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toxins
Article
Roles of Nutrient Limitation on Western Lake Erie CyanoHAB
Toxin Production
Malcolm A. Barnard 1, * , Justin D. Chaffin 2, Haley E. Plaas 1, Gregory L. Boyer 3, Bofan Wei 3,
Steven W. Wilhelm 4, Karen L. Rossignol 1, Jeremy S. Braddy 1, George S. Bullerjahn 5, Thomas B. Bridgeman 6,
Timothy W. Davis 5, Jin Wei 7, Minsheng Bu 7and Hans W. Paerl 1,*
Citation: Barnard, M.A.; Chaffin,
J.D.; Plaas, H.E.; Boyer, G.L.; Wei, B.;
Wilhelm, S.W.; Rossignol, K.L.;
Braddy, J.S.; Bullerjahn, G.S.;
Bridgeman, T.B.; et al. Roles of
Nutrient Limitation on Western Lake
Erie CyanoHAB Toxin Production.
Toxins 2021,13, 47. https://doi.org/
10.3390/toxins13010047
Received: 10 December 2020
Accepted: 6 January 2021
Published: 9 January 2021
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
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nal affiliations.
Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Institute of Marine Sciences, University of North Carolina at Chapel Hill, Morehead City, NC 28557, USA;
hplaas@live.unc.edu (H.E.P.); krossign@email.unc.edu (K.L.R.); jbraddy@email.unc.edu (J.S.B.)
2Stone Laboratory and Ohio Sea Grant, The Ohio State University, Put-In-Bay, OH 43456, USA;
chaffin.46@osu.edu
3Department of Chemistry, State University of New York College of Environmental Science and Forestry
Campus, Syracuse, NY 13210, USA; glboyer@esf.edu (G.L.B.); bwei101@syr.edu (B.W.)
4Department of Microbiology, University of Tennessee at Knoxville, Knoxville, TN 37996, USA;
wilhelm@utk.edu
5Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403, USA;
bullerj@bgsu.edu (G.S.B.); timdavi@bgsu.edu (T.W.D.)
6Lake Erie Center, University of Toledo, Oregon, OH 43616, USA; Thomas.Bridgeman@utoledo.edu
7Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Ministry of
Education, College of Environment, Hohai University, Nanjing 210098, China; weijin@hhu.edu.cn (J.W.);
hjxyym@hhu.edu.cn (M.B.)
*Correspondence: malcolm.barnard@unc.edu (M.A.B.); hans_paerl@unc.edu (H.W.P.);
Tel.: +1-252-726-6841 (ext. 254) (M.A.B.); +1-252-726-6841 (ext. 133) (H.W.P.);
Fax: +1-252-726-2426 (M.A.B. & H.W.P.)
Abstract:
Cyanobacterial harmful algal bloom (CyanoHAB) proliferation is a global problem im-
pacting ecosystem and human health. Western Lake Erie (WLE) typically endures two highly toxic
CyanoHABs during summer: a Microcystis spp. bloom in Maumee Bay that extends throughout the
western basin, and a Planktothrix spp. bloom in Sandusky Bay. Recently, the USA and Canada agreed
to a 40% phosphorus (P) load reduction to lessen the severity of the WLE blooms. To investigate
phosphorus and nitrogen (N) limitation of biomass and toxin production in WLE CyanoHABs, we
conducted in situ nutrient addition and 40% dilution microcosm bioassays in June and August 2019.
During the June Sandusky Bay bloom, biomass production as well as hepatotoxic microcystin and
neurotoxic anatoxin production were N and P co-limited with microcystin production becoming
nutrient deplete under 40% dilution. During August, the Maumee Bay bloom produced microcystin
under nutrient repletion with slight induced P limitation under 40% dilution, and the Sandusky Bay
bloom produced anatoxin under N limitation in both dilution treatments. The results demonstrate
the importance of nutrient limitation effects on microcystin and anatoxin production. To properly
combat cyanotoxin and cyanobacterial biomass production in WLE, both N and P reduction efforts
should be implemented in its watershed.
Keywords:
cyanotoxins; Maumee Bay; Sandusky Bay; Microcystis;Planktothrix; microcystin; anatoxin
Key Contribution:
Nutrient limitation of cyanobacterial harmful algal blooms (CyanoHABs) was
investigated with respect to the production of the cyanotoxins microcystin and anatoxin in Maumee
Bay and Sandusky Bay in Western Lake Erie. This is one of the first studies investigating nutrient
limitation effects on anatoxin production in Lake Erie and one of the first studies to evaluate the
effects of nutrient reduction on Western Lake Erie CyanoHABs using nutrient dilution assays. To
reduce CyanoHABs and their toxicity, both N and P reductions are needed in the Western Lake
Erie watershed.
Toxins 2021,13, 47. https://doi.org/10.3390/toxins13010047 https://www.mdpi.com/journal/toxins
Toxins 2021,13, 47 2 of 21
1. Introduction
Freshwater ecosystems are critical for sustaining life and supporting civilizations
throughout history [
1
]. As the global human population grows, increased urbanization,
agricultural and industrial productions, combined with insufficient wastewater treatment
practices, have led to a widespread increase in nutrient pollution of these ecosystems,
threatening clean and safe water supplies [
2
]. Excessive inputs of nitrogen (N) and phos-
phorus (P) have accelerated eutrophication, the process of increasing organic enrichment,
which is largely attributable to increased microalgal and aquatic macrophyte growth [
3
].
The major detrimental impacts of eutrophication include harmful algal bloom (HAB) for-
mation, decreased water transparency (increased turbidity), O
2
depletion, and reduced
biodiversity [
3
,
4
]. HAB formation has been a major water quality issue in the U.S. since
the 1960s, as noted in a 1965 White House Report indicating HABs as a major source
of environmental degradation [
5
]. Furthermore, nutrient-driven eutrophication of lakes
and rivers is one the most significant causes of water quality decline globally [
3
,
6
–
8
]. In
particular, there are growing concerns about the proliferation and diversification of N- and
P-based fertilizers, as they are potent stimulants of aquatic primary production along the
freshwater to marine continuum [
9
,
10
]. Additionally, climate change (e.g., warming and
changing precipitation patterns) is increasing the likelihood of more expansive blooms,
exposing human and animal populations (e.g., pets, wildlife, cattle, fish, birds) to water-
borne and aerosolized toxins [
7
,
11
–
14
]. Despite CyanoHAB toxicity being a major human
and ecosystem health hazard, the causes and controls of underlying toxicity mechanisms
remain poorly understood [15].
Blooms of cyanobacteria in Lake Erie, largely dominated by filamentous heterocystous
(N
2
-fixing) forms (Anabaena/Dolichospermum,Aphanizomenon), were common in the late
1950s through to the 1970s. These blooms dissipated following the signing in of the Great
Lakes Water Quality Agreement of 1972, which was updated in 2012. However, the blooms
returned as non-N
2
fixing Microcystis blooms in the early 2000s, which have continued
and perhaps worsened [
16
,
17
], leading to major environmental degradation and increased
human health risks [
7
]. In August 2014, a toxic Microcystis spp. bloom in Western Lake
Erie (WLE) created a water crisis, forcing public water supplies to be shut down for over
400,000 people in Toledo, OH, USA [
7
,
18
]. Nutrient runoff from agricultural nonpoint
sources has been a major factor promoting CyanoHABs in WLE [
7
]. Primary production
in Maumee Bay of Lake Erie (largely dominated by Microcystis spp. in the summer) shifts
from P-limitation to N-limitation with spatial nutrient limitation heterogeneity with N-
and P-limitation occurring several km apart [
19
–
21
]. Prior studies revealed that during the
summer months, N was often depleted in embayments such as Sandusky and Maumee
Bay [
22
–
26
], where summertime molar N:P ratios for Sandusky Bay remained below
the canonical Redfield ratio (16:1) [
26
–
28
]. This suggests the presence of strong N sinks,
mediated by denitrification and/or active N cycling and N uptake by high amounts of algal
biomass [
28
–
30
]. The primary fertilizers used in the agriculturally dominated drainage
basin of Lake Erie are inorganic fertilizers (ammonium nitrate, urea, and phosphate) and
manure, which has low N:P ratios (~5:1), is about 20% [31–34]. There is an urgent need to
determine the linkage between different bioreactive forms of N and P and the promotion
of toxic CyanoHABs, to establish the necessary reduction in these nutrient forms to ensure
the security of surface potable water. Nutrient reduction will likely need to be even greater
as climate change increases the N and P reduction thresholds required for CyanoHAB
mitigation [
35
,
36
]. The in situ bioassay-based study reported here is among the first to use
an experimental approach to investigate the response of a natural CyanoHAB community
dominated by either Microcystis (Maumee Bay) or Planktothrix (Sandusky Bay) to actual
reductions in N, P or both, under natural conditions in Lake Erie. Satellite and field images
of the 2019 WLE blooms can be seen in Figure 1.
