Environmental Management (2012) 50:10351046
DOI 10.1007/s00267-012-9936-0
Epilithic Community Metabolism as an Indicator of Impact
and Recovery in Streams Affected by Acid Mine Drainage
Dean M. DeNicola Lee Layton Tiffaney R. Czapski
Received: 8 February 2012 / Accepted: 2 August 2012 / Published online: 9 September 2012
Springer Science+Business Media, LLC 2012
Abstract We measured biomass and metabolism of epi-
lithic communities on five dates in different seasons at four
sites in a watershed that has received extensive restoration
for acid mine drainage (AMD) through the construction of
passive treatment systems. Chlorophyll a biomass and
productivity directly corresponded to AMD stress from
coal mining. The site downstream of extensive passive
treatment had significantly greater biomass and gross pri-
mary productivity rates than the site receiving only
untreated AMD, but values were below those for two ref-
erence sites, indicating incomplete recovery. The degree of
difference in these metrics among sites varied seasonally,
primarily related to differences in canopy cover changes,
but the ranking of sites in terms of stress generally was
consistent. Reference sites had a significantly greater
chlorophyll a/pheophytin ratio than untreated and treated
sites, also indicating AMD stressed the communities.
Community respiration was less affected by AMD stress
than productivity or chlorophyll a. Productivity measures
are not widely used to assess AMD impacts, and have been
shown to both increase and decrease with AMD stress. The
elimination of herbivores in AMD-impacted streams can
increase productivity in the benthic algal community. Our
study found productivity decreased with increasing AMD
stress. Although sites with AMD stress had reduced her-
bivore populations, light, nutrients and metal precipitates
appear to have limited growth of AMD-tolerant algal taxa.
D. M. DeNicola (&)
L. Layton
Department of Biology, Slippery Rock University, 1 Morrow
Way, Slippery Rock, PA 16057, USA
e-mail: dean.denicola@sru.edu
T. R. Czapski
Department of Biology, Duquesne University, Pittsburgh, PA,
USA
Therefore, it appears changes in food web structure due to
AMD stress had less of an effect on epilithic productivity
than environmental conditions within the stream.
Keywords Mine drainage
Passive treatment

