following [60], are reported in [25], and included TDP, SRP, TDN, NH4 ++NH3 (as NH4 + hereafter), and NO3 −+NO2 − (as NO3 − hereafter). Concentrations of urea [61] and dissolved organic carbon (DOC) [62] were determined at University of Regina Environmental Quality Analysis Laboratory using standard protocols reported in [25].

Numerical Analysis

Repeated measures analysis of variance (RM-ANOVA) was used to estimate the statistical significance of differences in phytoplankton abundance among treatments, as well as interactions between time and treatment effects [20], [25]. Briefly, data were transformed (log10 [x+1]) as necessary prior to analysis and appropriate critical F-statistics selected for experiments with four treatment levels, three replicates and five sampling events (i.e. F treatment = 4.07, F time = 2.78, F treatment×time = 2.18). Statistically-significant differences among treatments were tested using Tukey’s Honestly Significant Difference (HSD) post hoc test. No correction was made for the number of comparisons as one of our intentions was to quantify the general patterns of phytoplankton response to N amendments, although we recognize that this approach may inflate the number of apparently-significant algal responses. Least-squares regression analysis was used to quantify the linear relationship between microscopic- and pigment-based estimates of phytoplankton abundance. RM-ANOVA and linear regressions were conducted using software from SPSS version 11 (IBM, Armonk, NY, USA) and SYSTAT version 10 (SYSTAT Software Inc., Chicago, IL, USA), respectively.

Principal component analysis (PCA) was used to summarise the main patterns of phytoplankton community response to N fertilisation, and to evaluate how patterns of response varied with the taxonomic resolution of microscopic analysis, including division or class (division hereafter), genus and species. These levels of classification were selected because they are commonly used in limnological studies, but differ considerably in the total effort required for microscopic analysis [56]. An individual genus or species was included in PCA only if their mean biomass for the experiment was >1% of the total algal abundance in at least one of the twelve experimental enclosures. Estimates of algal abundance were log10 (x+1)-transformed prior to analysis using CANOCO version 4.5 software (Microcomputer Power, Ithaca, NY, USA). As our intention was to evaluate algal response to N fertilisation, categorical N treatments (e.g.,+or − urea) were included as passive variables in each PCA, whereas other environmental variables were not included in the ordinations.

This study obtained all necessary permits and approvals required by Environment Canada, Saskatchewan Environment, Transport Canada, and the Wascana Centre Authority (City of Regina) and adhered to all ethical and environmental regulations of the University of Regina and the Natural Sciences and Engineering Research Council of Canada.

Results

Lake and Mesocosm Conditions

As presented in [25], nutrient concentrations in Wascana Lake were elevated (∼125–175 µg P L-1, ∼1.2–1.6 mg N L-1), TDN : TDP mass ratios were low (∼10–15), and bottle bioassays revealed that phytoplankton growth exhibited instantaneous limitation by N supply during both August and September. As expected, mesocosm fertilisation elevated TDN to 15–20 mg N L-1, but had few measured effects on mesocosm water chemistry (pH, conductivity, oxygen, etc.) other than a rapid decline in SRP from ∼100 µg P L-1 to <5 µg P L-1, and a 50% decline in TDP, by day 14 in all N-amended enclosures. Further details and interpretations of water chemistry change are presented in [25].

Community Response to N fertilisation

Total algal biomass measured microscopically or using ubiquitous Chl a increased ∼200% and ∼350% when fertilised with NO3 − and chemically-reduced N (urea, NH4 +), respectively (Fig. 2). Biomass response to NH4 + fertilisation during August and all N amendments in September was statistically significant (p treatment <0.05) relative to control enclosures (Table 1), although there was no significant effect of treatment on the ratio of Chl a : biomass (p treatment >0.05).

Figure 2

Biomass responses of total algal response to fertilisation with nitrogen in mesocosms conducted in August and September.

Time series include; a) total phytoplankton biomass (mg wet mass L−1), b) Chl a (µg L−1) and c) the ratio of Chl a : total phytoplankton biomass. Symbols represent mean and standard error (± SE, n = 3) for each nitrogen treatment, including amendments with NH4 + (shaded triangle, coarse dashed line), NO3 − (shaded square, medium dashed line) and urea (shaded circle, fine dashed line), as well as unamended (control) mesocosms (solid circle, solid line).

Table 1

Repeated-measures analysis of variance of total phytoplankton biomass, chlorophyll a, chlorophyll a : biomass ratio, and biomass of major algal groups.

Response variable	August		September
	p	Post hoc	p	Post hoc
Total phytoplankton biomass
Treatment	0.041	NH, U, NO>U, NO, C	0.000	U, NH, NO>C
Interaction	0.042		0.000
Chlorophyll a
Treatment	0.043	NH, U, NO>U, NO, C	0.000	U, NO, NH>C
Interaction	0.016		0.000
Chlorophyll a : biomass ratio
Treatment	0.335	–	0.077	–
Interaction	0.457		0.000
Cyanobacteria
Treatment	0.110	–	0.000	NH, U, NO>C
Interaction	0.035		0.000
Chlorophytes
Treatment	0.048	NH, U, NO>U, NO, C	0.001	NH>U, NO, C
Interaction	0.053		0.000
Diatoms
Treatment	0.004	NO, U, C>C, NH	0.009	NO, U, C>U, C, NH
Interaction	0.000		0.000
Chrysophytes
Treatment	0.092	–	0.005	NH, U>U, C, NO
Interaction	0.032		0.004
Cryptophytes
Treatment	0.025	NO, U, C>U, C, NH	0.020	U, NO, C>NO, C, NH
Interaction	0.007		0.002
Dinoflagelates
Treatment	0.009	C, U, NO>NO, NH	0.345	–
Interaction	0.000		0.847

Probability (p) values were calculated for treatment and treatment-time interaction effects. Tukey’s HSD post hoc results represent mean treatment values ordered from largest to smallest and significant differences (>) at α = 0.05, for urea (U), nitrate (NO), ammonium (NH), and the control (C). If a treatment falls on both sides of a “>” this indicates no significant difference from the treatments on either side. All phytoplankton biomass (mg L−1) data, but not chlorophyll a concentrations (µg L−1), were log10(x+1) transformed prior to analysis to meet assumptions of normality. Probabilities were not corrected for number of comparisons. See Methods for additional information.

The biomass response of algal divisions varied substantially with both phylogenetic group and chemical form of added N (Fig. 3, Table 1). In particular, abundance of cyanobacteria (Fig. 3a), chlorophytes (Fig. 3b) and chrysophytes (Fig. 3d) increased 400–800% following fertilisation with NH4 + and secondarily urea, changes which were significant during September (p treatment <0.005), but only marginally so during August (0.05