Global Change Biology (2009) 15, 2779–2790, doi: 10.1111/j.1365-2486.2009.01897.x
Potential effects of elevated atmospheric carbon dioxide
on benthic autotrophs and consumers in stream
ecosystems: a test using experimental stream mesocosms
C H A D W . H A R G R AV E , K A I T L E N P. G A R Y and S A M I R K . R O S A D O
Department of Biological Sciences, Center for Biological Field Studies, Sam Houston State University, Huntsville, TX 77341 USA
Abstract
Elevated atmospheric carbon dioxide (eCO2) has been shown to have a variety of
ecosystem-level effects in terrestrial systems, but few studies have examined how
eCO2 might affect aquatic habitats. This limits broad generalizations about the effects
of a changing climate across biomes. To broaden this generalization, we used free air CO2
enrichment to compare effects of eCO2 (i.e., double ambient 720 ppm) relative to
ambient CO2 (aCO2 360 ppm) on several ecosystem properties and functions in large,
outdoor, experimental mesocosms that mimicked shallow sand-bottom prairie streams.
In general, we showed that eCO2 had strong bottom-up effects on stream autotrophs,
which moved through the food web and indirectly affected consumer trophic levels.
These general effects were likely mediated by differential CO2 limitation between the
eCO2 and aCO2 treatments. For example, we found that eCO2 decreased water-column pH
and increased dissolved CO2 in the mesocosms, reducing CO2-limitation at times of
intense primary production (PP). At these times, PP of benthic algae was about two times
greater in the eCO2 treatment than aCO2 treatment. Elevated PP enhanced the rate of
carbon assimilation relative to nutrient uptake, which reduced algae quality in the eCO2
treatment. We predicted that reduced algae quality would negatively affect benthic
invertebrates. However, density, biomass and average individual size of benthic invertebrates increased in the eCO2 treatment relative to aCO2 treatment. This suggested that
total PP was a more important regulator of secondary production than food quality in our
experiment. This study broadens generalizations about ecosystem-level effects of a
changing climate by providing some of the first evidence that the global increase in
atmospheric CO2 might affect autotrophs and consumers in small stream ecosystems
throughout the southern Great Plains and Gulf Coastal slope of North America.
Keywords: atmospheric CO2, ecosystem function, free air CO2 enrichment, global change, streams
Received 29 October 2008; revised version received 2 February 2009 and accepted 4 February 2009
Introduction
Human dependence on fossil fuels has caused an unprecedented increase of atmospheric carbon dioxide
(CO2) over the last century. Because global CO2 emissions will continue at a rapid rate, atmospheric CO2
could double within the next 50–100 years (Houghton
et al., 2001). This might have widespread impacts on
ecosystems by altering global climatic conditions. In
addition to broad-scale climatic changes, elevated atmospheric CO2 (eCO2) likely will alter local ecosystemlevel properties and functions by directly affecting basal
Correspondence: Chad W. Hargrave, tel. 1 1 936 294 1543, fax 1 1
936 294 3940, e-mail: [email protected]
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trophic levels (Bazzaz, 1990). For example, in terrestrial
ecosystems eCO2 affects plant biomass and primary
production (PP) (Rogers & Humphries, 2000; Niklaus
& Körner, 2004; Ainsworth & Long, 2005), soil nutrient
availability (Canadell et al., 1996; Lou et al., 2004, 2006),
soil respiration (Jastrow et al., 2000; Jin & Evans, 2007),
leaf litter quality (Strain & Bazaaz, 1983; Curtis et al.,
1996), decomposition (Norby et al., 2001), and microbial
productivity (Randlett et al., 1996). However, effects of
eCO2 on local energy, nutrient, and community dynamics are complex, changing with abiotic and biotic
context such as nutrient availability, species diversity
and community composition (He et al., 2002; Maestre &
Reynolds, 2007). Thus, testing the effects of eCO2 across
ecosystems with varying abiotic and biotic properties
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2780 C . W . H A R G R AV E et al.
will strengthen our ability to predict ecosystem-level
consequences in a rapidly changing climate.
Research addressing the effects of eCO2 on properties
and functions in aquatic ecosystems has received little
attention. This stems from the general argument that
aquatic ecosystems rarely are carbon (C) limited (Raven
& Johnston, 1991; Tortell et al., 2000). However, this
view has neglected the important interactions between
PP of aquatic biota and chemical characteristics of
water, namely pH and alkalinity (Riebesell et al., 1993;
Schippers et al., 2004). Based on the interactions between biotic and abiotic properties in aquatic ecosystems, autotrophs in these systems are likely to
experience C-limitation at individual and local scales.
At the individual scale, the degree of C-limitation in
aquatic autotrophs will depend on the algal species’
relative affinity for CO2 or bicarbonate (HCO3 ; Israel &
Hophy, 2002). Thus, an individual alga with a high
affinity for CO2 over HCO3 could experience C-limitation in the boundary layer of water surrounding the
organism when the CO2 concentrating mechanism for
photosynthesis (e.g., active transport of CO2 from the
medium into the cell) is active. Because eCO2 will
increase the rate of CO2 diffusion into this boundary
layer, it is likely that aquatic autotrophs with these
characteristics will respond positively to eCO2 (Hein &
Sand-Jensen, 1997). This hypothesis has been supported
in aquatic macrophytes (Titus & Andorfer, 1996), floating mats of cyanobacteria (Levitan et al., 2007), and
some attached algae (Sand-Jensen & Borum, 1991).