Toxins 2021,13, 47 3 of 21
Toxins 2021, 13, x FOR PEER REVIEW 3 of 22
Figure 1. Images of the 2019 WLE CyanoHABs. (a) Satellite imagery from the NASA Terra satellite of
the WLE CyanoHAB on 19 August 2019 as provided by NOAA MODIS [37]; (b) Maumee Bay Micro-
cystis-dominated cyanobacterial harmful algal bloom (CyanoHAB) on 4 August 2019 during sampling
for the August 2019 bioassays. Photo by H. Plaas; (c) Sandusky Bay Planktothrix-dominated bloom on 4
August 2019 during sampling for the August 2019 bioassays. Photo by H. Plaas.
Figure 1.
Images of the 2019 WLE CyanoHABs. (
A
) Satellite imagery from the NASA Terra satellite
of the WLE CyanoHAB on 19 August 2019 as provided by NOAA MODIS [
37
]; (
B
) Maumee Bay
Microcystis-dominated cyanobacterial harmful algal bloom (CyanoHAB) on 4 August 2019 during
sampling for the August 2019 bioassays. Photo by H. Plaas; (
C
) Sandusky Bay Planktothrix-dominated
bloom on 4 August 2019 during sampling for the August 2019 bioassays. Photo by H. Plaas.
Toxins 2021,13, 47 4 of 21
A recent review suggested that management efforts to reduce P pollution without
controlling N inputs have caused nutrient imbalances in eutrophic systems, which may
favor toxic CyanoHABs incapable of fixing atmospheric N
2
gas, i.e., requiring combined
N sources [
24
]. Prior to P load reductions in the 1970s, CyanoHABs in Lake Erie were
mostly the N
2
-fixers Aphanizomenon and Dolichospermum, formerly Anabaena [
38
]; now,
CyanoHABs are primarily non-N
2
-fixing Microcystis and Planktothrix [
16
]. In WLE, molecu-
lar analysis of the Microcystis community indicates a shift from toxic to non-toxic strains that
correlates with NO
3
availability [
39
], although there appears to be a temporal disconnect as
a multiyear analysis found no correlation between the proportion of microcystin-producing
genotypes of Microcystis and the concentration of microcystin [
40
]. Recent work has
strengthened links between N availability, dominant strain shifts, and toxicity by showing
seasonal trends in these patterns [
24
]. The inability of these cyanobacteria to fix atmo-
spheric N
2
, and their strong affinity for reduced N forms (e.g., NH
4
and urea), suggests that
N delivered through agricultural runoff and internal N recycling mechanisms play critical
roles in modulating total phytoplankton biomass, CyanoHAB community composition,
and toxicity [39,41].
The prominent cyanotoxins, microcystin and anatoxin, have molecular structures
containing N, suggesting that their syntheses may be linked to N availability; hence, there is
a need to investigate the potential roles N fertilizers (i.e., NH
4
, NO
3
, and urea) play in bloom
dynamics and toxin production in Lake Erie [
23
,
36
,
42
]. A recent study showed that there
are N concentration reduction thresholds at which bloom microcystin levels will decrease,
leading to further evidence that N limitation may play a role in controlling cyanotoxin
production in the WLE blooms [
41
]. Due to the shift to non-N
2
-fixing CyanoHABs, a
major unknown concerning this shift in nutrient limitation is how specific microcystin and
anatoxin production potentials are linked to nutrient input reductions.
The US Environmental Protection Agency (EPA) and Environment and Climate
Change Canada have recommended a 40% reduction in springtime P loading into WLE
to help control the blooms [
43
–
46
]. The 40% P load reduction was the result of a multiple
modeling exercise included in the Great Lakes Water Quality Agreement between the US
and Canada [
47
]. As both N and P have been shown to influence the WLE CyanoHABs,
it is crucial to investigate the effects of both 40% reductions in both N and P in addition
to the investigations of the effects of N and P addition. Here, we addressed the following
questions: (1) how do nutrients influence WLE microcystin and anatoxin production? (2)
Do the same nutrients limit toxin production and CyanoHAB biomass? (3) Will the 40% P
reduction as recommended by the US EPA be effective in reducing CyanoHAB microcystin
and anatoxin and biomass production in WLE? (4) Is P reduction alone enough to decrease
WLE CyanoHAB biomass and microcystin and anatoxin production, or is a combined N
and P reduction strategy needed? Given the relatively high content of N in the cyanotoxins
microcystin and anatoxin, we predicted that cyanotoxin production is N-limited and that
excessive N inputs promote toxicity of these non-N2-fixing CyanoHABs.
2. Results
2.1. June 2019 Experiement
June 2019 bioassay experiments were characterized by a late spring diatom bloom
shortly before the onset of a summer Microcystis bloom in Maumee Bay and the very early
Planktothrix bloom in Sandusky Bay. In both Maumee and Sandusky Bays, there were
high N concentrations—over 200
µ
mol L
−1
nitrate plus nitrite in Maumee Bay and over
100
µ
mol L
−1
nitrate plus nitrite in Sandusky Bay and relatively low P concentrations of
1–2 µmol L−1dissolved reactive phosphorus (DRP) (Table 1).
Toxins 2021,13, 47 5 of 21
Table 1.
Initial nutrient concentrations in the June 2019 bioassay water collected from control
Cubitainers. All data are n= 3.
Nutrient
Parameter
Maumee Bay Sandusky Bay
No Dilution 40% Dilution No Dilution 40% Dilution
NO3+ NO2
(µmol L−1)223.67 ±25.43 137.64 ±35.00 101.45 ±5.95 58.46 ±8.46
NH4
(µmol L−1)1.34 ±1.01 3.67 ±0.60 24.28 ±0.66 17.14 ±0.85
DRP
(µmol L−1)2.24 ±0.23 1.50 ±0.07 1.20 ±0.14 0.85 ±0.05
Silicate
(µmol L−1)139.14 ±12.23 100.46 ±2.53 130.42 ±19.50 78.88 ±16.20
In the June Maumee Bay experiment, growth rates significantly differed (p< 0.001)
among nutrient treatments, but there was no difference between the undiluted and diluted
treatments (p= 0.76). The +P and +P&N treatments resulted in a higher growth rate than
the control and +N treatments, indicating P-limited growth, in both the undiluted and 40%
dilution treatments, likely due to the high concentrations of N in the bay (Figure 2; Table 1,
Tables S1 and S2). Cyanotoxins were not detected in the June Maumee Bay experiment.
Toxins 2021, 13, x FOR PEER REVIEW 5 of 22
Table 1. Initial nutrient concentrations in the June 2019 bioassay water collected from control Cubitain-
ers. All data are n = 3.
Nutrient Parameter Maumee Bay Sandusky Bay
No Dilution 40% Dilution No Dilution 40% Dilution
NO
3
+ NO
2
(µmol L
−1
) 223.67 ± 25.43 137.64 ± 35.00 101.45 ± 5.95 58.46 ± 8.46
NH
4
(µmol L
−1
) 1.34 ± 1.01 3.67 ± 0.60 24.28 ± 0.66 17.14 ± 0.85
DRP
(µmol L
−1
) 2.24 ± 0.23 1.50 ± 0.07 1.20 ± 0.14 0.85 ± 0.05
Silicate
(µmol L
−1
) 139.14 ± 12.23 100.46 ± 2.53 130.42 ± 19.50 78.88 ± 16.20
In the June Maumee Bay experiment, growth rates significantly differed (p < 0.001)
among nutrient treatments, but there was no difference between the undiluted and diluted
treatments (p = 0.76). The +P and +P&N treatments resulted in a higher growth rate than the
control and +N treatments, indicating P-limited growth, in both the undiluted and 40% dilu-
tion treatments, likely due to the high concentrations of N in the bay (Figure 2; Tables 1, S1,
and S2). Cyanotoxins were not detected in the June Maumee Bay experiment.