Periphyton
Primary productivity
Reclamation
Introduction
Worldwide, mining activities have greatly increased over
the last 100 years to meet global demands for energy and
minerals. In particular, mining of coal has caused major
changes in land use in Appalachian ecoregions and had
major impacts on streams through the generation of acid
mine drainage (AMD) (Herlihy and others 1990; Palmer
and others 2010; Petty and others 2010). Formation of
AMD has been described in detail (Younger and others
2002; Johnson and Hallberg 2005). Briefly, exposure of
pyrite and other sulphidic minerals to air and water results
in a series of both abiotic and biotic oxidation reactions.
The result creates both chemical and physical stresses on
stream biota. Chemical stresses are primarily related to
large increases in acidity and concentrations of toxic dis-
solved metals. Physical stresses are created when substrata
become covered with metal hydroxide precipitates (Hogs-
den and Harding 2012a). Many studies have shown that
microbes, benthic algae, macroinvertebrates and fish are all
greatly impacted by AMD input. However, assessment of
AMD impacts on the biological condition of streams has
been similar to assessment of streams in general, in that the
emphasis has been on measurement of structural attributes
of the community, rather than functional attributes (Davies
and Jackson 2006). Reductions in taxonomic richness,
diversity and other measures of taxonomic structure appear
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to be sensitive indicators of AMD impacts (reviewed by
Hogsden and Harding 2012a). Despite their importance to
energy flow, element cycling and food web structure,
changes in ecosystem processes in streams affected by
AMD have received much less attention than structural
attributes (Niyogi and others 2002; Smucker and Vis 2011;
Hogsden and Harding 2012a). Although measurement of
benthic metabolism in streams contaminated with metals
has been shown to be as sensitive as acute toxicity tests
(Hill and others 1997), only five to six studies have
examined effects of AMD on primary productivity.
Niyogi and others (2002) proposed a hypothesis that
biodiversity metrics of benthic algae have a low threshold of
response to AMD stress, but biomass and functional mea-
sures (i.e., productivity) remain stable or increase under low
to moderate stress, and decrease only at high stress. In two
studies, biomass and productivity increased at low chemi-
cal-AMD stress primarily from the loss of sensitive, macr-
oinvertebrate herbivore populations (Crossey and La Point
1988; Niyogi and others 2002). Moreover, Niyogi and others
(2002) found high physical stress resulting from metal
precipitates decreased algal biomass and productivity, fur-
ther supporting the hypothesis. These studies were done in
open-canopy, mountain streams affected by hard rock
AMD, where a few AMD tolerant, highly productive, fila-
mentous-chlorophyte taxa dominated at low to moderate
chemical impacts in high light environments. There is some
evidence that physical stress has a greater effect than
chemical stress on periphyton in coal-associated, AMD
streams in Appalachia. However, these streams have dif-
ferent AMD chemistry, are usually more closed canopy, and
proliferation of filamentous chlorophytes is less common
(Verb and Vis 2001; Verb and Vis 2005). As a result, benthic
algal biomass at AMD sties is often low (ca. half that found
in Niyogi and others 2002), even when metal precipitate is
not extensive (Simmons and others 2005; Smucker and Vis
2011), and impacts are great enough to reduce macroin-
vertebrates (Simmons and others 2005). In these systems,
the relationship between algal productivity and AMD
stressors remains relatively unknown.
Remediation activities for streams affected by AMD are
occurring worldwide. Efforts to reduce the effects of AMD
on stream ecosystems involve either active or passive
treatment of the AMD source before it enters the stream.
Active treatment requires a continuous input of human
labor, energy and chemical reagents. The substantially
lower costs and maintenance of passive treatment, which
uses natural energy sources and materials, has made it the
preferred method for restoration. The specific type of
passive treatment system used depends on water chemistry
of the AMD source. Generally, passive treatment involves
raising pH and alkalinity with limestone, followed by
the precipitation and settling of metal compounds in an
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Environmental Management (2012) 50:10351046
oxidizing environment, such as a pond or on the surface of
a wetland (Younger and others 2002; Johnson and Hallberg
2005). Despite a large economic investment related to the
increasing use of passive treatment technology to restore
streams affected by AMD (PA DEP 2010), studies exam-
ining biological recovery are scarce, and to our knowledge
none have examined changes in primary productivity
related to treatment.
The objectives of this study were to examine the rela-
tionship between metabolism of epilithic communities and
AMD stress associated with coal mining, and to determine
how this functional aspect of the stream ecosystem has
responded to passive treatment. Specifically, we measured
biomass, community respiration (CR), net community
productivity (NCP) and gross primary productivity (GPP)
of epilithic communities at an untreated AMD site, a site
downstream of passive treatment, and two reference stream
sites on five dates at different seasons of the year. Based on
Niyogi and others (2002), our hypothesis was that biomass
and productivity would be lowest at the untreated AMD
site (moderate AMD chemical and physical stress), highest
at the treated AMD site (low to moderate chemical AMD
stress), with reference sites intermediate. Measurements of
canopy cover, water chemistry, and herbivore densities
were used to further explore mechanisms that underlie the
hypothesis. In addition, we examined the chlorophyll a/
pheophytin ratio as an indicator of algal health in the
communities. Pheophytin is a degradation product of
chlorophyll and is high in senescent cells (Steinman and
others 2007). The chlorophyll a/pheophytin ratio has been
found to decrease in benthic algal communities subjected
to high Fe precipitate (Sode 1983). As a result, we expected
this ratio to decrease with increased AMD stress.
Materials and Methods
Study Site
Slippery Rock Creek is a tributary within the upper Ohio
River Watershed that drains a 1,100 km2 area located in the
Western Allegheny Plateau ecoregion of Pennsylvania,
USA (Fig 1). The headwaters of the main stem (70 km2
watershed) have been severely impacted by AMD for more
than a century from activities associated with bituminous
coal mining. Streams are affected by more than 70 AMD
discharges that represented an average of 30 % of the total
flow out of the headwaters drainage, with most of the
impact coming from 35 discharges. The discharges range in
pH from 3.0 to 6.1 and in iron concentrations from 0.3 to
225 mg/L, and load approximately 1,289 kg/day of acidity
and 282 kg/day of iron into the stream network (PA DEP
1998). Since 1995, 15 passive treatment systems have been
Environmental Management (2012) 50:10351046 1037