At a larger, local scale, pH and alkalinity can interact
with rates of PP to influence C-limitation within a
habitat. For example, photosynthesis can drive down
free CO2 in the water column in aquatic ecosystems with
low alkalinity and high pH (Madsen & Maberly, 1991;
Raven & Johnston, 1991). This can induce C-limitation of
autotrophs with high affinities for CO2 throughout the
local habitat (Howell et al., 1990; Finlay & Jackson, 1991;
Finlay et al., 1999; Finlay, 2001, 2004). Under these
conditions, eCO2 can reduce CO2 depletion within the
local habitat and minimize periods of C-limitation, potentially benefiting aquatic autotrophs. In support of this
hypothesis, eCO2 has been shown to increase biomass
and PP of water column algae in both marine and lake
ecosystems (Riebesell et al., 1993; Hein & Sand-Jensen,
1997; Ibelings & Maberly, 1998; Schippers et al., 2004).
However, unlike terrestrial, marine and lake ecosystems to our knowledge no studies have tested the
effects of eCO2 on stream ecosystems, which limits
generalizations about the effects of changing atmospheric CO2 across biomes. This is probably because,
at the landscape scale, streams generally are a net
source of CO2 to the atmosphere, and, unlike terrestrial
ecosystems, have a negligible role in removing excess
CO2 from the atmosphere (Allen, 1995; Worrall & Burt,
2005; Cole et al., 2007). However, at local scales many
streams are net autotrophic, assimilating atmospheric CO2
at rapid rates (Busch & Fisher, 1981), so during periods of
intense PP stream autotrophs can regularly face C-limitation (Finlay et al., 1999; Finlay, 2001, 2003, 2004). Thus,
much like terrestrial, marine and lake ecosystems, it is
plausible to predict that under certain environmental
conditions increasing atmospheric CO2 will affect local
rates of PP and stimulate autotrophic biomass in streams.
In addition to stimulating PP, eCO2 can shift elemental ratios of autotrophs (Chen et al., 2005). This has been
demonstrated widely in terrestrial plants (Lou et al.,
2006). For example, many plant species grown under
eCO2 show greater C : N and more structural compounds (e.g., lignin) than plants grown at ambient
CO2 levels (aCO2). This is thought to reduce the nutritional quality of the plant material and affect rates of
herbivory (Levin, 1971; Cates & Rhoades, 1977; Swain,
1979). In stream ecosystems, terrestrial leaf material is
an important source of allochthonous C and nutrients
for consumers (Fisher & Likens, 1973). Thus, eCO2
might affect consumer dynamics in streams by altering
the nutritional quality of allochthonous resources
(Tuchman et al., 2002). Additionally, shifts in elemental
ratios of aquatic algae in response to eCO2 also are likely
to occur in stream ecosystems (Andersen et al., 2005;
Andersen & Andersen, 2006). Thus, much like terrestrial ecosystems, consumer dynamics linked to autochthonous energy and nutrient sources in streams (e.g.,
algae) are likely to be affected by increasing atmospheric CO2 (Stelzer & Lamberti, 2002).
In this study, we tested the effects of eCO2 on stream
ecosystem properties and functions using free air CO2
enrichment and experimental stream mesocosms. We
hypothesized that benthic algae in these experimental
mesocosms would temporarily experience CO2 limitation during periods of high PP, and therefore, we
predicted that eCO2 would (1) increase benthic algae
biomass, (2) increase benthic algae PP, and (3) reduce
benthic algae nutritional quality. We hypothesized that
the effects of eCO2 on benthic algae would move up the
food chain and affect benthic grazing invertebrates.
Specifically, we predicted that poorer algae quality in
the eCO2 treatment would (4) reduce benthic grazing
invertebrate density, biomass and individual size.
Materials and methods
Stream mesocosms
We tested the effects of eCO2 on stream ecosystem
properties and functions in 24 experimental stream
mesocosms located at the Sam Houston State University
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Center for Biological Field Studies (CBFS; Walker
County, TX, USA). The mesocosms used in this study
consisted of a single channel (1 m wide 4 m long) with
an 8 cm thick sand substrate (Fig. 1), which were designed to mimic small, shallow, sand-bottom streams
common throughout the southern Great Plains and Gulf
coastal slope of North America. All mesocosms were
under a shade structure that blocked about 40% of the
photosynthetic active radiation (PAR) to keep water
temperature cool during late summer by simulating a
sparse canopy cover. The design of these mesocosms
replicated many of the abiotic and biotic characteristics
of Harmon Creek – a nearby stream at the CBFS (Table
1). For example, physical properties linked to CO2
limitation such as flow, temperature, pH, and alkalinity
were similar between these the experimental mesocosms
and Harmon Creek (Table 1). Likewise, many of the
biotic properties of the mesocosms that could be affected
by CO2 limitation (e.g., chlorophyll-a and benthic invertebrates) also fell within the range of conditions
observed in a natural system (Table 1). As with other
experimental stream mesocosms that have provided
realistic conditions to test a variety of ecosystem, community, and population-level questions in stream ecology (e.g., Matthews et al., 2006), we believe the
mesocosms used in this study provided a set of reasonably realistic conditions for testing the effects of eCO2 on
ecosystem processes and functions occurring at local
scales within small, sand-bottom temperate streams.