Figure 2. Growth rates of phytoplankton, as determined by chlorophyll a accumulation during the course of incubation in the
June 2019 bioassays. (a) Undiluted Maumee Bay water (also see Table S1); (b) undiluted Sandusky Bay water (also see Table
S1); (c) 40% dilution Maumee Bay water (also see Table S1); (d) 40% dilution Sandusky Bay water (also see Table S1); (e)
Maumee Bay growth rates under the various nutrient addition treatments at the two locations of T3 compared to T0 (also see
Table S2). Error bars are standard error; (f) Maumee Bay growth rates under the various nutrient addition treatments at the
two locations of T3 compared to T0 (also see Table S2). Error bars are standard error. Significances between treatments for (e)
and (f) are from two-factor ANOVAs.
In the June Sandusky Bay experiment, nutrient enrichment did not impact growth rates
(p = 0.68), but growth rates were lower in the 40% diluted treatments (p = 0.013); Figure 2;
Figure 2.
Growth rates of phytoplankton, as determined by chlorophyll aaccumulation during the course of incubation in
the June 2019 bioassays. (
A
) Undiluted Maumee Bay water (also see Table S1); (
B
) undiluted Sandusky Bay water (also see
Table S1); (
C
) 40% dilution Maumee Bay water (also see Table S1); (
D
) 40% dilution Sandusky Bay water (also see Table S1);
(
E
) Maumee Bay growth rates under the various nutrient addition treatments at the two locations of T3 compared to T0 (also
see Table S2). Error bars are standard error; (
F
) Maumee Bay growth rates under the various nutrient addition treatments at
the two locations of T3 compared to T0 (also see Table S2). Error bars are standard error. Significances between treatments
for (E,F) are from two-factor ANOVAs.
In the June Sandusky Bay experiment, nutrient enrichment did not impact growth
rates (p= 0.68), but growth rates were lower in the 40% diluted treatments (p= 0.013);
Figure 2
; Tables S1 and S2), which indicates nutrient-replete conditions. The initial undi-
Toxins 2021,13, 47 6 of 21
luted total microcystin concentration was 0.136
µ
g/L and total anatoxin concentration
was 0.053
µ
g/L (Tables S3 and S5). Microcystin concentrations increased throughout
the experiment in the no dilution treatments but not the 40% dilution (Figure 3). The
Sandusky Bay microcystin production rate was slightly yet not significantly affected by
nutrient enrichment (
p= 0.067
), becoming significant in the biomass-normalized analyses
(p< 0.001). However, the production rate was lower in the 40% diluted treatments (Figure 3
and Tables S3—S6). The June 2019 Sandusky Bay anatoxin production rate was not affected
by dilution treatment with a slight nutrient effect in the biomass-normalized analyses
(p< 0.01) (Figure 4).
Toxins 2021, 13, x FOR PEER REVIEW 6 of 22
Tables S1 and S2), which indicates nutrient-replete conditions. The initial undiluted total mi-
crocystins concentration was 0.136 µg/L and total anatoxins concentration was 0.053 µg/L (Ta-
bles S3 and S5). Microcystin concentrations increased throughout the experiment in the no
dilution treatments but not the 40% dilution (Figure 3). The Sandusky Bay microcystin pro-
duction rate was slightly yet not significantly affected by nutrient enrichment (p = 0.067), be-
coming significant in the biomass-normalized analyses (p < 0.001). However, the production
rate was lower in the 40% diluted treatments (Figure 3 and Tables S3, S4, S5, and S6). The June
2019 Sandusky Bay anatoxin production rate was not affected by dilution treatment with a
slight nutrient effect in the biomass-normalized analyses (p < 0.01) (Figure 4).
Figure 3. Production rates of microcystin during the June 2019 bioassays. Only Sandusky Bay produced microcystin in June.
(a) Undiluted Sandusky Bay microcystin concentrations (also see Table S3); (b) undiluted Sandusky Bay biomass-normalized
microcystin concentrations (also see Table S4); (c) 40% dilution Sandusky Bay microcystin concentrations (also see Table S3);
(d) 40% dilution Sandusky Bay biomass-normalized microcystin concentrations (also see Table S4); (e) Maumee Bay microcys-
tin production rates under the various nutrient addition treatments at the two locations of T3 compared to T0 (also see Table
S5); (f) Maumee Bay biomass-normalized microcystin production rates under the various nutrient addition treatments at the
two locations of T3 compared to T0 (also see Table S6). Error bars are standard error. Significance for (e) and (f) is from n-
factor ANOVA analysis due to unbalanced data sets.
Figure 3.
Production rates of microcystin during the June 2019 bioassays. Only Sandusky Bay produced microcystin in
June. (
A
) Undiluted Sandusky Bay microcystin concentrations (also see Table S3); (
B
) undiluted Sandusky Bay biomass-
normalized microcystin concentrations (also see Table S4); (
C
) 40% dilution Sandusky Bay microcystin concentrations
(also see Table S3); (
D
) 40% dilution Sandusky Bay biomass-normalized microcystin concentrations (also see Table S4);
(
E
) Maumee Bay microcystin production rates under the various nutrient addition treatments at the two locations of T3
compared to T0 (also see Table S5); (
F
) Maumee Bay biomass-normalized microcystin production rates under the various
nutrient addition treatments at the two locations of T3 compared to T0 (also see Table S6). Error bars are standard error.
Significance for (E,F) is from n-factor ANOVA analysis due to unbalanced data sets.
Toxins 2021,13, 47 7 of 21
Toxins 2021, 13, x FOR PEER REVIEW 7 of 22
Figure 4. Chlorophyll a-based production rates of anatoxin during the June 2019 bioassays. Only Sandusky Bay produced
anatoxin in June. (a) Undiluted Sandusky Bay anatoxin concentrations (also see Table S7); (b) undiluted Sandusky Bay bio-
mass-normalized anatoxin concentrations (also see Table S8); (c) 40% dilution Sandusky Bay anatoxin concentrations (also see
Table S7); (d) 40% dilution Sandusky Bay biomass-normalized anatoxin concentrations (also see Table S8); (e) Maumee Bay
anatoxin production rates under the various nutrient addition treatments at the two locations of T3 compared to T0 (also see
Table S9); (f) Maumee Bay biomass-normalized anatoxin production rates under the various nutrient addition treatments at
the two locations of T3 compared to T0 (also see Table S10). Error bars are standard error. Significance for (e) and (f) is from
n-factor ANOVA analysis due to unbalanced data sets.
2.2. August 2019 Experiment
August experiments were characterized by dense blooms of Microcystis in Maumee Bay
and Planktothrix in Sandusky Bay. Maumee Bay had high N concentrations—over 100 µmol
L
−1
nitrate plus nitrite—but Sandusky Bay had low N concentrations with 6.5 µmol L
−1
nitrate
plus nitrite (Table 2). Both Maumee and Sandusky Bay had low P concentrations of 0.03 to
0.20 µmol L
−1
DRP (Table 2).
Table 2. Initial concentrations of nutrients in the August 2019 bioassay water taken from T0 control
Cubitainers. All data are n = 3.
Nutrient Parameter Maumee Bay Sandusky Bay
No Dilution 40% Dilution No Dilution 40% Dilution
NO
3
+ NO
2
(µmol L
−1
) 127.12 ± 10.82 60.10 ± 12.94 6.59 ± 0.29 6.61 ± 0.05
NH
4
(µmol L
−1
) 0.70 ± 0.42 1.74 ± 1.58 1.05 ± 0.69 1.04 ± 0.06
Urea
(µmol L
−1
) 3.45 ± 0.61 1.59 ± 1.15 2.91 ± 1.46 3.99 ± 1.09
DRP
(µmol L
−1
) 0.20 ± 0.20 0.05 ± 0.01 0.03 ± 0.01 0.10 ± 0.07
Figure 4.
