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Research Article

Associations between chlorophyll a and various microcystin-LR health advisory concentrations

[version 1; peer review: 1 approved, 2 approved with reservations]
PUBLISHED 09 Feb 2016
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Abstract

Cyanobacteria harmful algal blooms (cHABs) are associated with a wide range of adverse health effects that stem mostly from the presence of cyanotoxins. To help protect against these impacts, several health advisory levels have been set for some toxins. In particular, one of the more common toxins, microcystin-LR, has several advisory levels set for drinking water and recreational use. However, compared to other water quality measures, field measurements of microcystin-LR are not commonly available due to cost and advanced understanding required to interpret results. Addressing these issues will take time and resources. Thus, there is utility in finding indicators of microcystin-LR that are already widely available, can be estimated quickly and in situ, and used as a first defense against high concentrations of microcystin-LR. Chlorophyll a is commonly measured, can be estimated in situ, and has been shown to be positively associated with microcystin-LR. In this paper, we use this association to provide estimates of chlorophyll a concentrations that are indicative of a higher probability of exceeding select health advisory concentrations for microcystin-LR. Using the 2007 National Lakes Assessment and a conditional probability approach, we identify chlorophyll a concentrations that are more likely than not to be associated with an exceedance of a microcystin-LR health advisory level. We look at the recent US EPA health advisories for drinking water as well as the World Health Organization levels for drinking water and recreational use and identify a range of chlorophyll a thresholds. A 50% chance of exceeding one of the microcystin-LR advisory concentrations of 0.3, 1, 1.6, and 2 g/L is associated with chlorophyll a concentration thresholds of 23.4, 67.0, 83.5, and 105.8, respectively. When managing for these various microcystin-LR levels, exceeding these reported chlorophyll a concentrations should be a trigger for further testing and possible management action.

Keywords

Harmful Algal Blooms, Cyanotoxins, National Lakes Assessment, Conditional Probability Analysis, Cyanobacteria

Introduction

Over the last decade, numerous events and legislative activities have raised the public awareness of harmful algal blooms13. In response the US Environmental Protection Agency (US EPA) has recently released suggested microcystin-LR (one of the more common toxins) concentrations that would trigger health advisories46. Additionally, the World Health Organization (WHO) has proposed microcystin advisory levels for drinking water and a range of recreational risk levels7,8. While these levels and associated advisories are likely to help mitigate the impacts from harmful algal blooms, they are not without complications.

One of these complications is that they rely on available measurements of microcystin-LR. While laboratory testing (e.g., chromatography) remains the gold standard for quantifying microcystin-LR concentrations in water samples, several field test kits have been developed. Even though field tests provide a much needed means for rapid assessment, they are not yet widely used and are moderately expensive (approximately $150–$200 depending on the specific kit) with a limited shelf life (typically one year)9,10. Additionally, each technique requires nuanced understanding of the detection method (e.g., limit of detection, specific microcystin variants being measured, and sampling protocol).

Fortunately, microcystin-LR has been shown to be associated with several, more commonly measured and well understood components of water quality that are readily assessed in the field. For instance, there are small or hand held fluorometers that measure chlorohpyll a. Additionally, chlorophyll a is a very commonly measured component of water quality that is also known to be positively associated with microcystin-LR concentrations11,12. Yuan et al. (2014) explore these associations in detail and control for other related variables12. In their analysis they find that total nitrogen and chlorophyll a show the strongest association with microcystin. Furthermore, they identify chlorophyll a and total nitrogen concentrations that are associated with exceeding 1 μg/L of microcystin. Given these facts, it should be possible to identify chlorophyll a concentrations that would be associated with the new US EPA microcystin-LR health advisory levels for drinking water. Identifying these associations would provide another tool for water resource managers to help manage the threat to public health posed by cHABs and would be especially useful in the absence of measured microcystin-LR concentrations.

In this paper we build on past efforts and utilize the National Lakes Assessment (NLA) data and identify chlorophyll a concentrations that are associated with higher probabilities of exceeding several microcystin-LR health advisory concentrations6,8,13. We add to past studies by exploring associations with newly announced advisory levels and by also applying a different method, conditional probability analysis. Utilizing different methods strengthens the evidence for suggested chlorophyll a levels that are associated with increased risk of exceeding the health advisory levels as those levels are not predicated on a single analytical method. So that others may repeat or adjust this analysis, the data, code, and this manuscript are freely available via https://github.com/USEPA/microcystinchla.

Methods

Data

We used the 2007 NLA chlorophyll a and microcystin-LR concentration data13. These data represent a snapshot of water quality from the summer of 2007 for the conterminous United States and were collected as part of an ongoing probabilistic monitoring program13. Data on chlorophyll a and microcystin-LR concentrations are available for 1028 lakes. These data are available for download from the National Lake Assessment Data Site.