To prepare the mesocosms for the experiment, we
drained and cleaned, and homogenized sediments
among units on July 7, 2007. Mesocosms remained
dry until July 16, 2007 when they were refilled with
well water to a depth of about 18 cm. After filling, we
inoculated each mesocosm with natural periphyton and
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microbial taxa by adding 1 L of an algae/biofilm slurry
collected from Harmon Creek. Following the algae/
biofilm inoculation, recirculating flow was created in
each mesocosm with a 3500 L h 1 submersible pump.
Mesocosms remained unaltered for 15 days to allow
establishment of a periphyton and biofilm assemblage
and to allow colonization of aquatic insect larvae by
ovipositing terrestrial adults (e.g., Hargrave, 2006).
Atmospheric CO2 enrichment
On July 30, 2007 (day 0), we implemented an eCO2
treatment in which we doubled atmospheric CO2
( 720 ppm) in 12 randomly selected mesocosms. The
remaining 12 mesocosms were maintained at aCO2
levels ( 360 ppm). The CO2 enrichment lasted 90 days
and was implemented by releasing pure CO2 gas (from
110 kg CO2 tanks) above the water surface with a CO2
emitter system. The CO2 emitter system consisted of
four gas regulators each attached to a CO2 tank. Each
regulator released CO2 into PVC piping which delivered
the gas to four distributors. Each distributor supplied
CO2 to three mesocosms via six flexible tubes (two tubes
per mesocosm). The tubes were suspended about 10 cm
above the water surface in each mesocosm. The walls of
the mesocosm provided a space, somewhat protected
from wind, in which the pure CO2 gas could mix with
the air and produce a CO2 enriched atmosphere above
the water surface (Fig. 1). Because the mesocosms were
open to the environment, we checked atmospheric CO2
levels above the water surface daily with a handheld
Bacharachs Model 2810 atmospheric CO2 analyzer (Bacharach Inc., New Kensington, PA, USA). We adjusted
the gas regulators as necessary to keep CO2 concentrations above the water surface about double ambient.
Water column chemistry
Fig. 1 Longitudinal section (upper illustration) and plan (lower
illustration) of experimental stream mesocosms located at the
Sam Houston State University Center for Biological Field Studies, near Huntsville, TX, Walker Co., USA.
At 15-day intervals, beginning on day 0, we measured
temperature, pH, and alkalinity in each mesocosm. The
temperature and pH were measured every 2–3 h for a
24-h period with a YSI model 85 multiparameter meter
and a handheld WTW model pH-15 pH meter (WTW,
Weilheim, Germany) respectively. We estimated alkalinity using Gran titration from a 250 mL water sample
taken at mid-day from each mesocosm. Using temperature, pH, and the alkalinity, we calculated dissolved
CO2(aq) concentration in the water column of each
mesocosm every 15 days throughout the experiment.
On days 0, 30, 60, and 90, we took additional water
samples to measure water-column nutrient concentrations from each mesocosm. A 250 mL water sample was
collected from each mesocosm, stored on ice and returned to the laboratory for immediate analysis of total
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Table 1 Abiotic and biotic parameters estimated at mid-day from experimental stream mesocosms and Harmon creek from
August 1, 2008 to October 29, 2008 (90 days)
Experimental mesocosms
Harmon creek
Parameter
Average
SD
Max
Min
Average
SD
Min
Max
Width (m)
Depth (cm)
Light (mmol cm 2 s 1)
Flow (m s 1)
Dissolved oxygen (mg L 1)
Temperature (C)
Conductivity (ms)
pH
Alkalinity (mg L 1)
Carbon dioxide (mg L 1)
Turbidity (NTU)
Chlorophyll a (mg cm 2)
Invertebrate density (m 2)
Invertebrate biomass (mg m 2)
1.0
18.4
603.5
0.3
7.8
24.4
450.5
8.3
14.3
0.6
0.3
8.6
4176.0
171.0
0.0
2.3
87.5
0.1
0.6
4.1
24.9
0.4
0.4
0.4
0.2
4.0
1491.0
56.0
1.0
14.0
513.1
0.25
6.30
10.2
394.0
7.4
11.2
0.1
0.1
3.8
6321.0
279.0
1.0
25.0
768.5
0.3
9.4
33.7
496.0
8.7
17.2
2.3
0.6
19.0
1034.0
114.0
3.5
12.2
497.3
0.2
7.1
24.1
426.0
8.5
12.3
2.4
5.7
10.0
2368.0
791.0
0.9
3.6
127.5
0.1
0.4
2.8
117.0
0.1
5.6
1.1
1.3
0.8
1084.0
1439.0
2.3
1.5
4.9
0.0
6.4
18.3
258.0
8.4
5
1.0
4.1
0.2
229.0
9.0
5.0
20.0
615.9
0.3
7.5
29.1
575.0
8.8
23
3.5
7.8
23.4
4943.0
4548.0
Measurements were taken from six randomly selected stream mesocosms with no experimental manipulation (i.e., controls) and
from six randomly selected, fixed, stations in Harmon creek. Reported are averages, standard deviations (SD), minimum (Min), and
maximum (Max) values based on weekly measurements in each of the systems.
phosphorus (P) and total nitrogen (N), using persulfate
digestion followed by the ascorbic acid method and
cadmium reduction method for total P and N respectively (APHA, 1998).