Fig. 1 Map of the headwaters

restoration area of Slippery
Rock Creek. U, T and SR refer
to sample locations. Sample site
LR is not on the map and
located about 15.5 km west of
the headwaters area. Letters
AO correspond to locations of
treatment. Types of treatment
and date of construction: A land
reclamation 1995; B anoxic
limestone drain (ALD) 1995;
C aerobic wetland 1996;
D reducing alkalinity producing
system (RAPS) 1997; E two
RAPS 1997; F land reclamation
1998; G ALD 1998; H RAPS
1998; I two RAPS and one
horizontal flow limestone bed
(HFLB) 2000; J two RAPS and
one HFLB 2000; K four RAPS
2001; L oxic limestone drain
2001; M one RAPS and one
HFLB 2002; N three ALD and
one HFLB 2002; O two RAPS
2003

built in the watershed that treat approximately 30 AMD
discharges (approximately 2.8 9 106 m3/year) before they
enter the streams (PA DEP 1998). This represents about
10 % of the base flow at the bottom of the headwaters
drainage. The types of treatment systems included: aerobic
wetlands/ponds; anoxic limestone drains; reducing alka-
linity producing systems; horizontal flow limestone beds;
and oxic limestone drains (Fig. 1) (see reviews by Younger
and others 2002, and Johnson and Hallberg 2005 for details
on specific types of systems). Significant increases in
stream, flow-adjusted pH and alkalinity within the stream
network from 19962008 have indicated moderate
improvement in the ecosystem from treatment. In addition,
benthic algal community composition at small-order
stream sites below treatment in the watershed, have
become more similar to their corresponding reference site.
Although flow-adjusted aqueous metal concentrations have
generally declined slightly below treatment, the trend is
inconsistent, and not significant (D.M. DeNicola and M.G.
Stapleton, personal communication). Macroinvertebrate,
herbivore-densities at sites below treatment have remained
substantially lower than at reference sites (DeNicola and
Stapleton 1999, 2002a).
Sampling and Productivity Measurements
Biomass and metabolism of epilithic communities were
measured at four sites (Fig. 1). One site was at the head-
waters of the main stem of Slippery Rock Creek. It is
upstream of all treatment (U), and receives untreated
AMD. One site was located on a third order section of the
main stem, downstream of eight treatment systems (T).
Two sites were unimpacted reference streams of different
sizes. The first was a first order tributary (SR) on the main
stem within the watershed. The other was a large-order
reference stream site (LR), Wolf Creek, a fourth order
stream within the Slippery Rock Creek watershed but not
in the headwaters area. It served as the large reference
stream because there are no larger order streams within the
headwaters area that are not affected by AMD. Water
chemistry and epilithic communities at three of the sites
(U, T and LR) were sampled in October (fall) 2005. All
four sites were sampled in April (spring) 2009, August
(summer) 2009, October (fall) 2009, and April (spring)
2010. Stream water temperature, pH, total alkalinity, and
total acidity (titration endpoints of 4.5 and 8.3, respec-
tively) were determined at the sites on each date using
standard methods (American Public Health Association
1998). Total P was determined colorimetrically using the
molybdate reaction (Lind 1985). Nitrate was determined
using a Lazar Model 43, NO3 electrode (Lazar Research
Laboratories Inc., Los Angeles, CA). Terrestrial canopy
cover, as percent sky, was determined for each season at
the sites using a LI-COR, LAI-2000 plant canopy analyzer
(LI-COR, Lincoln, NE) (DeNicola and others 1992). These
four sites are part of a larger 12-year study of recovery in
the watershed, and other water chemistry parameters
determined from samples taken in spring 2005 and spring
2008 (the dates that most closely correspond to the dates
of metabolism measurements) were used to further