Chlorophyll a-based production rates of anatoxin during the June 2019 bioassays. Only Sandusky Bay produced
anatoxin in June. (
A
) Undiluted Sandusky Bay anatoxin concentrations (also see Table S7); (
B
) undiluted Sandusky Bay
biomass-normalized anatoxin concentrations (also see Table S8); (
C
) 40% dilution Sandusky Bay anatoxin concentrations
(also see Table S7); (
D
) 40% dilution Sandusky Bay biomass-normalized anatoxin concentrations (also see Table S8); (
E
)
Maumee Bay anatoxin production rates under the various nutrient addition treatments at the two locations of T3 compared
to T0 (also see Table S9); (
F
) Maumee Bay biomass-normalized anatoxin production rates under the various nutrient addition
treatments at the two locations of T3 compared to T0 (also see Table S10). Error bars are standard error. Significance for (
E
,
F
)
is from n-factor ANOVA analysis due to unbalanced data sets.
2.2. August 2019 Experiment
August experiments were characterized by dense blooms of Microcystis in Maumee
Bay and Planktothrix in Sandusky Bay. Maumee Bay had high N concentrations—over
100
µ
mol L
−1
nitrate plus nitrite—but Sandusky Bay had low N concentrations with
6.5
µ
mol L
−1
nitrate plus nitrite (Table 2). Both Maumee and Sandusky Bay had low P
concentrations of 0.03 to 0.20 µmol L−1DRP (Table 2).
Table 2.
Initial concentrations of nutrients in the August 2019 bioassay water taken from T0 control
Cubitainers. All data are n= 3.
Nutrient
Parameter
Maumee Bay Sandusky Bay
No Dilution 40% Dilution No Dilution 40% Dilution
NO3+ NO2
(µmol L−1)127.12 ±10.82 60.10 ±12.94 6.59 ±0.29 6.61 ±0.05
NH4
(µmol L−1)0.70 ±0.42 1.74 ±1.58 1.05 ±0.69 1.04 ±0.06
Urea
(µmol L−1)3.45 ±0.61 1.59 ±1.15 2.91 ±1.46 3.99 ±1.09
DRP
(µmol L−1)0.20 ±0.20 0.05 ±0.01 0.03 ±0.01 0.10 ±0.07
Silicate
(µmol L−1)124.33 ±7.13 95.44 ±6.24 51.12 ±12.23 64.91 ±18.52
Toxins 2021,13, 47 8 of 21
Chlorophyll aconcentrations decreased throughout incubation of the very dense
bloom in the August Maumee Bay experiment, coinciding with negative growth rates
(Figure 5). The diluted treatments had reduced algal mortality compared to the undiluted
treatments likely due to lower initial starting biomass (p< 0.001). The growth rate was
significantly affected by nutrients (p< 0.001) and the interaction between nutrients and
dilution (p= 0.012), but there was no discernable pattern, leading to a lack of ecological
significance. The initial undiluted total microcystin concentration in the August Maumee
Bay experiment was 18.06
µ
g/L. Microcystin concentration and production rates (Figure 6)
followed a similar pattern to chlorophyll with a non-significant nutrient effect (p= 0.14) and
significant dilution effect without biomass-normalization (p< 0.001) and a non-significant
effect in biomass-normalized analysis (p= 0.5452). Anatoxin was not detected in the
Maumee Bay August experiment.
Toxins 2021, 13, x FOR PEER REVIEW 8 of 22
Silicate
(µmol L
−1
) 124.33 ± 7.13 95.44 ± 6.24 51.12 ± 12.23 64.91 ± 18.52
Chlorophyll a concentrations decreased throughout incubation of the very dense bloom
in the August Maumee Bay experiment, coinciding with negative growth rates (Figure 5). The
diluted treatments had reduced algal mortality compared to the undiluted treatments likely
due to lower initial starting biomass (p < 0.001). The growth rate was significantly affected by
nutrients (p < 0.001) and the interaction between nutrients and dilution (p = 0.012), but there
was no discernable pattern, leading to a lack of ecological significance. The initial undiluted
total microcystin concentration in the August Maumee Bay experiment was 18.06 µg/L. Mi-
crocystin concentration and production rates (Figure 6) followed a similar pattern to chloro-
phyll with a non-significant nutrient effect (p = 0.14) and significant dilution effect without
biomass-normalization (p < 0.001) and a non-significant effect in biomass-normalized analysis
(p = 0.5452). Anatoxin was not detected in the Maumee Bay August experiment.
Figure 5. Growth rates of phytoplankton in the August 2019 bioassays. (a) Undiluted Maumee Bay Chlorophyll a (also see
Table S1); (b) undiluted Sandusky Bay Chlorophyll a (also see Table S1); (c) undiluted Maumee Bay Chlorophyll a (also see
Table S1); (d) 40% dilution Sandusky Bay Chlorophyll a (also see Table S1); (e) Maumee Bay growth rates under the various
nutrient addition treatments at the two locations at T3 compared to T0 (also see Table S2). Error bars are standard error. Sig-
nificance for (e) is from two-factor ANOVA analysis; (f) Maumee Bay growth rates under the various nutrient addition treat-
ments at the two locations at T3 compared to T0 (Table S2). Error bars are standard error. Significance for (f) is from n-factor
ANOVA analysis due to unbalanced data sets.
Figure 5.
Growth rates of phytoplankton in the August 2019 bioassays. (
A
) Undiluted Maumee Bay Chlorophyll a(also see
Table S1); (
B
) undiluted Sandusky Bay Chlorophyll a(also see Table S1); (
C
) undiluted Maumee Bay Chlorophyll a(also see
Table S1); (
D
) 40% dilution Sandusky Bay Chlorophyll a(also see Table S1); (
E
) Maumee Bay growth rates under the various
nutrient addition treatments at the two locations at T3 compared to T0 (also see Table S2). Error bars are standard error.
Significance for (
E
) is from two-factor ANOVA analysis; (
F
) Maumee Bay growth rates under the various nutrient addition
treatments at the two locations at T3 compared to T0 (Table S2). Error bars are standard error. Significance for (
F
) is from
n-factor ANOVA analysis due to unbalanced data sets.
Toxins 2021,13, 47 9 of 21
Toxins 2021, 13, x FOR PEER REVIEW 9 of 22
Figure 6. Production rates of microcystin during the August 2019 bioassays. Only Maumee Bay produced microcystin in all
samples. (a) Undiluted Maumee Bay microcystin concentrations (also see Table S3); (b) undiluted Maumee Bay biomass-nor-
malized microcystin concentrations (also see Table S4); (c) 40% dilution Maumee Bay microcystin concentrations (also see
Table S3); (d) 40% dilution Maumee Bay biomass-normalized microcystin concentrations (also see Table S4); (e) Maumee Bay
microcystin production rates under the various nutrient addition treatments at the two locations of T3 compared to T0 (also
see Table S5); (f) Maumee Bay biomass-normalized microcystin production rates under the various nutrient addition treat-
ments at the two locations of T3 compared to T0 (also see Table S6). Error bars are standard error. Significance for (e) and (f)
is from 2-factor ANOVA analysis.
In the August Sandusky Bay experiment, chlorophyll concentration increased through-
out the incubation in the three N-only treatments and the + N&P treatment, while it declined
in the control and P-only treatment in both the diluted and non-diluted treatments (Figure 5),
which indicates N was the primary limiting nutrient. The various forms of N did not exert a
discernable difference on growth rates. The highest growth rates were measured in the +N&P
treatments, which indicates a secondary P limitation. The dilution effect was also significant
(p = 0.004). The initial undiluted anatoxin concentration was 0.596 µg/L (Figure 7). Anatoxin
production was primarily N-limited both with and without biomass normalization (p < 0.001),
like growth rates, but P was not secondarily limiting. Unlike chlorophyll, which decreased
throughout incubation in the control and P-only treatment, anatoxin concentrations in the
control and P-only treatment remained constant throughout the incubation due to production
rates of anatoxin increasing throughout the incubation.
Figure 6.
Production rates of microcystin during the August 2019 bioassays. Only Maumee Bay produced microcystin in all
samples. (
A
) Undiluted Maumee Bay microcystin concentrations (also see Table S3); (
B
) undiluted Maumee Bay biomass-
normalized microcystin concentrations (also see Table S4); (
C
) 40% dilution Maumee Bay microcystin concentrations (also
see Table S3); (
D
) 40% dilution Maumee Bay biomass-normalized microcystin concentrations (also see Table S4); (
E
) Maumee
Bay microcystin production rates under the various nutrient addition treatments at the two locations of T3 compared to T0
(also see Table S5); (
F
) Maumee Bay biomass-normalized microcystin production rates under the various nutrient addition
treatments at the two locations of T3 compared to T0 (also see Table S6). Error bars are standard error. Significance for (
E
,
F
)
is from 2-factor ANOVA analysis.
