High microcystin concentrations occur only at low nitrogen-to-phosphorus ratios in nutrient-rich Canadian lakes

Diane M. Orihel,a David F. Bird,b Michael Brylinsky,c Huirong Chen,d Derek B. Donald,e Dorothy Y. Huang,f Alessandra Giani,g David Kinniburgh,f Hedy Kling,h Brian G. Kotak,i Peter R. Leavitt,e Charlene C. Nielsen,a Sharon Reedyk,j Rebecca C. Rooney,a Sue B. Watson,k Ron W. Zurawell,l Rolf D. Vinebrookea

aDepartment of Biological Sciences, University of Alberta, 11455 Saskatchewan Drive, Edmonton, AB T6G 2E9, Canada.

bDépartement des Sciences Biologiques, Université du Québec à Montréal, C.P. 8888, Succursale Centre-ville, Montréal, QC H3C 3P8, Canada.

cAcadia Centre for Estuarine Research, 23 Westwood Avenue, Acadia University, Wolfville, NS B4P 2R6, Canada.

dShenzhen University, Shenzhen, Guangdong Province, P.R. China.

eLimnology Laboratory, Department of Biology, University of Regina, 3737 Wascana Parkway, Regina, SK S4S 0A2, Canada

fAlberta Centre for Toxicology, Department of Physiology & Pharmacology, Faculty of Medicine, University of Calgary, Calgary, AB T2N 4N1 Canada.

gDepartemento de Botânica, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, C.P. 48631270-010, Belo Horizonte, MG, Brazil.

hAlgal Taxonomy and Ecology Inc., 31 Laval Dr., Winnipeg, MB R3T 2X8, Canada.

iAlgalTox International, P.O. Box 268, Pine Falls, MB R0E 1M0, Canada.

jAgri-Environment Services Branch, Agriculture and Agri-Food Canada, 9700 Jasper Avenue, Edmonton, AB T5J 4C3, Canada.

kAquatic Ecosystem Management Research Division, Science & Technology Branch, Environment Canada, National Water Research Institute, 867 Lakeshore Road, Burlington, ON L7R 4A6, Canada.

lWater Policy Branch, Alberta Environment, 7th Floor Oxbridge Place, 9820-106 Street, Edmonton, AB T5K 2J6, Canada.

Corresponding author: Diane Orihel (e-mail: Diane Orihel (e-mail: orihel@ualberta.ca ).



Paper handled by associate editor Ralph E.H. Smith

Received March 10, 2012. Accepted July 16, 2012.

Canadian Journal of Fisheries and Aquatic Sciences, 2012, 69(9): 1457-1462, https://doi.org/10.1139/f2012-088

In this paper Top of page References Abstract Although the cyanobacterial toxin microcystin has been detected in Canadian fresh waters, little is known about its prevalence on a national scale. Here, we report for the first time on microcystin in 246 water bodies across Canada based on 3474 analyses. Over the last 10 years, microcystins were detected in every province, often exceeding maximum guidelines for potable and recreational water quality. Microcystins were virtually absent from unproductive systems and were increasingly common in nutrient-rich waters. The probable risk of microcystin concentrations exceeding water quality guidelines was greatest when the ratio of nitrogen (N) to phosphorus (P) was low and rapidly decreased at higher N:P ratios. Maximum concentrations of microcystins occurred in hypereutrophic lakes at mass ratios of N:P below 23. Our models may prove to be useful screening tools for identifying potentially toxic “hotspots” or “hot times” of unacceptable microcystin levels. A future scientific challenge will be to determine whether there is any causal link between N:P ratios and microcystin concentrations, as this may have important implications for the management of eutrophied lakes and reservoirs.

The occurrence of cyanobacterial toxins in Canadian fresh waters is a serious environmental and public health concern (Kotak and Zurawell 2007). One common class of cyanobacterial toxins, the microcystins, are potent inhibitors of eukaryotic protein phosphatases. In controlled experiments, microcystins have been shown to cause liver haemorrhage and promote tumour development in mammals. Furthermore, microcystins have been implicated in illnesses and deaths of domestic animals, wildlife, and humans following exposure to these toxins in the natural environment. Concentrations of microcystins are highly dynamic on both temporal and spatial scales, and understanding the factors that drive this variation in and among freshwater systems is key to developing effective water management tools and policies. In the current study, we report the likelihood of microcystin concentrations exceeding water quality guidelines in eutrophic lakes, ponds, and reservoirs in Canada increases as the mass ratio of nitrogen (N) to phosphorus (P) declines.