Analytical methods

We used a conditional probability analysis (CPA) approach to explore associations between chlorophyll a concentrations and World Health Organization (WHO) and US EPA microcystin-LR health advisory levels14. Many health advisory levels have been suggested (Table 1), but lakes with higher microcystin-LR concentrations in the NLA were rare. Only 1.16% of lakes sampled had a concentration greater than 10 μg/L. Thus, for this analysis we focused on the microcystin concentrations that are better represented in the NLA data. These were the US EPA children’s drinking water advisory level of 0.3 μg/L (US EPA child), the WHO drinking water advisory level of 1 μg/L (WHO drinking), the US EPA adult drinking water advisory level of 1.6 μg/L (US EPA adult), and the WHO recreational, low probability of effect advisory level of 2 μg/L (WHO recreational)68.

Conditional probability analysis provides information about the probability of observing one event given another event has also occurred. For this analysis, we used CPA to examine how the conditional probability of exceeding one of the health advisories changes as chlorophyll a increases in a lake. We expect to find higher chlorophyll a concentrations to be associated with higher probabilities of exceeding the microcystin-LR health advisory levels. We also calculated 95% confidence intervals (CI) using 1000 bootstrapped samples. Thus, to identify chlorophyll a concentrations of concern we identify the value of the upper 95% CI across a range of conditional probabilities of exceeding each health advisory level. Using the upper confidence limit to identify a threshold is justified as it ensures that a given threshold is unlikely to miss a microcystin exceedance.

Table 1. Various suggested microcystin-LR health advisory concentrations from the US EPA and World Health Organization.

SourceTypeConcentration
US EPAAdult drinking water advisory1.6 μg/L
US EPAChild drinking water advisory0.3 μg/L
WHODrinking water1 μg/L
WHORecreational: High probability of effect20–2000 μg/L
WHORecreational: Low probability of effect2–4 μg/L
WHORecreational: Moderate probability of effect10–20 μg/L
WHORecreational: Very high probability of effect>2000 μg/L

As both microcystin-LR and chlorophyll a values were highly skewed right, a log base 10 transformation was used. Additional details of the specific implementation are available at https://github.com/USEPA/microcystinchla. A more detailed discussion of CPA is beyond the scope of this paper, but see Paul et al.15 and Hollister et al.16 for greater detail. All analyses were conducted using R version 3.2.2 and code and data from this analysis are freely available as an R package at https://github.com/USEPA/microcystinchla.

Lastly, we assess the ability of these chlorophyll a thresholds to predict microcystin exceedance. We use error matrices and calculate total accuracy as well as the proportion of false negatives. Total accuracy is the total number of correct predictions divided by total observations. The proportion of false negatives is the total number of lakes that were predicted to not exceed the microcystin guidelines but actually did, divided by the total number of observations.

Results

In the 2007 NLA, microcystin-LR concentrations ranged from 0.05 to 225 μg/L. Microcystin-LR concentrations of 0.05 μg/L represent the detection limits. Any value greater than that indicates the presence of microcystin-LR. Of those lakes with microcystin, the median concentration was 0.51 μg/L and the mean was 3.17 μg/L. Of all lakes sampled, 21% of lakes exceeded the US EPA child level, 8.8% of lakes exceeded the US EPA adult level, 11.7% of lakes exceeded the WHO drinking level, and 7.3% of lakes exceeded the WHO recreational level. For chlorophyll a, the range was 0.07 to 936 μg/L. All lakes had reported chlorophyll a concentrations that exceeded detection limits. The median concentration was 7.79 μg/L and the mean was 29.63 μg/L. The associations between chlorophyll a and the upper confidence interval across a range of conditional probability values are shown in Table 2. Specific chlorophyll a concentrations that are associated with greater than even odds of exceeding the advisory levels were 23.4, 67.0, 83.5, and 105.8 μg/L for 0.3, 1.0, 1.6, and 2.0 μg/L advisory levels, respectively (Table 2 & Figure 1).

Table 2. Chlorophyll a concentrations that are associated with various conditional probabilities of exceeding a microcystin-LR health advisory concentration.

Conditional
probability
US EPA
child
WHO
drink
US EPA
adult
WHO
recreational
0.100.070.070.071.22
0.200.074.4311.8020.00
0.302.9820.4331.4053.64
0.4010.6742.2466.9682.22
0.5023.3666.9683.52105.84
0.6038.30103.20133.20155.52
0.7065.60166.63871.20216.00
0.80117.50338.40871.20871.20
0.90167.04516.00871.20871.20
87842f24-b8d6-4201-a304-ddc715e914f1_figure1.gif

Figure 1. Conditional probability plots showing association between the probability of exceeding various microcystin-LR (MLR) health advisory levels.