Algae biomass, production, and quality
Beginning on day 0, we measured biomass (as chlorophyll-a), PP, and nutritional quality of benthic algae at
15-day intervals in both CO2 treatments. Using scintillation vials, we took four randomly placed core samples
(8 cm2 by 0.5 cm deep) from the sand substrate in each
mesocosm to estimate chlorophyll-a. These core samples were immediately placed on ice, and returned to
the lab where they were frozen for at least 24 h. After
that time period, 10 mL of 90% acetone was added to
each vial, the vial was vortexed for several seconds, and
returned to the freezer for a 24 h extraction period. The
concentration of chlorophyll-a in the extract was measured after 24 h with a Turner IR-Model fluorometer
(Turner Designs Inc., Sunnyvale, CA, USA) (APHA,
1998). The four chlorophyll-a subsamples were averaged for each mesocosm within a sample period. On
days 45 and 90, we took two additional core samples
per mesocosm. We preserved the contents of these core
samples with 2% formalin and, using a compound
microscope, classified the algae assemblages in terms
of proportion of diatoms, bluegreen algae, and green
algae for each mesocosm.
Every 15 days, beginning on day 0, we estimated PP
(as gross PP 5 net PP 1 respiration) from diel oxygen
curves for each stream mesocosm using the open-system single-station approach (Owens, 1974; Bott, 1996).
We first turned off circulation pumps in each mesocosm, and, using a YSI model 85 oxygen meter, (YSI
Inc., Yellow Springs, OH, USA) we measured dissolved
oxygen concentration in each mesocosm every 2–3 h
over a 24-h period (Owens, 1974). We estimated gas
exchange with the atmosphere using the surface renewal model, and assumed this value was constant across
all experimental mesocosms. PAR was measured during each sample period using a handheld LI-COR
LI250A light meter (LI-COR Inc., Lincoln, NE, USA).
We did not correct PP with PAR because these measurements were similar across sample days.
We estimated benthic algae quality from that growing
on unglazed clay tiles in each mesocosm. On day 0, four
unglazed clay tiles were placed on the substrate in each of
the mesocosms. Then on days 45 and 90, two tiles were
removed from each mesocosm and the algae from both
tiles were scraped into a single Nalgene container. We
estimated C, P, and N content of the algae from these
samples. C was estimated as ash free dry mass (AFDM) by
combustion, and P and N were determined using the same
methodology described above for water-column nutrients.
Benthic invertebrates
To estimate benthic invertebrate density, biomass and
average individual size, we took one randomly placed
core sample (750 cm2 by 5 cm deep) from each mesocosm on days 0, 30, 60, and 90. Invertebrates were
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washed from sediments using a 250 mm sieve, counted,
and identified to family or genus under a dissecting
microscope. All invertebrates from each core for each
taxonomic group were placed into a preweighed aluminum-weighing pan. The pans with invertebrates were
dried at 60 1C to a constant mass (4 days), and total dry
mass of all invertebrates per core sample was determined
to the nearest microgram using a CAHN, Model C-33
microbalance (Thermo Fisher Scientific Inc., Waltham,
MA, USA). To estimate the average individual mass of
invertebrates from each mesocosm we divided the total
dry mass of all invertebrates from the sample by number
of individuals from that same sample.
Terrestrial inputs
We estimated the rates of terrestrial inputs to evaluate
whether this variable differed between CO2 treatments
and thus could have affected response in benthic algae
or aquatic invertebrates. On days 0, 30, 60, and 90 we
placed a single, circular pan trap (730 cm2, 6 cm deep) on
the stream surface in each mesocosm unit. The pan traps
were filled with a solution of soap and water and left in
the mesocosms for 48 h. After this time period, the
contents from each pan trap were collected in a Nalgene
container, immediately placed on ice, and returned to
the laboratory where we filtered the contents from each
pan trap through Gelman A/E glass fiber filters (Gelman Sciences Inc., Pensacola, FL, USA). This filtrand
was dried to a constant mass and combusted to determine AFDM of terrestrial input on each sample day.
Statistical analyses
We analyzed all data using a repeated measures analysis of variance, with sample day as the repeated measures and CO2 concentration as the treatment. We tested
for an overall treatment effect, a time effect, and a time
by treatment interaction on each of the 13 response
variables. We used a sequential Bonferroni’s correction
to limit the overall experimentwise error rate across
analyses. Finally, we used separate univariate one-way
ANOVAs to test for differences between treatments within each sample day. All statistics were calculated with
SAS Institute, SAS Version 9.1, SAS Institute, Cary, NC,
USA, 2003 using the PROC GLM procedure.