Smith (1983) first described a strong relationship between the relative amounts of N and P in surface waters and cyanobacterial blooms. In this seminal paper, Smith argued that diazotrophic cyanobacteria should be superior competitors under conditions of N-limitation because of their unique capacity for N-fixation. The hypothesis that low N:P ratios favor cyanobacteria has been intensely debated (Lampert 1999) and challenged for its poor performance predicting cyanobacterial dominance (Downing et al. 2001). The dominance of N-fixing cyanobacteria at low N:P ratios has been decisively demonstrated in mesocosm- and ecosystem-scale experiments in prairie and boreal lakes (Schindler et al. 2008, and references therein). Nonetheless, some still question whether these experimental results can be generalized to hypereutrophic lakes with a long history of anthropogenic nutrient loading (e.g., Paerl et al. 2011).

The main goal of our investigation was to test for a relationship between the mass ratio of N to P and microcystin concentrations in water bodies across Canada. Given that production of microcystins is exclusive to certain members of the phylum Cyanophyta (Cyanobacteria), the presence of microcystins in lakes should theoretically be higher under low N:P ratios if cyanobacteria dominate under conditions of relative N deficiency. Noteably, some cyanobacteria that produce microcystins are capable of N-fixation (e.g., Anabaena ), but many microcystin producers are non-diazotrophic (e.g., Microcystis ). The dominance of cyanobacteria does not necessarily predicate the occurrence of microcystins, because not all cyanobacterial species are capable of synthesizing microcystins, and not all strains of known toxin-producing species are toxic. As such, this hypothesis does not imply a simple, linear, negative relationship exists between N:P ratios and microcystin concentrations, but rather that microcystin concentrations are potentially elevated at low N:P ratios. A negative relationship between N:P ratio and microcystin concentration has previously been reported in three Albertan lakes (Kotak et al. 2000), four US states (Graham et al. 2004), and Lake Erie (Rinta-Kanto et al. 2009), but has not been tested at the broad geographic extent examined in the current study.

We compiled measurements of microcystin concentrations in fresh waters collected in Canada between 2001 and 2011. A comprehensive program for monitoring cyanobacterial toxins in Canada does not exist, and thus we collated data from academics, government agencies, and private companies. (Details of their respective sampling protocols and analytical methods are provided online in Supplemental Table S11

Our consolidated data set of microcystin measurements includes 246 water bodies situated in British Columbia, Alberta, Saskatchewan, Manitoba, Ontario, Quebec, Newfoundland and Labrador, New Brunswick, Nova Scotia, and Prince Edward Island. In most cases, concentrations of total microcystins were determined by protein phosphatase inhibition assay or enzyme-linked immunosorbent assay. Where microcystins were measured by high-performance liquid chromatography or liquid chromatography–(electrospray ionization) tandem mass spectrometry, the sum of individual variants was assumed to approximate total microcystin concentration. In samples below limits of detection (0.005–0.22 µg·L–1), total microcystin concentrations were assigned a value of half the limit of detection. Total P and total N concentrations were also included in our database if these parameters were measured on the same sample as microcystins (or samples collected on the same day). We considered each water body as one sampling site, with the exception of three large systems (i.e., Lake Erie: 43 sites; Lake of the Woods: 32 sites; and Lake Winnipeg: 87 sites). In total, our data set includes 405 sites and 3474 microcystin measurements, of which 982 measurements have associated P and N data. We excluded lakes with only one sample from all statistical analyses.

Microcystins were detected in fresh waters in every province in Canada from coast to coast (Fig. 1). Concentrations ranged from below minimum detection limits to a maximum of 2153 µg·L–1. Notably, all regions contained lakes where toxin concentrations reached levels of concern. Although the sampling effort was unequal across the country, the highest concentrations tended to occur in the Canadian prairie provinces, particularly in clusters in central Alberta and southwestern Manitoba. Prairie lakes are typically shallow and warm, with high nutrient concentrations (as a result of high external and internal nutrient loading) — conditions ideal for the proliferation of cyanobacteria. In our national database, 18% of samples and 41% of lakes exceeded the World Health Organization’s drinking water quality guideline of 1.0 µg·L–1. The Canadian drinking water guideline of 1.5 µg·L–1 was exceeded in 14% of samples and 35% of lakes, while 1.3% of samples and 9% of lakes surpassed the proposed Canadian guideline for recreational waters of 20 µg·L–1.

»View larger version Fig. 1. Microcystin concentrations in lakes, ponds, and reservoirs in Canada. The map of Canada indicates sampling locations for microcystins. Inset maps (a–e) indicate the maximum concentration of microcystin recorded in each water body (or at each sampling site, in the case of Lake Erie, Lake of the Woods, and Lake Winnipeg).