In the August Sandusky Bay experiment, chlorophyll concentration increased through-
out the incubation in the three N-only treatments and the + N&P treatment, while it de-
clined in the control and P-only treatment in both the diluted and non-diluted treatments
(Figure 5), which indicates N was the primary limiting nutrient. The various forms of N did
not exert a discernable difference on growth rates. The highest growth rates were measured
in the +N&P treatments, which indicates a secondary P limitation. The dilution effect was
also significant (p= 0.004). The initial undiluted anatoxin concentration was 0.596
µ
g/L
(Figure 7). Anatoxin production was primarily N-limited both with and without biomass
normalization (p< 0.001), like growth rates, but P was not secondarily limiting. Unlike
chlorophyll, which decreased throughout incubation in the control and P-only treatment,
anatoxin concentrations in the control and P-only treatment remained constant throughout
the incubation due to production rates of anatoxin increasing throughout the incubation.
Toxins 2021,13, 47 10 of 21
Toxins 2021, 13, x FOR PEER REVIEW 10 of 22
Figure 7. Production rates of anatoxin during the August 2019 bioassays. Only Sandusky Bay produced anatoxin in August.
(a) Undiluted Sandusky Bay anatoxin concentrations (also see Table S7); (b) undiluted Sandusky Bay biomass-normalized
anatoxin concentrations (also see Table S8); (c) 40% dilution Sandusky Bay anatoxin concentrations (also see Table S7); (d) 40%
dilution Sandusky Bay biomass-normalized anatoxin concentrations (also see Table S8); (e) Maumee Bay anatoxin production
rates under the various nutrient addition treatments at the two locations of T3 compared to T0 (also see Table S9); (f) Maumee
Bay biomass-normalized anatoxin production rates under the various nutrient addition treatments at the two locations of T3
compared to T0 (also see Table S10). Error bars are standard error. Significance for (e) and (f) is from 2-factor ANOVA analysis.
3. Discussion
Given that CyanoHABs and their associated cyanotoxins have led to adverse human and
ecosystem health outcomes in WLE [18], it is important to clarify the major driver(s) of Cya-
noHAB toxicity. This study investigated nutrient limitation on biomass production and cy-
anotoxin production, focusing on microcystin and anatoxin. We found that high concentra-
tions of both major nutrients, P and N, drove CyanoHAB growth and microcystin and ana-
toxin production in WLE. We also found times when the 40% reduction in nutrients could
slow microcystin production during nutrient replete conditions (Figures 3E, and 3F).
We found that the June 2019 late spring diatom bloom in Maumee Bay was P-limited,
which was induced in both the undiluted and 40% dilution samples due to high ambient N
concentrations (>100 µmol/L), while the June 2019 Sandusky Bay Planktothrix bloom was not
affected by nutrient addition, but growth was slowed following a 40% reduction in nutrients.
This is possibly explained by the rapid growth associated with the early bloom, with the 40%
reduction in nutrients dropping below the threshold needed to support this bloom [48]. Dur-
ing the bloom maxima in August 2019, the Maumee Bay Microcystis bloom was nutrient re-
plete under both undiluted and 40% dilution treatments, with less of a decline in the biomass
due to the 40% lower starting biomass following dilution. Additionally, ammonium concen-
tration was higher in the initial 40% dilution than the undiluted sample in both the June and
Figure 7.
Production rates of anatoxin during the August 2019 bioassays. Only Sandusky Bay produced anatoxin in August.
(
A
) Undiluted Sandusky Bay anatoxin concentrations (also see Table S7); (
B
) undiluted Sandusky Bay biomass-normalized
anatoxin concentrations (also see Table S8); (
C
) 40% dilution Sandusky Bay anatoxin concentrations (also see Table S7); (
D
)
40% dilution Sandusky Bay biomass-normalized anatoxin concentrations (also see Table S8); (
E
) Maumee Bay anatoxin
production rates under the various nutrient addition treatments at the two locations of T3 compared to T0 (also see Table
S9); (
F
) Maumee Bay biomass-normalized anatoxin production rates under the various nutrient addition treatments at the
two locations of T3 compared to T0 (also see Table S10). Error bars are standard error. Significance for (
E
,
F
) is from 2-factor
ANOVA analysis.
3. Discussion
Given that CyanoHABs and their associated cyanotoxins have led to adverse human
and ecosystem health outcomes in WLE [
18
], it is important to clarify the major driver(s)
of CyanoHAB toxicity. This study investigated nutrient limitation on biomass production
and cyanotoxin production, focusing on microcystin and anatoxin. We found that high
concentrations of both major nutrients, P and N, drove CyanoHAB growth and microcystin
and anatoxin production in WLE. We also found times when the 40% reduction in nutrients
could slow microcystin production during nutrient replete conditions (Figure 3E,F).
We found that the June 2019 late spring diatom bloom in Maumee Bay was P-limited,
which was induced in both the undiluted and 40% dilution samples due to high ambient N
concentrations (>100 µmol/L), while the June 2019 Sandusky Bay Planktothrix bloom was
not affected by nutrient addition, but growth was slowed following a 40% reduction in
nutrients. This is possibly explained by the rapid growth associated with the early bloom,
with the 40% reduction in nutrients dropping below the threshold needed to support
this bloom [
48
]. During the bloom maxima in August 2019, the Maumee Bay Microcystis
bloom was nutrient replete under both undiluted and 40% dilution treatments, with less
of a decline in the biomass due to the 40% lower starting biomass following dilution.
Additionally, ammonium concentration was higher in the initial 40% dilution than the
undiluted sample in both the June and August 2019 Maumee Bay, likely due to an initial
die off in the subsample, leading to increased regenerated N as ammonium. These results
are likely due to bottle effects attributable to the very high biomass; restricted exchange of
gases and nutrients [
49
–
51
]. The August Sandusky Bay Planktothrix bloom was N-limited
Toxins 2021,13, 47 11 of 21
in both the 40% reduction and the undiluted samples. All nutrient concentrations in the
August 2019 Sandusky Bay 40% dilution were higher than concentrations in the undiluted
treatment, likely due to the rapid growth of the Planktothrix bloom using up more nutrients
in the undiluted control group prior to sample filtration, when compared to the reduced
biomass in the 40% dilution. Differences between the effects of the different N species
were not significant at either location during either experimental period, which has been
seen previously in strongly N-limited blooms in WLE [
52
], but differs from past findings
in WLE during periods of weaker N-limitation [
22
,
53
–
55
]. This could be due to the high
ambient concentrations of NO
3
paired with low NH
4
(Tables 1and 2). Our findings of
N limitation contradict the previous assumption that P availability exclusively controls
CyanoHABs [
56
–
59
]. Instead, these findings support the paradigm shift to also consider N
input reductions to mitigate CyanoHABs [19,29,60,61].
During the early Sandusky Bay Planktothrix bloom (June 2019), microcystin production
shifted from between N and P co-limitation in the undiluted samples to nutrient deplete
conditions in the 40% dilution samples. This is likely due to the bloom’s use of nutrient
resources early on to support biomass production rather than produce secondary metabo-
lites, e.g., cyanotoxins, possibly due to the genetic inability of the June populations to
produce the microcystin as seen in prior years [
62
,
63
]. Alternatively, the cells could have
lysed due to viral or other processes and the dissolved microcystin was not captured on the
0.7
µ
m porosity GF/F filters or degraded [
64
,
65
]. At its peak in August 2019, the Microcystis
bloom in Maumee Bay was the only bloom that produced microcystin. This production of
microcystin occurred under nutrient replete conditions, with less of a decline in microcystin
concentrations with slight P limitation in the diluted samples and no apparent nutrient
limitation in the undiluted samples. Neither experiments showed significant effects of the
various forms of N.