Results
Water column physicochemical factors
We used temperature, alkalinity, and pH measurements
to calculate the concentration of CO2(aq) in the water
column for each mesocosm. Average daily temperature
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across mesocosms in August, September, and October
was 28.2 0.3, 24.3 0.2, and 20.3 0.3 1C, respectively. Alkalinity remained constant throughout the
experiment and did not differ between CO2 treatments
[average for eCO2 12.25 1.2 (SD) mg L 1, aCO2
15.25 3.2 mg L 1]. Water column pH significantly increased with time in all mesocosms (Table 2), but
remained about 0.5 U lower in the eCO2 treatment then
the aCO2 treatment (Fig. 2). These measurements indicated that, although CO2(aq) decreased throughout this
experiment, CO2 in the enriched atmosphere was indeed dissolving into the water column and enhancing
CO2(aq) in the eCO2 treatment. Thus, CO2(aq) in the aCO2
treatment approached very low concentrations
(o0.1 mg L 1) by day 45, while CO2(aq) in the eCO2
treatment remained about 2–6.5 times greater throughout the course of this experiment (Fig. 2).
We measured water column nutrients in all mesocosms throughout the experiment to determine if this
variable could have affected ecosystem response to
eCO2. Although total P and N spiked in the water
column on day 60 (Fig. 3), these nutrients did not differ
between CO2 treatments (Table 2). This suggests that the
effects of eCO2 on PP and benthic invertebrates were not
mediated by differences in water column nutrients
between CO2 treatments.
Benthic algae biomass, production, and quality
The eCO2 treatment had no effect on the composition of
the benthic algae in these mesocosms, which consisted
primarily of diatoms (aCO2 87 11.1%; eCO2 83 5.0%
of the assemblages), with bluegreen (aCO2 10 1.1%;
eCO2 13 4.2%) and filamentous algae comprising a
smaller proportion of the assemblages (aCO2 3 3%;
eCO2 7 6.1%). Benthic algae biomass (chlorophyll-a)
also did not differ between CO2 treatments, but changed with time (Table 2). The temporal trends in algae
biomass likely reflected population increases followed
by a period of self-shading and sloughing (Fig. 4).
However, in support of our predictions, PP was significantly greater in the eCO2 vs. aCO2 treatment (Table
2). This significant difference in PP between these
treatments was transient however, occurring on sampling days when benthic algae biomass was lowest (Fig.
4). For example, PP was about 1.6, 1.9, 2.5, and 1.3 times
greater in the eCO2 treatment than in the aCO2 treatment on days 30, 45, 60, and 75, respectively.
We predicted that elevated PP would result in a
reduction in benthic algae quality, i.e., greater C : P
and C : N. As predicted, C : P of algae was on average
about 2 and 1.5 times greater in the eCO2 treatment than
that in the aCO2 treatment on days 45 and 90, respectively, but C : N did not differ between treatments
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Table 2 Results from repeated measure ANOVA, testing effects of elevated atmospheric CO2, time, and time by CO2 interaction on 13
response variables
CO2 time
Time
CO2
Response variable
df
F
P
df
F
P
df
F
P
Water column P
Water column N
Alkalinity
Water column pH
Water column CO2
Benthic chlorophyll-a
Primary production
Algae quality C : P
Algae quality C : N
Chironomid density
Chironomid biomass
Chironomid size
Terrestrial input
1,22
1,22
1,22
1,22
1,22
1,22
1,22
1,22
1,22
1,22
1,22
1,22
1,22
0.23
0.01
0.77
135.50
26.27
0.09
16.03
6.40
0.86
40.70
85.28
19.32
0.13
0.636
0.944
0.390
o0.001
o0.001
0.773
0.001
0.019
0.362
o0.001
o0.001
o0.001
0.717
3,20
3,20
6,17
6,17
6,17
6,17
6,17
1,22
1,22
3,20
3,20
3,20
3,20
5.33
15.24
0.80
204.90
81.57
229.61
189.51
0.83
1.20
63.23
62.72
65.22
2.35
0.013
o0.001
0.380
o0.001
o0.001
o0.001
o0.001
0.373
0.291
o0.001
o0.001
o0.001
0.103
3,20
3,20
6,17
6,17
6,17
6,17
6,17
1,22
1,22
3,20
3,20
3,20
3,20
0.09
0.38
1.27
39.82
12.37
1.70
4.23
1.27
0.88
15.18
20.66
4.66
1.21
0.911
0.685
0.272
o0.001
o0.001
0.181
0.009
0.272
0.358
o0.001
o0.001
0.013
0.331
Bolded values indicate significance (at a 5 0.05 level) after adjusting P with a sequential Bonferroni’s correction. Noncorrected Pvalues are shown in table.
Fig. 2 Mean ( 1 SE; n 5 12) water column pH (a) and dissolved CO2(aq) (b) measured every 15 days (beginning day 0) in
mesocosms receiving eCO2 (open circles) and aCO2 (filled circles). Asterisks indicate significant difference at each sample day
(P 0.05) based on separate one-way ANOVAs.
Fig. 3 Mean ( 1 SE; n 5 12) total phosphorus (a) and total
nitrogen (b) measured every 30 days (beginning day 0) in
mesocosms receiving eCO2 (open circles) and aCO2 (filled
circles).
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Fig. 4 Mean ( 1 SE; n 5 12) benthic chlorophyll-a (a) and
primary production (b) measured every 15 days (beginning
day 0) in mesocosms receiving eCO2 (open circles) and aCO2
(filled circles). Asterisks indicate significant difference at each
sample day (P 0.05) based on separate one-way ANOVAs.
