Summary of Research on the Effects of Sulfate and Sulfide on Wild Rice Sediment Chemistry, Growth,
and Seed Production
Prof. John Pastor
Prof. Nathan Johnson
Dept. of Biology
Dept. of Civil Engineering
University of Minnesota Duluth
University of Minnesota Duluth
January 29, 2014
1
Background
Minnesota currently has a water quality standard of “10 mg/L sulfate - applicable to water used
for production of wild rice during periods when the rice may be susceptible to damage by high sulfate
levels.” (Minn. R. 7050.0224, subpart 2). This standard was adopted in 1973 based on a study published
in 1945 by Dr. John Moyle, a botanist with the Department of Natural Resources1. Moyle found that wild
rice populations were of low plant density and uncommon or even absent in waters with sulfate
concentrations between 10 and 50 mg/L. However, questions have recently arisen as to the generality of
Moyle’s research and also whether sulfate was itself toxic to wild rice or whether sulfide, to which sulfate
is transfromed in some anaerobic environments, is the operative toxic agent. Some clarification of the
standard’s applicability occurred in 1997, but the standard was not, at that time, proposed for revision and
has not been widely enforced. Accordingly, in 2010 the MPCA initiated a multi-year effort, known as the
Wild Rice Sulfate Standard Study, to begin to understand the mechanisms of transformation of sulfate in
aquatic ecosystems and whether sulfate or any of its transformed products adversely affect wild rice
growth. The primary hypothesis has been that elevated sulfate depresses the growth of wild rice when it is
transformed into hydrogen sulfide by microbes in the wild rice’s rooting zone, and that elevated iron
could mitigate the toxicity of the sulfide by forming insoluble iron sulfide compounds.
The study consisted of a three-prong approach: (1) a field study of wild rice habitats across numerous
lakes and rivers, including many sites previously sampled by Moyle, to determine physical and chemical
conditions correlated with the presence or absence of wild rice stands, including concentrations of sulfate
in surface water and sulfide in the rooting zone; (2) outdoor container (mesocosm) experiments in which
wild rice is grown in natural sediments from a wild rice lake to determine its response to a range of sulfate
concentrations in the surface water and associated sulfide in the rooting zone across the growing season.
(3) controlled laboratory hydroponic experiments to determine the effect of elevated sulfate and sulfide on
early stages of wild rice growth and development. These studies were completed by scientists at the
Limnological Research Center at the University of Minnesota Twin Cities campus and the Departments of
Civil Engineering and Biology at the University of Minnesota Duluth campus. The studies were
completed and final reports were delivered to the Minnesota Pollution Control Agency in late December
2013. These reports and the data are available to the public online at
ftp://files.pca.state.mn.us/pub/tmp/wildRice/. This brief report summarizes our current interpretation of
these results; it should not be taken as an official statement of policy by the MPCA.
1
Moyle, J. B. 1945. Some chemical factors influencing the distribution of aquatic plants in Minnesota.
American Midland Naturalist 34: 402-420.
2
Results
Sulfide concentrations in sediments of natural waters are correlated with sulfate concentrations in
overlying waters. This correlation is stronger in lakes, where anaerobic conditions in the sediment are
conducive to the biological transformation of sulfate to sulfide, than in rivers, where flowing water is
more likely to aerate the sediments and therefore prevent sulfate transformation to sulfide. Therefore,
sulfide concentrations in sediment pore waters are high only when sulfate concentrations in the overlying
water are high, but under certain circumstances overlying sulfate concentrations can be high without
leading to sulfide in sediment pore waters. Wild rice is uncommon and sparse in most natural waters with
sulfate concentrations greater than 10-40 mg/L, except in waters flowing over sediments that may be
aerobic. These results broadly corroborate those of Moyle (1948) but go further in making a distinction
between aerobic and anaerobic sediments and in quantifying concomitant rooting zone geochemistry.
In laboratory hydroponic experiments, sulfate had no effect on wild rice seed germination and
seedling growth in hydroponic solution across a broad range of concentrations from 1 to 1600 mg/L. This
range is broader than the levels of sulfate concentrations in natural waters, even those impacted from
mines in Minnesota. Sulfide also had no effect on seed germination. However, the growth of alreadygerminated seedlings was severely decreased by sulfide at low concentrations (Fig. 1). These low sulfide
concentrations would be produced by as little as 1 mg/L of sulfate if 100% of the sulfate were
transformed to sulfide.
Significant decline (p < 0.05)
Fig. 1 Sulfide severely reduces growth of wild rice seedlings at concentrations above 9 – 10 μM
(equivalent to 1 mg/L of sulfate if 100% of the sulfate was transformed to sulfide).
During the third year of controlled experiments in outdoor mesocosms with wild rice populations
growing in wild rice sediments, additions of sulfate to surface water were accompanied by increased
sulfide concentrations in the upper 5 cm of sediments (Fig. 2). The concentrations of sulfide increased
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over the growing season and were highest in July and August when microbial activity was presumably
greatest because of warmer water and sediment temperatures. High-resolution measurements of pore
water chemistry revealed that sulfate concentrations declined along with a rise in sulfide concentration
from the sediment surface down to 5 cm depth (Fig. 3) and greater sulfate amendments increased the peak
sulfide concentration in the sediments. These depths are within the rooting zone of wild rice seedlings.
The sulfide concentrations measured in the pore waters of sulfate-amended outdoor mesocosms exceed
the sulfide concentration which severely reduced growth of seedlings in the hydroponic experiments. Iron
concentrations were depressed in the pore fluids of sulfate amended tanks, showing that the transport and
reaction of sulfur from the overlying water is consuming iron in the sediments.