Our results support the current hypothesis that microcystin concentrations increase with lake trophic status (Kotak and Zurawell 2007). First, we examined the correlation between concentrations of nutrients and microcystins in Canadian lakes (Supplemental Fig. S11). Microcystin concentrations were positively correlated with concentrations of P (Spearman rank correlation, r = 0.36, p < 0.01, n = 937) and N (Spearman rank correlation, r = 0.39, p < 0.01, n = 937). However, the strength of these correlations imply that variables other than nutrient concentrations most certainly played a role in determining microcystin concentrations. Next, we calculated minimum thresholds for P and N (independently) as the nutrient concentration above which 95% of values exceeding a specific level of microcystin occurred (Supplemental Table S21). For example, 95% of the instances where microcystin concentrations exceeded the World Health Organization’s drinking water guideline of 1 µg·L–1 occurred when P concentrations were above 26 µg·L–1 and N concentrations were above 658 µg·L–1. Minimum thresholds for P and N were successively higher for microcystin concentrations of 1, 2, 5, and 10 µg·L–1 (Supplemental Table S21). Therefore, correlation analyses and minimum threshold determinations clearly demonstrated that microcystins were primarily a concern in eutrophic and hypereutrophic systems.

As expected, based on our hypothesis, our meta-analysis revealed that microcystin concentrations in Canadian fresh waters were high only at low N:P ratios and conversely were consistently low at high N:P ratios (Figs. 2a, 2b). These observations were especially apparent for lakes in Alberta (Fig. 2a), but also held true for systems in other provinces (Fig. 2b). Note that N:P ratio was, as anticipated, weakly correlated with microcystin concentration (Spearman rank correlation, r = –0.09, p = 0.004, n = 937). While N:P ratios are not suitable for predicting absolute concentrations of microcystins, we propose N:P ratios are useful for estimating the “risk” of elevated microcystin concentrations. We performed a simple numerical analysis to quantify the probability of microcystin exceeding specific toxin thresholds at four N:P categories (<20, 20–40, 40–60, and >60; Supplemental Table S21). Probabilities were calculated by dividing the number of samples above a specific toxin threshold by the total number of samples in each N:P category. The probability of microcystin concentrations exceeding all toxin thresholds was highest when N:P ratios were less than 20 and decreased at higher N:P ratios (Fig. 2c). Importantly, the probability of levels of concern for recreational contact dropped to near zero above an N:P ratio of 40, and similarly, that for drinking water was negligible above an N:P ratio of 60.

»View larger version Fig. 2. Microcystin concentrations in Canadian fresh waters in relation to the mass ratio of total nitrogen (N) to total phosphorus (P). (a) Scatterplot of N:P ratio versus microcystin concentration, with data for each province denoted by a different symbol. Dashed lines indicate N:P ratios of 20, 40, and 60. The inset (b) shows the same plot on a smaller y-axis scale, and with data points for water bodies in Alberta excluded. (c) Probability of exceeding specific microcystin concentrations (1, 2, 5, or 10 µg·L–1) at four N:P categories. (d) Scatterplot of N concentration versus P concentration with symbols denoting microcystin concentration classes (µg·L–1). Dashed lines are as in panel (a). Solid lines indicate 95% nutrient thresholds, calculated independently for P and N, above which microcystin concentrations exceed 1, 2, 5, or 10 µg·L–1.

Relating microcystin concentration in lakes to nutrient ratios (Fig. 2a in this study, and similar figures in Kotak et al. 2000 and Graham et al. 2004) ignores the important influence of nutrient concentrations, and thus, a more informative approach is to consider the relationship between microcystin concentrations and both nutrient ratios and concentrations. To begin, we visualized these associations in a scatterplot of N versus P concentration, with microcystin levels displayed categorically with different symbols and different N:P ratios depicted by diagonal lines (Fig. 2d). Shown in this manner, it is evident that microcystin concentrations in Canadian lakes in our study were elevated only in nutrient-rich waters at low N:P ratios. Note that minimum nutrient thresholds for P and N concentrations (Supplemental Table S21) corresponding to microcystin levels of 1, 2, 5, and 10 µg·L–1 are shown graphically (Fig. 2d).