2785
Fig. 5 Mean ( 1 SE; n 5 12) C : P (a) and C : N (b) of benthic
algae on days 45 and 90 from mesocosms receiving eCO2 (open
bars) and aCO2 (filled bars). Asterisks indicate significant difference at each sample day (P 0.05) based on separate one-way
ANOVAs.
(Fig. 5). However, the statistical significance of this effect
changed after a Bonferroni’s correction, leaving the importance of this trend open for interpretation (Table 2).
2.5 times greater in the eCO2 treatment than in the aCO2
treatment on days 30, 60, and 90, respectively. Biomass
was about 4, 3, and 3 times greater in the eCO2 treatment than in the aCO2 treatment on days 30, 60 and 90,
respectively. Average individual mass was about two
times greater on days 30 and 60 (Fig. 6).
Benthic invertebrates
Terrestrial inputs
Benthic invertebrate assemblages were similar between
CO2 treatments and were composed primarily of dipteran (aCO2: 95 3%; eCO2 98 5%) and odonate larvae (aCO2: 5 1%; eCO2: 2 0.5%). We analyzed only
density, biomass, and individual size of dipterans (i.e.,
Chironomidae; Chironomus sp.) because they comprised
most of the invertebrate assemblages. We predicted that
reduced benthic algae quality would negatively affect
benthic invertebrates by reducing density, biomass, and
average individual size of these organisms. Contrary to
our predictions, eCO2 had positive effects on benthic
invertebrates, significantly increasing chironomid density, biomass, and average individual size (Table 2).
Specifically, chironomid density was about 3, 5, and
We monitored terrestrial inputs to determine if this
property varied between CO2 treatments, and, thus,
could have potentially affected the benthic algae and
aquatic invertebrates in this experiment. Terrestrial inputs were composed exclusively of insects because the
shade structures prevented leaf litter from entering the
mesocosms. The rate of terrestrial insect input into the
mesocosms did not differ between CO2 treatments
(Table 2), averaging about 0.2 0.03 g C m 2 days 1 on
days 0, 30, and 90 and about 0.4 0.11 g C m 2 days 1
on day 60 (Fig. 7). The increase in terrestrial inputs on
day 60 resulted from a Lovebug (Plecia nearctica) emergence. On this sample day, mats of insects were observed floating on the water surface and decomposing
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2786 C . W . H A R G R AV E et al.
Fig. 7 Mean ( 1 SE; n 5 12) terrestrial insect input measured
every 30 days (beginning day 0) from mesocosms receiving eCO2
(open circles) and aCO2 (filled circles). Asterisks indicate significant difference at each sample day (P 0.05) based on
separate one-way ANOVAs.
influence on the response in benthic algae and invertebrates to eCO2.
Discussion
Fig. 6 Mean ( 1 SE; n 5 12) chironomid density (a), total
chironomid biomass (b), and average individual mass per chironomid (c) measured every 30 days (beginning day 0) from
mesocosms receiving eCO2 (open circles) and aCO2 (filled circles). Asterisks indicate significant difference at each sample day
(P 0.05) based on separate one-way ANOVAs.
on the substrate in each of the mesocosms. The increase
in terrestrial input on day 60 likely caused the associated spike in total P and N in the water column of
each mesocosm observed on this sample day (Fig. 2).
Although terrestrial inputs varied temporally and likely
influenced water column nutrients, these changes were
consistent between treatments and likely had little
Although eCO2 has been shown to have a variety of
ecosystem-level effects in terrestrial systems (e.g., Idso,
1999; Long et al., 2004), generalizations about the effects
of this greenhouse gas are limited across ecosystem
types. In part, this is because experiments testing the
effects of eCO2 are rare for aquatic systems, particularly
freshwater streams. Herein, we present data that shows
eCO2 can affect several ecosystem properties and functions in freshwater experimental stream mesocosms. We
believe our data provide evidence that the global increase in atmospheric CO2 might affect stream ecosystems at local scales by altering properties and functions
of the benthic algae and consumer trophic levels.
When atmospheric CO2 dissolves into water it reacts
with the medium, forming carbonic acid (H2CO3). The
H2CO3 quickly dissociates into biocarbonate HCO3 and
carbonate CO23 ions, resulting in a dissolved inorganic
carbon (DIC) pool of three species CO2, HCO3 and
CO23 . Because the relative proportion of these species
varies with pH, abiotic or biotic processes that affect pH
can shift this equilibrium (Allen, 1995). In our experiment, eCO2 likely affected DIC dynamics within the
experimental mesocosms in two ways that subsequently affected the benthic algae. First, the mesocosms
with eCO2 had greater CO2(aq), which increased the total
DIC pool. Second, the greater amount of CO2(aq) formed
more H2CO3 and reduced pH. This lower pH shifted
the CO2(aq), HCO3 and CO23 equilibrium toward greater CO2(aq). The increase in productivity of benthic algae
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E L E VA T E D A T M O S P H E R I C C O 2 A F F E C T S S T R E A M S
in the eCO2 treatment suggests that DIC (either CO2(aq)
or HCO3 ) was limiting to benthic algae at some point
during this experiment.