A. Plot for US EPA child (0.3 μg/L). B. Plot for WHO drinking (1 μg/L). C. Plot for US EPA adult (1.6 μg/L). D. Plot for WHO recreational (2 μg/L).

The chlorophyll a cutoffs may be used to predict whether or not a lake exceeds the microcystin-LR health advisories. Doing so allows us to compare the accuracy of the prediction as well as evaluate false negatives. Total accuracy of the four cutoffs predicting microcystin-LR exceedances were 75% for the US EPA children’s advisory, 86% for the WHO drinking water advisory, 89% for the US EPA adult advisory, and 91% for the WHO recreational advisory (Table 3Table 6). However, total accuracy is only one part of the prediction performance with which we are concerned.

Table 3. Confusion matrix comparing chlorophyll a predicted exceedances (rows) versus real exceedances (columns) for the US EPA children’s drinking water advisory.

Not exceedExceedRow totals
Not exceed64996745
Exceed162121283
Column totals8112171028

Table 4. Confusion matrix comparing chlorophyll a predicted exceedances (rows) versus real exceedances (columns) for the WHO drinking water advisory.

Not exceedExceedRow totals
Not exceed83977916
Exceed6844112
Column totals9071211028

Table 5. Confusion matrix comparing chlorophyll a predicted exceedances (rows) versus real exceedances (columns) for the US EPA adult drinking water advisory.

Not exceedExceedRow totals
Not exceed88357940
Exceed543488
Column totals937911028

Table 6. Confusion matrix comparing chlorophyll a predicted exceedances (rows) versus real exceedances (columns) for the WHO recreational water advisory.

Not exceedExceedRow totals
Not exceed91052962
Exceed432366
Column totals953751028

When using the chlorophyll a cutoffs as an indicator of microcystin-LR exceedances, the error that should be avoided is predicting that no exceedance has occurred when in fact it has. In other words, we would like to avoid Type II errors and minimize the proportion of false negatives. For the four chlorophyll a cut-offs we had a proportion of false negatives of 9%, 7%, 6%, and 5% for the US EPA children’s, the WHO drinking water, the US EPA adult, and the WHO recreational advisories, respectively. In each case we missed less than 10% of the lakes that in fact exceeded the microcystin-LR advisory. While this method performs well with regard to the false negative percentage, it is possible that is a relic of the NLA dataset and testing with additional data would allow us to confirm this result.

Discussion

The association between Log10 microcystin-LR and Log10 chlorophyll a shows a wedge pattern (Figure 2). This indicates that, in general, higher concentrations of microcystin-LR almost always co-occur with higher concentrations of chlorophyll a yet the inverse is not true. Higher chlorophyll a is not necessarily predictive of higher microcystin-LR concentrations; however, chlorophyll a may be predictive of the probability of exceeding a certain threshold.

87842f24-b8d6-4201-a304-ddc715e914f1_figure2.gif

Figure 2. Scatterplot showing association between chlorophyll a and microcystin-LR.

This is the case as the probability of exceeding each of the four tested health advisory levels increases as a function of chlorophyll a concentration (Figure 1). We used this association to identify chlorophyll a concentrations that are associated with a range of probabilities of exceeding a given health advisory level (Table 2). For the purposes of this discussion we focus on a conditional probability of 50% or greater (i.e., greater than even odds to exceed a health advisory level). The 50% conditional probability chlorophyll a thresholds represent 27.9%, 11.3%, 9%, and 6.9% of sample lakes for the US EPA child, the WHO drinking, the US EPA adult, and the WHO recreational levels, respectively.

There are numerous possible uses for the chlorophyll a and microcystin-LR advisory cut-off values. First, in the absence of microcystin-LR measurements, exceedance of the chlorophyll a concentrations could be a trigger for further actions. Given that there is uncertainty around these chlorophyll a cutoffs the best case scenario would be to monitor for chlorophyll a and in the event of exceeding a target concentration take water samples and have those samples tested for microcystin-LR.

A second potential use is to identify past bloom events from historical data. As harmful algal blooms are made up of many species and have various mechanisms responsible for adverse impacts (e.g., toxins, hypoxia, odors), there is no single definition of a bloom. For cHABs, one approach has been to identify an increase over a baseline concentration of phycocyanin17. This is a useful approach for targeted studies, but phycocyanin is also not always available and measures the predominance of cyanobacterial pigments and not toxins. Using our chlorophyll a cutoffs provides a value that is more directly associated with microcystin-LR and can be used to classify lakes, from past surveys, as having bloomed.

Lastly, using chlorophyll a is not meant as a replacement for testing of microcystin-LR or other toxins. It should be used when other, direct measurements of cyanotoxins are not available. In those cases, which are likely to be common at least in the near future, using a more ubiquitous measurement, such as chlorophyll a will provide a reasonable proxy for the probability of exceeding a microcystin-LR health advisory level and provide better protection against adverse effects in both drinking and recreational use cases.