Next, we performed a regression tree analysis to identify the nutrient conditions under which N:P ratios are most strongly associated with high microcystin concentrations. Regression trees explain the variation of a single response variable by repeatedly partitioning the data into homogenous groups using explanatory variables, which is a powerful technique for analyzing complex ecological data with nonlinear relationships and higher-order interactions. Using the TREES module in SYSTAT 13 (Systat Software Inc., Chicago, Illinois), we constructed a regression tree to explain microcystin concentrations in Canadian water bodies based on P concentrations, N concentrations, and N:P ratios (Supplemental Fig. S21; total proportional reduction in error = 0.46; n = 937). This analysis indicated that microcystin concentrations remained low (mean ± standard deviation (SD) = 0.39 ± 0.99 µg·L–1) as long as N concentrations were under 2600 µg·L–1. When N concentrations exceeded this threshold, microcystin concentrations were higher when N:P ratios were below 23 (5.6 ± 8.4 µg·L–1) in comparison with when ratios were above 23 (1.5 ± 2.3 µg·L–1). In other words, regression tree analysis supported our hypothesis that high microcystin concentrations occur under low N:P ratios and stipulated this hypothesis is applicable under high nutrient concentrations. In keeping with our findings in Canadian water bodies, high N and P concentrations coupled with low N:P ratios were recently determined to favor the growth of toxigenic strains of Microcystis in hypereutrophic Lake Taihu, China (Otten et al. 2012).

While the correspondence between high microcystin concentrations and low N:P ratios we observed in Canadian lakes is consistent with our original hypothesis (i.e., conditions of relative N deficiency favor the dominance of cyanobacteria, and thus enable the potential for microcystin production), there are several alternative explanations for this observation. First, N:P ratios may physiologically cue cyanobacteria to produce microcystins and thereby increase their intracellular microcystin content (Lee et al. 2000). Second, N:P ratios may be indicative of the nature of nutrient sources to oligotrophic versus eutrophic ecosystems (Downing and McCauley 1992), and therefore, the association between N:P and microcystin results from the covariation of N:P ratios and lake trophic status. Third, low N:P ratios may be the consequence, rather than the cause, of cyanobacterial blooms. Many planktonic cyanobacteria have a benthic life stage where they engage in “luxury uptake” of P from sediments, and consequently, episodes of cyanobacterial recruitment from sediments can dramatically decrease the N:P ratio in the water column (Xie et al. 2003).

In summary, our meta-analysis of microcystins in Canadian fresh waters has revealed that microcystins are now an issue of national concern, as these toxins were detected in every province, and concentrations exceeded water quality guidelines for drinking water, and sometimes for recreational waters, in many eutrophic ecosystems. Consolidating data on microcystin concentrations in water bodies across Canada allowed us to determine nationally relevant minimum thresholds for P and N concentrations corresponding to microcystin exceedences, probabilities of microcystin exceedances at different N:P ratios, and a regression tree partitioning variance in microcystin concentrations based on nutrient concentrations and ratios. Once externally validated, these models may become applicable as screening tools for identifying potentially toxic “hotspots” or “hot times” of unacceptable concentrations of microcystins based on simple chemical parameters that are routinely measured in water quality monitoring programs. Our survey revealed that microcystin concentrations in Canadian fresh waters were elevated only under low mass ratios of N:P, and while this observation is not likely to be disputed, its interpretation and potential ramifications may prove contentious. Is the association between N:P ratios and microcystin concentrations mere coincidence or driven by a biogeochemical mechanism? The present study is a mensurative experiment, and hence we cannot invoke a “cause-and-effect” argument, or identify the causal mechanisms, to explain why high microcystin concentrations coincide with low N:P ratios. We recommend that subsequent experiments should manipulate N:P ratios — at scales relevant to ecosystem management — as the outcome may be germane to the ongoing debate regarding the need for a “dual-nutrient management strategy” (Paerl et al. 2011).

Acknowledgements Sincere thanks are extended to Val H. Smith for encouraging this research, and to David W. Schindler and his graduate students, and several anonymous reviewers, for providing helpful comments on earlier drafts of this paper. The Manitoba Department of Conservation and Water Stewardship, New Brunswick Department of Environment, Newfoundland and Labrador Department of Environment and Conservation, and Ontario Ministry of the Environment graciously contributed data to this meta-analysis. This research was supported by Acadia University, Agriculture and Agri-Food Canada, Alberta Environment, Alberta Ingenuity Fund, Alberta Water Research Institute, AlgalTox International, Algal Taxonomy and Ecology Inc., Canada Foundation for Innovation, Environment Canada, Fonds de Recherche du Québec – Nature et Technologies, le Ministère du Développement Économique, Innovation et Exportation du Québec, Natural Sciences and Engineering Research Council of Canada, University of Alberta, University of Calgary, and University of Regina.

In this paper Top of page References

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