Even though cyanobacteria require N to produce N-rich microcystin, P is also re-
quired for cellular growth to allow for higher microcystin concentrations. As the ratio of
microcystin to chlorophyll ain both June and August was nearly linear (Figures 3and 6),
we conclude that the primary bloomers—Planktothrix in Sandusky Bay and Microcystis in
Maumee Bay—were the primary producers of microcystin. The P requirement for micro-
cystin production has been observed in prior studies in Lake Erie, and in several German
lakes [
66
]. This deviates from previous studies that clearly demonstrated links between N
availability and higher N:P and bloom toxicity in microcystin-producing blooms [
7
,
67
–
69
].
This could be due to microcystin being an “N bargain” with a C:N ratio of 4.9:1 compared to
the average of 3.6:1 in a survey of 2000 proteins [
69
]. However, P-limitation of microcystin
production has been shown to occur in chemostat experiments [
70
] and in a transcriptome
experiment on Lake Erie blooms [
40
]. The microcystin congener pattern observed in these
experiments followed what was expected for North American lakes, including Lake Erie,
with microcystin LR, YR, RR being the dominant congeners [18].
We observed anatoxin production in the Sandusky Bay Planktothrix bloom during
both early and peak blooms. This is the first study showing anatoxin production in Lake
Erie, although it has been shown that anatoxin production can occur during Planktothrix
blooms accompanied by other cyanobacteria, including Cuspidothrix issatschenkoi, which
has previously been identified in Sandusky Bay [
23
,
71
–
75
]. This was likely the case, as
the biomass normalized anatoxin production mirrors the anatoxin production in the non-
normalized analysis (Figures 4and 7), meaning that secondary cyanobacterial species may
be driving the anatoxin production in Sandusky Bay. During the early Planktothrix bloom in
June 2019, there was no apparent nutrient limitation in the undiluted treatments. However,
there was co-limitation by both N and P in the diluted treatments. During the peak
bloom in August 2019, anatoxin production was N-limited in both the undiluted and 40%
diluted samples. While no differences were found between forms of N added in the June
bioassay, during the peak bloom in August, NO
3
additions led to higher concentrations
of anatoxin compared to NH
4
and urea additions. Additionally, N limitation of anatoxin
production has been shown previously [
76
]. As observed in this experiment, higher overall
Toxins 2021,13, 47 12 of 21
N concentrations lead to higher anatoxin concentrations, with NO
3
enrichment leading
to the largest increase in anatoxin production, which parallels results from other limnetic
anatoxin-producing CyanoHABs [
77
–
81
]. Anatoxin production in Sandusky Bay and other
Planktothrix-dominated bodies of water needs further examination, given the neurotoxicity
and potential developmental toxicity of anatoxin [
82
,
83
] as well as its multiple deleterious
environmental effects [84,85].
Nutrient concentrations were very high during both the early and peak 2019 bloom
in Maumee Bay with 223.67
±
25.43
µ
g L
−1
NO
3
and 2.224
±
1.008
µ
g L
−1
DRP in June
and 127.12
±
10.82
µ
g L
−1
combined NO
3
and NO
2
in August and 0.203
±
0.199
µ
g L
−1
DRP. Similar to Maumee Bay, Sandusky Bay exhibited high nutrient concentrations in June
with 101.45
±
5.95
µ
g L
−1
NO
3
and 0.203
±
0.138
µ
g L
−1
DRP in June, but had lower
nutrient concentrations in August with 127.12
±
10.82
µ
g L
−1
NO
3
in August and 0.032
±
0.012
µ
g L
−1
DRP. This is likely due to larger nutrient loads from the Maumee River than
from the Sandusky River, as seen previously in 2007 [
86
]. The high nutrient loads were
exacerbated by elevated precipitation associated with a very wet winter in 2019 [
87
], which
will likely continue to be an issue as high precipitation events are predicted to continue in
the future [
88
–
90
]. Denitrification and assimilation draw down nitrate to concentrations
below the threshold of detection (<0.5
µ
mol/L) throughout summer and fall in western
Lake Erie and Sandusky Bay [
28
,
91
], which is a pattern that occurs independent of tributary
nutrient loads [
19
]. Our Maumee Bay experiments occurred before nitrate depletion, and
therefore, we would expect to have observed N-limited growth and microcystin production
following the N depletion [
25
]. However, it remains to be seen how a 40% dilution in
nutrients (N and P) would affect N-limited Microcystis in late summer. Therefore, nutrient
input reductions need to target both N and P rather than just P as recommended by the
US EPA and Environment and Climate Change Canada [
43
–
45
,
92
]. While P reduction is
actively pursued [93], N management strategies are required as well [35,94,95].
4. Conclusions
Our results suggest that nutrient dynamics play a crucial role in the WLE CyanoHABs
for both biomass production as well as microcystin and anatoxin production in the eu-
trophic Sandusky and Maumee Bays. During the peak bloom periods when microcystin
and anatoxin concentrations are highest, microcystin production was nutrient deplete
and anatoxin production was N-limited. Maumee Bay biomass shifted from P-limited
immediately prior to the Microcystis bloom to nutrient deplete during peak bloom, while
the Sandusky Bay Planktothrix bloom shifted from nutrient deplete to N-limited from
early bloom to peak bloom. A 40% reduction in N and P led to a slight reduction in
biomass and microcystin and anatoxin production. However, further studies are needed
to investigate the long-term nutrient reduction thresholds needed to control CyanoHABs.
With N and P enrichment stimulating the WLE CyanoHABs, there is a need to constrain
external loads of both N and P, and impose stricter nutrient-limited conditions in order
to help mitigate the CyanoHAB problem in WLE [
52
,
96
–
98
]. Our study took place only
in eutrophic bays and we showed that a 40% reduction might not be enough in Maumee
and Sandusky Bay because growth and toxin production could still be nutrient-saturated.
Future studies are needed to determine if a 40% reduction is adequate for the open waters
of WLE. Furthermore, an adaptive management approach is needed to determine if the
40% reduction goal needs to be adjusted with changes in land use practices and climate
change [
99
]. Additionally, future studies should focus on drawing direct functional links
between nutrient enrichment and cyanotoxin production, e.g., Krausfeldt et al. [
36
]. Lastly,
anatoxin should be more closely monitored in WLE, as it is a potent neurotoxin with human
health-associated implications [100].
Toxins 2021,13, 47 13 of 21
5. Materials and Methods
5.1. Bioassay Methods
We performed experimental manipulations of natural Maumee Bay (Oregon, OH,
USA) and Sandusky Bay (Sandusky, OH, USA) phytoplankton communities that were
collected from nearshore docks (Figure 8; Table S11). Water was pumped from 1 m below
the surface into pre-cleaned (flushed with lake water) 20 L carboys using a non-destructive
diaphragm pump and was transported to The Ohio State University Stone Laboratory on
South Bass Island (Put-in-Bay, OH, USA) (Figure 8).
This experiment deployed in situ bioassays, using 4 L pre-cleaned polyethylene
Cubitainers to which natural lake water was added from Maumee and Sandusky Bays
using the methodology described in Paerl et al. [
101
] and Xu et al. [
102
]. Microcosm
treatments were individually amended with either 100
µ
M N of NO
3
(as KNO
3
), 100
µ
M N
of NH
4
(as NH
4
Cl), 6
µ
M PO
4
(as KH
2
PO
4
), 100
µ
M N and 6
µ
MP added as a combined
addition of 50
µ
M NO
3
, 50
µ
M NH
4
, and 6
µ
M PO
4
, and, in August 2019, urea (50
µ
M
urea to achieve 100
µ
M N), yielding similar total dissolved nutrient concentrations (for
each treatment) and falling within a range matching riverine dissolved inorganic nutrient
discharge into Lake Erie nearshore waters. To avoid silica or dissolved inorganic carbon
limitation in Cubitainers during the incubation period, we added 50
µ
M Si as Na
2
SiO
3
and
10 mg L
−1
(83.25
µ
M) DIC as NaHCO
3
based on previous Si and DIC values from Hanson
et al. [
103
] and Rockwell et al. [
104
]. We used a major ion solution (MIS) specific to WLE
to provide 40% dilutions to mimic the EPA-recommended reductions in P inputs to WLE
as well as a parallel 40% reduction in N, as both N and P have been shown to influence
WLE CyanoHAB bloom dynamics [
22
,
39
,
43
]. The 40% dilution control investigated a 40%
reduction in both N and P. Incubations were run for 72 h at a lake site near the Stone
Laboratory at ambient lake water temperatures and light conditions [
23
,
101
,
102
]. Based on
previous work on eutrophic Lake Taihu, China [
95
], a 72 h maximum incubation period was
chosen to minimize “bottle effects”, while having ample time to examine phytoplankton
growth, microcystin, and anatoxin production responses.