Many algae can use both CO2 and HCO3 as their
main inorganic C source (Falkowski & Raven, 2007).
Because HCO3 is typically the more common species
present in natural freshwaters with pH between 6 and
10, the degree to which freshwater algae will respond to
CO2 enrichment is dependent upon their relative affinity for CO2 vs. HCO3 (Raven & Johnston, 1991). If their
affinity for HCO3 is high, then CO2 enrichment likely
will have no effect on PP (Israel & Hophy, 2002).
However, some algae have higher affinities for CO2
and might lack the molecular mechanisms to use
HCO3 when CO2 is unavailable (Madsen & Maberly,
1991; Schippers et al., 2004). Thus, ecosystems dominated by such algae should experience CO2 limitation
more frequently than ecosystems with algae that can
use HCO3 (Hein & Sand-Jensen, 1997; Schippers et al.,
2004). Furthermore, dissolved CO2 in most aquatic
ecosystems is low compared with the concentrations
required within the cell to saturate ribulose biphosphate
carboxylase-oxygenase – the carboxylase responsible
for CO2 fixation in autotrophs. As a result, algae often
actively transport CO2 into the cell, concentrating it for
photosynthesis (Falkowski & Raven, 2007). Because
alga cells are governed by low Reynolds numbers
(Vogel, 1994), the boundary layer of water around the
cell can easily become depleted of CO2 during times of
high PP (Hein & Sand-Jensen, 1997).
In our experiment, the benthic algae assemblages
were dominated by diatoms that covered the surface
of the sand sediments in each mesocosm. Their dominant presence in these mesocoms likely resulted from
the relatively unstable sand sediments in the mesocosms that prevented large filamentous forms and kept
algae assemblages at early successional stages. Marine
and freshwater planktonic diatoms are highly productive with high CO2 affinities (Cushing, 1989; Falkowski
& Raven, 2007). This makes them susceptible to CO2
limitation within the boundary layer of water surrounding the cell (Falkowski & Raven, 2007). Not surprisingly,
research has demonstrated that CO2 enrichment in the
water column enhances diatom productivity and biovolume (Riebesell et al., 1993). Our data support these
trends, suggesting that the diatom assemblages in our
mesocosms likely experienced differential degrees of
CO2 limitation at the sediment–water interface between
the two CO2 treatments. Thus, eCO2 likely increased
CO2 diffusion across this boundary layer, enhancing
individual CO2 uptake and rates of PP. Diatoms are
common taxa in streams in early stages of succession
and with high disturbance frequencies (e.g., sand bottom streams, grazed streams, etc.). Thus, the eCO2
2787
effects discussed here are plausible across the landscape. However, it is necessary to directly test the CO2
affinities of various stream algae to thoroughly identify
the exact mechanisms driving the response of benthic
algae to a changing climate.
In addition to the potential CO2 limitation experienced at the fine, boundary-layer scale, our data suggest
that CO2 also was limiting at the larger, local scale (i.e.,
within each mesocosm). This was evident by the consistent increase in water column pH and subsequent
decrease in CO2(aq) in both CO2 treatments throughout
this experiment. Primary productivity of the benthic
algae in these mesocosms drove down CO2(aq); however, the supply of CO2 to the water surface at twice the
ambient rate in the eCO2 treatment caused a significant
increased the CO2(aq) in the water column, reducing
overall CO2 limitation and stimulating PP at the larger
local scale. These dynamics were in part influenced by
artificial factors inherent to our mesocosms, e.g., recirculating flow, lack of nutrient exports, lack of ground water
inputs etc. Despite these limitations, we believe that the
eCO2 effect on PP observed in our experiment can scale
up to natural streams under some specific contexts.
The contexts that are likely to promote CO2 limitation
in natural streams include low canopy cover or high
solar irradiance (high PAR), and seasons when water is
warm, flow is low, and PP is high. Streams throughout
the Great Plains exhibit these characteristics during
summer and autumn months (Lamberti & Steinman,
1997). The South Canadian River (Oklahoma), for example, is a shallow, sand-bottom, prairie stream. It is
dominated by a diatom algae assemblage and likely
experiences local CO2 limitation during periods of intense PP and low flow. Matthews (1976) sampled physicochemical properties in a 600 m reach of this river at
about 15 min intervals across five time periods during a
single year. Average daily pH of the water column was
about 9.0 during August and October and about 8.3
during the rest of the year. The high pH in this river
suggests strong CO2 limitation during these time periods, and provides strong support that the physicochemical conditions in our experimental mesocosms exist, at
least temporarily, in some prairie streams. Likewise,
Finlay et al. (1999) showed that several streams in
California can experience periods of high pH (8.7 at
day vs. 7.7 at night) and low CO2(aq) (9.7 mM at day vs.
19.9 mM at night). Using C13/C12 ratios, Finlay et al.
(1999) also showed that current velocity within and
across streams can influence degree of CO2 limitation.
Thus, conditions in our mesocosms were not unrealistic,
and likely reflect summer conditions of many prairie
rivers (e.g., Matthews, 1976), as well as along shorelines,
pools, and backwater habitats in other rivers throughout the terrestrial landscape (Finlay, 2004).
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2788 C . W . H A R G R AV E et al.