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characterize environmental conditions at them. On these
two dates, conductivity and sulfate were measured fol-
lowing standard methods (American Public Health Asso-
ciation 1998). Digested water samples were measured for
total Fe and Mn, the major metals that impact these sites,
using a Perkin Elmer 400 inductively coupled plasma
spectrophotometer (Perkin Elmer, Waltham, MA) (Amer-
ican Public Health Association 1998). Macroinvertebrate,
herbivore densities were determined by three replicate
Surber samples taken at each of the four sites in spring
2005 and summer 2007. Taxa were identified to genus and
classified as herbivores (scrapers) using Merritt and others
(2008).
To measure epilithic metabolism, 1820 rocks were
collected at random from riffle areas at each sample site on
each of the five dates, and transferred submerged in stream
water in coolers to the laboratory within 1 h of collection.
Four to six rocks were placed in recirculating chambers
with stream water for metabolic measurements, with three
replicate chambers for each site. In addition, a stream water
only chamber was used as a control blank. Chambers were
34 9 21.5 9 11 cm (5.1 L volume) clear plexiglass boxes
with a center divider containing openings at each end.
Water was circulated around the periphery of the sealed
chambers (i.e., no air space) by a Mini-jet, model 404
submersible pump (Marineland Supplies, Los Angeles,
CA). One to 15 h incubations in both the light and dark
were done in a Percival model 1-35 LLVL growth chamber
(Percival, Perry, IA) with full spectrum lights. Stream
water temperature was similar at each site on each sample
date, and each incubation chamber remained within 23 C
of ambient stream temperature. The photon flux density
(PFD) for all light incubations was 110 lmol/m2/s. Thus
production values in the light are a standardized estimate of
productivity of the epilithic communities rather than in situ
production under ambient light conditions. Differences in
environmental conditions among communities should
affect production standardized for light, because they are
reflected in community biomass and species composition
(DeNicola and others 2003; Vadeboncoeur and others
2008). A PFD of 110 lmol/m2/s is generally below satu-
ration level for periphyton photosynthesis, but higher than
intensities often found at the sample sites with high canopy
cover (D.M. DeNicola, personal communication). Changes
in dissolved oxygen were measured 23 times during each
incubation using an Orion model three Star dissolved
oxygen meter (Thermo Scientific, Beverly, MA), with an
auto-stir, polarographic probe inserted into a resealable
port of the chamber. Incubations in the light were ended
before oxygen saturation was reached. Following oxygen
readings for dark and light incubations, the surface area of
the rocks, water volume of the chamber, and oxygen
changes in the control chambers were used to calculate
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Environmental Management (2012) 50:10351046
changes in dissolved oxygen m2/h for the epilithic com-
munities in each chamber. Net community productivity
was determined from production of oxygen in the light.
Community respiration was measured as the consumption
of oxygen in the dark. Gross primary productivity was
equal to NCP ? CR. Metabolism was not determined at
site U in spring 2009 because of leaks in two of the
chambers.
Following metabolism measurements, epilithic algae for
chlorophyll a analysis was scraped from a known area
(5.3 cm2) of each rock in each chamber in fall 2005 and
spring 2009, and from the whole upper surface of the rocks
in summer 2009, fall 2009 and spring 2010. Scraped algae
was filtered onto glass fiber filters, and pigments extracted
in 90 % buffered acetone overnight at 4 C. Chlorophyll
a and pheophytin concentrations were determined spec-
trophotometrically by measuring absorbance before and
after acidification (Steinman and others 2007).
Data Analysis
Data from fall 2005 were analyzed separately from the
20092010 data because there were a different number of
sites. Site differences in mean chlorophyll a, CR, NCP, and
GPP for fall 2005 were analyzed by 1-factor Analysis of
Variance (ANOVA), respectively. Parameters for the 4,
20092010 sample dates were analyzed by 2-factor
ANOVA with site and sample date as the factors. To
compare the 2005 to the 20092010 results, fall 2005 and
fall 2009 values for U, T and LR for the above parameters
were analyzed as a 2-factor ANOVA with site and sample
year as the factors. Means of the chlorophyll a/pheophytin
ratio were analyzed by 2-factor ANOVA, but only for last
three sample dates. Pheophytin measurements for the first
two sample dates were inconsistent because of too small of
an epilithon sample was scraped on those dates. In all
cases, if the main factor effects were significant, posteriori,
pair-wise comparison of means was done using Fishers
Protected Least Significant Differences (FPLSD). For all
tests, the level of significance was P \ 0.05. When needed,
transformations were used to normalize the data prior to
ANOVA. All statistical analyses were done using Systat
(Systat Software, San Jose, CA).
Results
Stream water temperatures ranged from 8 C in the spring
to 21 C in the summer. Median stream pH was lowest at U
and highest in LR (Table 1). The major aqueous metals in
the streams, total Fe and Mn, were highest at U (Table 2).
This also was the only site to have a visible coating of
metal precipitate covering the substrata (ca. 23 mm thick,
Environmental Management (2012) 50:10351046 1039