To perform nutrient dilutions, we developed a major ion solution (MIS) for WLE,
which provided a N- and P-free dilution media to minimize hypertonic and hypotonic
effects on the organisms in the samples by balancing major dissolved ions in the system
(Table 3). As an example, artificial seawater is the MIS for the open ocean. For WLE, we
based the ambient ion concentrations on a past study by Chapra et al. [
105
]. As there is
substantial natural variability due to rainfall and evaporative effects and the ions in the MIS
are in micromolar concentrations and pulse events change the ions in WLE, these deviations
are considered reasonable. The compounds used in the MIS are found in Table S12.
Table 3.
Concentrations of major ions in the ambient Lake Erie water and the major ion solution (MIS) used for the dilutions
in the bioassays.
Ion 1
Average Ambient
Concentration (mg/L)
[105]
MIS 1
Concentration
(mg/L)
MIS 1
Concentration
(µM)
Percent Difference between
Chapra et al. [105] and MIS
Concentrations
Ca 2+ 32.11 32 800 −0.34%
Mg 2+ 8.89 8.88 370 −0.11%
Na +8.58 4.6 200 −46.39% 2
K+1.431 1.56 40 9.01% 3
Cl –14.58 16.33 460 12.00% 3
SO42- 22.81 43.2 450 89.39% 3
1
Constituents of MIS can be found in Table S12;
2
lower concentration compared to ambient concentration;
3
higher concentration compared
to average ambient concentrations.
Toxins 2021,13, 47 14 of 21
Toxins 2021, 13, x FOR PEER REVIEW 13 of 22
and climate change [99]. Additionally, future studies should focus on drawing direct func-
tional links between nutrient enrichment and cyanotoxin production, e.g., Krausfeldt et al.
[36]. Lastly, anatoxin should be more closely monitored in WLE, as it is a potent neurotoxin
with human health-associated implications [100].
5. Materials and Methods
5.1. Bioassay Methods
We performed experimental manipulations of natural Maumee Bay (Oregon, OH, USA)
and Sandusky Bay (Sandusky, OH, USA) phytoplankton communities that were collected
from nearshore docks (Figure 8; Table S11). Water was pumped from 1 m below the surface
into pre-cleaned (flushed with lake water) 20 L carboys using a non-destructive diaphragm
pump and was transported to The Ohio State University Stone Laboratory on South Bass Is-
land (Put-in-Bay, OH, USA) (Figure 8).
Figure 8. Map of the sampling sites and the location of the incubation. Maumee Bay water was collected off a bulkhead dock
near the University of Toledo Lake Erie Center in Oregon, OH, USA. Sandusky Bay sampling took place at a dock outside the
Paper District Marina in Sandusky, OH, USA. Incubation took place at The Ohio State Stone Laboratory on South Bass Island
(Put-In-Bay, OH, USA). GPS coordinates for the sampling and incubation sites can be found in Table S11. This figure was
created with www.simplemappr.net [106].
This experiment deployed in situ bioassays, using 4 L pre-cleaned polyethylene Cubi-
tainers to which natural lake water was added from Maumee and Sandusky Bays using the
methodology described in Paerl et al. [101] and Xu et al. [102]. Microcosm treatments were
individually amended with either 100 µM N of NO
3
(as KNO
3
), 100 µM N of NH
4
(as NH
4
Cl),
6 µM PO
4
(as KH
2
PO
4
), 100 µM N and 6 µMP added as a combined addition of 50 µM NO
3
,
50 µM NH
4
, and 6 µM PO
4
, and, in August 2019, urea (50 µM urea to achieve 100 µM N),
yielding similar total dissolved nutrient concentrations (for each treatment) and falling within
a range matching riverine dissolved inorganic nutrient discharge into Lake Erie nearshore
waters. To avoid silica or dissolved inorganic carbon limitation in Cubitainers during the in-
cubation period, we added 50 µM Si as Na
2
SiO
3
and 10 mg L
−1
(83.25 µM) DIC as NaHCO
3
based on previous Si and DIC values from Hanson et al. [103] and Rockwell et al. [104]. We
used a major ion solution (MIS) specific to WLE to provide 40% dilutions to mimic the EPA-
Figure 8.
Map of the sampling sites and the location of the incubation. Maumee Bay water was collected off a bulkhead
dock near the University of Toledo Lake Erie Center in Oregon, OH, USA. Sandusky Bay sampling took place at a dock
outside the Paper District Marina in Sandusky, OH, USA. Incubation took place at The Ohio State Stone Laboratory on
South Bass Island (Put-In-Bay, OH, USA). GPS coordinates for the sampling and incubation sites can be found in Table S11.
This figure was created with www.simplemappr.net [106].
5.2. Phytoplankton Biomass Determination
Chlorophyll a, as an indicator of phytoplankton biomass, was measured on subsam-
pled samples by filtering 50 mL of sample water onto Whatman glass fiber filters (GF/F).
Filters were frozen at
−
20
◦
C and subsequently extracted using a tissue grinder in 90%
acetone [
107
,
108
]. Chlorophyll ain extracts was measured using the non-acidification
method of Welschmeyer [
109
] on a Turner Designs Trilogy fluorometer calibrated with
pure Chlorophyll astandards (Turner Designs, Sunnyvale, CA, USA).
5.3. Nutrient Concentration Determination
Nutrient samples were collected in 50 mL Falcon tubes by collecting the GF/F filtered
water from the chlorophyll asample collection and frozen at
−
20
◦
C. A continuous seg-
mented flow auto-analyzer (QuAAtro SEAL Analytical Inc., Mequon, WI, USA) was used
to quantify nitrate, nitrite, ammonium, dissolved reactive P, and silicate using standard
U.S. EPA methods [
110
]. Urea concentration (as urea-N) was determined spectrophotomet-
rically [52,111,112].
5.4. Anatoxin and Microcystin Determinations
Cyanotoxins were measured on subsampled samples by filtering 50 mL of the sample
water onto Whatman GF/F. Filters were frozen at
−
20
◦
C until extraction with ultrasonic
sonication in 5 mL of 50% methanol and 1% acetic acid. Samples were centrifuged at
14,000
×
gfor 10 min at 4
◦
C. The supernatants were filtered through 0.45
µ
m pore-size
nylon syringe filters (Corning, CLS431225) and stored at
−
20
◦
C until analysis. Microcystin
was quantified via coupled liquid chromatography/mass spectrometry using methods
modified from Boyer [
113
] and Peng et al. [
114
]. Reverse-phase liquid chromatography
using a Waters 2695 solvent delivery system (Waters, Milford, MA, USA) coupled to a
Waters ZQ4000 mass spectrometer (Waters, Milford, MA, USA) (m/z 500–1250 amu) and a
2996 photodiode array detector (Waters, Milford, MA, USA) (210 to 400 nm wavelength)
was used to screen for molecular ions of 22 common microcystin congeners (RR, dRR,
mRR, H4YR, hYR, YR, LR, mLR, zLR. dLR, meLR, AR, FR, WR, LA, dLA, mLA, LL, LY, LW,
Toxins 2021,13, 47 15 of 21
LF, WR). Separation conditions used an ACE 5 C18, 150
×
3.0 mm column and a 30–70%
aqueous acetonitrile gradient containing 0.1% formic acid at a flow rate of 0.3 mL min.
Individual congener concentrations were quantified using the peak area of the extracted
ion relative to standards of microcystin-LR (Enzo Life Sciences, Ann Arbor, MI, USA).
This allows quantification of congeners where standards are not available. Detection of
congeners was validated by co-occurring presence of the diagnostic UV signature from
the ADDA group. Full methodological details and the standard operating protocols are
available from Protocols.io [115].