In terrestrial ecosystems, eCO2 can shift elemental
ratios of primary producers by stimulating C-assimilation relative to nutrient uptake (Curtis et al., 1996).
Because algae nutrient ratios often reflect that of their
environment (Sterner & Elser, 2002), we predicted that
eCO2 would reduce benthic algae quality by increasing
C : P and C : N. In our experiment, water-column nutrients were likely maintained by a constant flux of
terrestrial insects into the mesocosms, which did not
differ between CO2 treatments. Moreover, the lack of
buildup of nutrients over time in this experiment suggests that P and N were being assimilated into the
benthic biota relatively quickly and potentially leaving
the mesocosms with insect emergence or denitrification
processes. The increase in C : P of the benthic algae in
the eCO2 treatment supports our predictions, suggesting that algae quality was reduced because of greater C
assimilation in mesocosms with more CO2(aq). Our data
contribute to results from terrestrial, marine, and lake
ecosystems that also have demonstrated that C : P and
C : N of autotrophs can vary with atmospheric CO2
concentration (Andersen & Andersen, 2006; Levitan
et al., 2007). Further, the extremely high C : P measured
in this study indicated that the benthic algae were
‘severely’ nutrient deficient (Healey & Hendzel, 1980),
and thus we believe eCO2 has high potential to affect
quality of autochthonous food sources in stream ecosystems. Because secondary production in consumers
often is linked the quality of food resources (Sterner &
Hessen, 1994), our experiment broadens the hypothesis
that eCO2 might affect secondary production in food
webs by altering basal resource quality (Sterner & Elser,
2002).
With respect to the above hypothesis, we predicted
that reduced algae quality in response to eCO2 would
negatively affect grazers. Our data did not support this
prediction. Rather, benthic invertebrate density, biomass, and average individual size were greater in the
eCO2 treatment than in the aCO2 treatment. Thus,
benthic invertebrates apparently were decoupled from
food resource quality in this experiment. One possible
explanation is that grazers increased foraging rates in
response to reduced food quality (e.g., Raubenheimer,
1992). The increase in grazing rate by these benthic
invertebrates could explain why benthic algae biomass
did not accumulate despite the greater PP in the eCO2
treatment. Contrary to the dominating hypotheses in
the literature, our study suggests that eCO2 might have
positive, bottom-up effects on secondary production in
some stream food webs.
The benthic invertebrate assemblages were dominated by chironomids, and assemblages were simplistic
compared with those from natural streams (Hershey &
Lamberti, 2001). This likely was because of the lack of
substrate heterogeneity used in our mesocosms and the
lack of other structure such as root wads, large leaf
packs, rocks, etc. However, chironomids are highly
ubiquitous and often are numerically dominant in
stream ecosystems worldwide. Chironomids forage on
a variety of food resources, including algae (Hershey &
Lamberti, 2001). Therefore, chironomid abundance and
biomass can be linked to autochthonous production in
many stream ecosystems. For example, we have documented the positive response in benthic chironomid
density, biomass, and size to increased PP via nutrient
stimulation in other studies in these mesocosms (C. W.
Hargrave, unpublished results). Thus, we feel that,
although simple, the invertebrate assemblages in these
mesocosm provide a realistic indication of the potential
effects of eCO2 on grazer dynamics in other stream
ecosystems.
It is generally accepted that at the landscape scale
stream ecosystems are a net source of CO2 to the atmosphere, and, therefore, likely are not C-limited (Worrall
& Burt, 2005; Cole et al., 2007). However, we argue that
at the very fine, boundary layer scale and the larger,
local scale (i.e., within reaches, pools, or small backwaters), benthic algae can be highly productive, reducing the CO2 pool, and temporarily experience Climitation (Power, 1992; Finlay, 2001). Thus, C-limitation
is likely to interact with temperature, flow, and PP
within habitats in a stream reach (Finlay et al., 1999;
Finlay, 2004). Moreover, longitudinal location of the
stream within the watershed also can affect CO2 availability. For example, algae in high-order streams with
large watersheds and depleted CO2 levels tend to show
evidence of CO2 limitation (Finlay, 2003). These documented patterns from the literature combined with our
mesocosm data suggest that eCO2 might have strong
bottom-up effects on ecosystem-dynamics at local scales
in many streams (Madsen & Maberly, 1991). We believe
the effects of eCO2 are likely going to be most important
in streams with high PAR, warm water, low flows, high
PP, and with algae assemblages dominated by species
with high CO2 affinities. Thus, this study and the large
literature from terrestrial and marine ecosystems suggests that future atmospheric CO2 concentrations are
likely to have broad reaching effects on autotrophs and
consumers across terrestrial and aquatic biomes.
Acknowledgements
We thank R. Deaton and M. Rowe for critically reviewing this
manuscript, members of the Hargrave-Deaton lab group for
critical discussion of this project, and L. Shoemaker for assistance
in setting up the experiment, fabrication of CO2 emitters and
moving CO2 cylinders throughout the study. Funding for this
research was provided by a faculty research grant awarded to C.
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E L E VA T E D A T M O S P H E R I C C O 2 A F F E C T S S T R E A M S
Hargrave by Office of Research and Sponsored Programs at Sam
Houston State University.
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