Overlying water sulfate [mg/L]
100
200
300
0
Porewater sulfide [uM] (0-5cm)
1000
100
10
Jul/Aug Average
1
May/Jun Average
0
0
1000
2000
3000
Overlying water sulfate [uM]
Fig. 2 Porewater sulfide concentrations in 0-5 cm of sulfate amended mesocosms. Note log scale. Sulfide
is clearly related to overlying water sulfate amendment and increases over the course of the summer.
10 mg/L Sulfate tank
50 mg/L Sulfate tank
Iron [uM]
150 300 450 600 750
0
125
250
Iron [uM], Sulfate [mg/L]
375
0
500
-4
-4
-2
-2
-2
0
0
0
2
2
2
4
6
8
Depth [cm]
-4
Depth [cm]
Depth [cm]
0
300 mg/L sulfate tank
Iron [uM]
4
6
8
8
10
12
12
12
14
14
10
15
20
25
Sulfide [uM], Sulfate [mg/L]
Iron
Sulfide
Sulfate
0
25
50
75
100
Sulfide [uM], Sulfate [mg/L]
Iron
Sulfide
Sulfate
300
6
10
5
200
4
10
0
100
14
0
250
500
750
Sulfide [uM]
Sulfate
Iron
Sulfide
Fig. 3 Depth profiles in August 2013 for pore water sulfate, sulfide, and iron in mesocosms amended
with nominally (a) 10 mg/L, (b) 50 mg/L and (c) 300 mg/L sulfate in the overlying water. 0 depth
represents the sediment-water interface. Sulfide is clearly higher and pore water iron is depleted in
porewaters of amended mesocosms.
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Wild rice seedling survival in these mesocosms declined with increased sulfate and hence sulfide
concentrations in the sediments (Fig. 4). This decreased survival of seedlings is consistent with the results
of the hydroponic experiments (Fig. 1) and the increased sulfide concentrations within the rooting zone in
mesocosms amended with sulfate (Fig. 3).
Plants grown in the control tanks had white or light tan roots, but plants in the tanks amended
with sulfate had blackened roots (Fig. 5). Visual estimates of the proportion of blackened roots increased
progressively from approximately 50% in the tanks with 50 mg · L-1 SO4 to 100% in tanks with 300 mg/l
sulfate. Examination of these roots by scanning X-ray showed that this blackened deposit is almost
entirely iron sulfide which precipitated on the surface of the roots.
Fig. 4 Decline in wild rice seedling survival with increased sulfate concentrations.
Fig. 5 Blackened roots from plants grown in tanks with 300 mg SO4 · L-1 (left) compared to tan roots
from plants grown in control tanks (right).
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We hypothesize that these iron sulfide deposits may have inhibited late season uptake of nutrients
which the wild rice plant uses to fill out seeds because the proportion of viable seeds and the mean seed
weight declined with sulfate ammendments (Fig. 6). Therefore plants grown with enhanced sulfate
concentrations produced fewer viable seeds and those seeds which were viable had less food reserves to
support germination. These effects of sulfide on seed production resulted in lower germination rates the
following year. The decline in germination rate with increased sulfide concentrations in the sediments
does not appear to be a direct effect of sulfide on the germinating seeds, since we found no effect of
sulfide on seed germination in the hydroponic experiments. Rather reduced germination in the outdoor
mesocosms appears to be due to an indirect effect of sulfide on seed production by the parent plant.
Fig. 6 Declines in seed production associated with increased sulfate concentrations in the water columns.
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Summary
Wild rice populations are uncommon and sparse in lakes with concentrations of sulfate in
overlying waters greater than 10-30 mg/L. Sulfide concentrations in sediments of these lakes also
increased with overlying water sulfate concentrations. In controlled hydroponic experiments, enhanced
sulfate concentrations had no effect on wild rice seed germination and seedling growth, but sulfide
concentrations severely inhibited seedling growth at very low concentrations. In controlled experiments in
mesocosms with wild rice growing in wild rice sediments, sulfate amendments increased sediment sulfide
concentrations within the seedling rooting zone to levels above those observed to be toxic to seedlings
seen in the hydroponic experiments. Seedling survival also declined in these mesocosms with increased
sulfate amendments. Iron sulfide precipitates on roots of mature plants are associated with reduced
production of viable seeds and reduced average seed weight. This may have indirectly decreased seed
germination the following year.
Future Research
We are currently consulting with scientists at the MPCA to synthesize the results of these studies
into a final report which will be completed by MPCA at the end of February 2014. In that final report,
MPCA will answer two questions: 1. Should the current standard of 10 mg sulfate/L for wild rice waters
in Minnesota be changed? 2. Should the standard concentration be increased or decreased? The final
report will not specify a new standard. If a change to the existing standard is recommended and a new
standard developed, it would be adopted into Minnesota Rules via the administrative rulemaking process
and subject to U.S. EPA approval before the changes could be implemented. In this case, several more
years of research will likely be needed to specify what the new standard should be.
We have also received a $200,000 award from Minnesota Sea Grant to continue this research
along with our colleague Prof. James Cotner of the Dept. of Ecology, Evolution, and Behavior on the
Twin Cities campus. This award will be supplemented by a matching award from the Natural Resource
Management Division of the Fond du Lac Band of Lake Superior Ojibway. This new research will further
quantify the geochemical interactions between iron and sulfide in the sediments, examine whether the
iron sulfide precipitates on roots impede nutrient uptake, and determine whether rates of these processes
are modified during long term population cycles of wild rice. New field sites will be established at the
University of Minnesota’s Itasca Biological Station. The mesocosm experiment at the University of
Minnesota Duluth’s Field Studies Research Station will also be expanded.
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