Anatoxin-a, dihydro-anatoxin-a and homoanatoxin-a were determined by LC-MS/MS
using one quantification ion and two confirmation ions for each compound. Separation
was achieved with an ACE 5 4.6
×
150 mm column (MacMod Analytical, Chadds Ford,
PA, USA) assembly with solvent flow of 0.5 mL/min from a Waters Alliance 2695 solvent
system (Waters, Milford, MA, USA). The solvent system was: A, 0.1% formic acid in water;
B, 0.1% formic acid in acetonitrile. The separation gradient was: 0 to 20% B from 0 to
10 min, 20% to 80% B from 10 to 20 min, and 80% to 100% B from 20 to 23 min, followed by
equilibration back to 0% B for 7 min. Toxins were identified using a Waters Acquity TQD
mass spectrometer (Waters, Milford, MA, USA) operated in positive mode with capillary
voltage 3.5 kV, desolvation and cone gasses at 30 and 800 Lh
−1
, respectively, desolvation
and source temperatures of 400 and 150
◦
C, respectively. Retention times and fragmentation
patterns were determined using anatoxin-a (BioMOL International, Farmingdale, NY, USA),
homoanatoxin-a isolated from natural sources and
α
and
β
dihydroanatoxin synthesized
by catalytic hydrogenation/reduction of anatoxin-a [
116
]. Calibration was performed
with anatoxin-a; dihydro-anatoxin-a and homoanatoxin-a concentrations were estimated
using the anatoxin-a standard curve. A phenylalanine standard was run with each set to
confirm the baseline resolution between anatoxin-a and phenylalanine. Multiple reaction
monitoring quantitation transitions were: anatoxin-a (166.09 > 131.00, collision energy (CE)
15 eV), dihydro-anatoxin-a (168.20 > 43.10, CE 23 eV), homoanatoxin-a (180.10 > 163.10,
CE 15 eV). Confirmation transitions were: anatoxin-a (166.09 > 148.90, CE 15 eV; 166.09 >
90.90, CE 17 eV), dihydro-anatoxin-a (168.20 > 55.90, CE 22 eV; 168.20 > 67.00, CE 26 eV),
homoanatoxin-a (180.10 > 145.10, CE 15 eV; 180.10 > 105.00, CE 17 eV).
5.5. Data Transformation and Analysis
To remove biomass effects on toxin to better measure nutrient effects on microcystin
and anatoxin production, microcystin and anatoxin concentrations are normalized to
biomass as proxied by chlorophyll a. Microcystin:chl aand anatoxin:chl aratios are calcu-
lated using Equation (1):
toxin :chl a ratio µg microcystin or anatoxin µg chlorophyll a−1=[toxin]
[biomass](1)
where [toxin] is the concentration of either microcystin or anatoxin (in
µ
g L
−1
) and
[biomass] is the concentration of chlorophyll a(in µg L−1).
For comparison between dilution treatments, we calculated production rates from
the chlorophyll aand biomass-normalized microcystin and biomass-normalized anatoxin
concentrations. Production rate (d
−1
) is a method to ln normalize the changes in concen-
trations, where a production of 0.693 d
−1
is a doubling of the concentration per day, a
production of 0.0 d
−1
indicates no change, and a production of
−
0.693 d
−1
represents a
halving of the concentration. Production is calculated using Equation (2):
Production d−1=lnµT3
µT0∗1
t(2)
where
µT0
is the average value of the measurement for the initial time point (T0),
µT3
is
the average value of the measurement for the time point of 3 days (T3), and t is the time
difference between the samplings (in days), which in this case is t= 3 days. To calculate the
Toxins 2021,13, 47 16 of 21
standard deviation for the production, propagated standard deviation is used, as calculated
by Equation (3):
Propagated Standard Deviation =sσT0
µT02
+σT3
µT32
(3)
where
µT0
is the average value of the measurement for T0,
ςT0
is the standard deviation
for the measurement at T0,
µT3
is the average value of the measurement for T3, and
ςT3
is
the standard deviation for the measurement at T3. For error bars, standard error is used,
which is calculated using Equation (4):
Standard Error =σ
√n(4)
where
ς
is the standard deviation for Figure 2a–d, Figure 3a–d, Figure 4a–d, Figure 5a–d,
Figure 6a–d, and Figure 7a–d,
ς
is the propagated standard deviation for Figure 2e–f,
Figure 3e–f, Figure 4e–f, Figure 5e–f, Figure 6e–f, and Figure 7e–f, and nis the number of
data points. The standard errors are available in the WLE_Barnard_et_al_Toxins GitHub
repository [117].
5.6. Statistical Analysis
To evaluate the source of the variation between the treatments, ANOVA analyses were
performed. For this experiment, two-factor ANOVA analyses were run on balanced data
sets (all data n= 3), and n-factor ANOVA analyses were run on unbalanced data sets (one
or more treatments were characterized as n= 1 or n= 2) using MATLAB ver. R2018b [
118
].
Both the two-factor and n-factor ANOVA analyses calculate degrees of freedom (d.f.) as
the number of treatments (n) minus one (d.f. = n
−
1). The homogeneity of variances was
tested for with Levene’s Absolute test using MATLAB ver. R2018b [
118
]. All data and
corresponding n-values are in Tables S1, S3, S4, S7, and S8.
Supplementary Materials:
The following are available online at https://www.mdpi.com/2072-6
651/13/1/47/s1, Table S1: Chlorophyll adata, Table S2: Chlorophyll aproduction rates, Table S3:
Microcystin data, Table S4: Biomass-normalized microcystin data, Table S5: Microcystin produc-
tion rates, Table S6: Biomass-normalized microcystin production rates, Table S7: Anatoxin data,
Table S8: Biomass-normalized anatoxin data, Table S9: Anatoxin production rates, Table S10: Biomass-
normalized anatoxin production rates, Table S11: GPS coordinates of the Western Lake Erie sampling
sites, Table S12: Compounds comprising the major ion solution. The following are available online
at www.doi.org/10.5281/zenodo.4281127, Code used to produce Figures 2–7, importable data file
formatted for the code, Key to the importable data file.
Author Contributions:
Conceptualization, M.A.B. and H.W.P.; data curation, M.A.B.; formal analysis,
M.A.B., G.L.B. and B.W.; funding acquisition, M.A.B., J.D.C., G.L.B., S.W.W., G.S.B., T.B.B. and T.W.D.;
investigation, M.A.B., J.D.C., H.E.P., G.L.B., B.W., S.W.W., K.L.R., J.S.B., G.S.B., T.B.B., T.W.D., J.W.,
M.B. and H.W.P.; methodology, M.A.B., J.D.C., H.E.P., G.L.B., B.W., K.L.R., J.S.B., G.S.B., T.B.B., T.W.D.,
J.W., M.B. and H.W.P.; project administration, G.S.B.; supervision, J.D.C., G.L.B., S.W.W., G.S.B., T.B.B.,
T.W.D. and H.W.P.; writing-original draft, M.A.B.; writing-review and editing, J.D.C., H.E.P., G.L.B.,
B.W., S.W.W., K.L.R., J.S.B., G.S.B., T.B.B., T.W.D., J.W., M.B. and H.W.P.; all authors have read and
agreed to the published version of the manuscript. All authors have read and agreed to the published
version of the manuscript.
Funding:
This research was funded by the United States National Science Foundation (OCE 0812913,
OCE 0825466, OCE 1840715, CBET 0826819, IOS 1451528 and DEB 1831096), the United States
National Institutes of Health (NIEHS P01ES028939), a Grant-in-Aid of Research from Sigma Xi,
The Scientific Research Society (G201903158412545) [MAB], a Kenan Graduate Student Award from
the University of North Carolina at Chapel Hill Department of Marine Sciences [MAB], and the
NOAA/North Carolina Sea Grant Program R/MER-43, R/MER-47 [MAB, HEP, KLR, JSB, HWP].
Institutional Review Board Statement: Not applicable.
Toxins 2021,13, 47 17 of 21
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data formatted for analysis and executable MATLAB code used to
produce Figures 2–7can be found on GitHub at www.doi.org/10.5281/zenodo.4281127 [
117
]. The
data presented in this study are also available in table form in the accompanying supplementary
material: https://www.mdpi.com/2072-6651/13/1/47/s1.
Acknowledgments:
We thank R. Sloup, N. Hall, and B. Abare of the UNC Institute of Marine Sciences,
as well as laboratory technicians and students from The Ohio State University and University of
Toledo Lake Erie Center at the for their help with experimental work.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
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