Data and software availability

Data and latest source code

https://github.com/USEPA/microcystinchla

Archived data and source code at time of publication

http://dx.doi.org/10.5281/zenodo.4531718

License

Creative Commons Zero 1.0: http://creativecommons.org/publicdomain/zero/1.0/

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Hollister JW and Kreakie BJ. Associations between chlorophyll a and various microcystin-LR health advisory concentrations [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Research 2016, 5:151 (https://doi.org/10.12688/f1000research.7955.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
VERSION 1
PUBLISHED 09 Feb 2016
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Reviewer Report 11 Apr 2016
Zofia E Taranu, Department of Biological Sciences, Université de Montréal, Montreal, QC, Canada 
Approved
VIEWS 18
General comments
This study provides an elegant framework to predict the severe impairment of U.S. lakes and reservoirs by cyanobacterial harmful algal blooms. I especially appreciated the clever use of conditional probability analysis to identify chlorophyll a threshold above which MC ... Continue reading
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Taranu ZE. Reviewer Report For: Associations between chlorophyll a and various microcystin-LR health advisory concentrations [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Research 2016, 5:151 (https://doi.org/10.5256/f1000research.8562.r12719)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 13 Jun 2016
    Jeffrey Hollister, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Atlantic Ecology Division, US Environmental Protection Agency, Narragansett, USA
    13 Jun 2016
    Author Response
    Thank you for your review.  We have just submitted our revisions and expect the new version to be available in the next few days.  Below are our responses to the ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 13 Jun 2016
    Jeffrey Hollister, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Atlantic Ecology Division, US Environmental Protection Agency, Narragansett, USA
    13 Jun 2016
    Author Response
    Thank you for your review.  We have just submitted our revisions and expect the new version to be available in the next few days.  Below are our responses to the ... Continue reading
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Reviewer Report 05 Apr 2016
Jason W. Marion, Department of Environmental Health Science, Eastern Kentucky University, Richmond, KY, USA 
Approved with Reservations
VIEWS 22
Overview: The manuscript/article addresses a critical question applicable to recreational and drinking water managers: Can we rapidly predict potentially harmful cyanobacteria blooms using traditional water quality methods? This question is likely to become more relevant according to the most current ... Continue reading
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HOW TO CITE THIS REPORT
Marion JW. Reviewer Report For: Associations between chlorophyll a and various microcystin-LR health advisory concentrations [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Research 2016, 5:151 (https://doi.org/10.5256/f1000research.8562.r12688)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 13 Jun 2016
    Jeffrey Hollister, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Atlantic Ecology Division, US Environmental Protection Agency, Narragansett, USA
    13 Jun 2016
    Author Response
    Thank you for your review.  We have just submitted our revisions and expect the new version to be available in the next few days.  Below are our responses to the ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 13 Jun 2016
    Jeffrey Hollister, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Atlantic Ecology Division, US Environmental Protection Agency, Narragansett, USA
    13 Jun 2016
    Author Response
    Thank you for your review.  We have just submitted our revisions and expect the new version to be available in the next few days.  Below are our responses to the ... Continue reading
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39
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Reviewer Report 22 Feb 2016
Alan E. Wilson, School of Fisheries, Aquaculture, and Aquatic Sciences, College of Agriculture, Auburn University, Auburn, AL, USA 
Approved with Reservations
VIEWS 39
Title and Abstract:
For clarity, the authors might consider replacing “various” with “World Health Organization and U.S. Environmental Protection Agency”.  
 
Article content:
Using publicly available data produced from the 2007 USEPA National Lakes Assessment, the authors use conditional probability analysis ... Continue reading
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CITE
HOW TO CITE THIS REPORT
Wilson AE. Reviewer Report For: Associations between chlorophyll a and various microcystin-LR health advisory concentrations [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Research 2016, 5:151 (https://doi.org/10.5256/f1000research.8562.r12330)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 13 Jun 2016
    Jeffrey Hollister, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Atlantic Ecology Division, US Environmental Protection Agency, Narragansett, USA
    13 Jun 2016
    Author Response
    ## I think the authors need to more broadly consider the existing literature and describe how their findings relate to and build from past studies. Below, I provide some related ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 13 Jun 2016
    Jeffrey Hollister, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Atlantic Ecology Division, US Environmental Protection Agency, Narragansett, USA
    13 Jun 2016
    Author Response
    ## I think the authors need to more broadly consider the existing literature and describe how their findings relate to and build from past studies. Below, I provide some related ... Continue reading

Comments on this article Comments (0)

Version 2
VERSION 2 PUBLISHED 09 Feb 2016
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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