Table 1 Water temperature, percent sky, pH, alkalinity and acidity at the sample sites at the time of metabolism measurements
Site Date Water temp (C) Percent sky pH Alkalinity (mg/L CaCO3) Acidity (mg/L CaCO3)

Untreated Fall 05 10 nd 6.0 9.2 10.6

Sp 09 8 49 6.4 8.8 6.6
Sum 09 17 5 7.2 4.0 5.7
Fall 09 11 20 7.0 8.0 8.1
Sp 10 14 nd 6.4 13.0 5.0
Mean 25 6.4* 8.6 7.2
Small reference Fall 05 nd nd nd nd nd
Sp 09 8 44 5.9 5.5 6.9
Sum 09 17 1 7.6 7.5 3.3
Fall 09 11 39 7.5 3.3 3.0
Sp 10 14 nd 6.8 7.0 8.3
Mean 28 7.2* 5.8 5.4
Treated Fall 05 10 nd 6.1 3.0 17.0
Sp 09 10 81 6.6 6.9 8.3
Sum 09 21 96 7.1 21.2 7.2
Fall 09 13 nd 7.2 6.1 3.7
Sp 10 15 nd 6.9 7.0 8.3
Mean 88.3 7.0* 8.8 8.9
Large reference Fall 05 13 nd 8.0 70.0 1.6
Sp 09 10.0 59 7.8 60.0 4.7
Sum 09 21.0 44 8.3 125.0 19.0
Fall 09 13.0 nd 8.2 130.0 1.2
Sp 10 15.0 nd 8.8 68.0 0.0
Mean 51 8.2* 90.6 5.3
* Median, nd no data

Table 2 Other water chemistry parameters of the sites based on spring 2005 and spring 2008 samples
Untreated Small reference Treated Large reference
2005 2008* 2005 2008* 2005 2008* 2005 2008*

Total Fe (mg/L) 1.4 1.7 0.4 0.2 0.7 0.6 0.1 0.4
Total Mn (mg/L) 1.0 1.2 0.1 0.2 0.6 0.6 \0.1 \0.1
SO4 (mg/L) 80 58 32 42 104 80 60 40
Conductivity (mS/cm) 0.3 0.2 0.1 0.1 0.6 0.2 0.5 0.3
Herbivore density (individuals/m2) 4 (0.3) 14 (0.3) 72 (2) 166 (4) 29 (2) 14 (0.3) 148 (2) 806 (30)
NO3 (mg/L) 0.69 0.67 0.71 0.43 0.34 0.59 1.00 1.55
Total P (lg/L) 1.1 6.8 3.0 3.0 3.0 4.9 83.7 21.8
Mean (1 SE) given for macroinvertebrate herbivore densities (n = 3)
*Summer 2007 for herbivore data

primarily Fe(OH)3). Total Al was similar at all sites and
was generally less than 1 mg/L. Acidity and SO4 (an
indicator of AMD but not reduced by treatment) concen-
trations were highest at site U and T. Conductivity was
lowest at SR. High conductivity at U and T was due pri-
marily to SO4-, whereas higher conductivity at LR was
most likely due to NO3- and Cl-. Alkalinity was much
higher in LR, which has limestone bedrock in the upper
strata of its basin. This site also has more agricultural
inputs, resulting in much higher nutrient concentrations
than the other sites (Tables 1, 2). T and LR are higher order
streams, and thus have a more open canopy on average
than U and SR, particularly in summer. At the latter two
sites, percent sky was \5 % in the summer when the
canopy was closed, and 2049 % in the fall and spring
when leaves were off the trees (Table 1). On average,

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macroinvertebrate, herbivore-densities were about an order
of magnitude greater at the two reference sites than for the
untreated and treated sites (Table 2).
Chlorophyll a concentrations were significantly differ-
ent among the three sites sampled in fall 2005. Mean
concentrations were highest at LR, followed by T, with
lowest concentrations at site U (Fig. 2). Post comparison
tests indicated all three sites were significantly different
from each other. For the 20092010 samples, chlorophyll
a concentration was significantly affected by site, and there
was a significant site by sample date interaction. Across all
dates, mean concentrations in LR were about an order of
magnitude higher, and significantly greater, than for sites
U, T and SR (Fig. 2). This difference was smaller in spring
2009, when the canopy was more open at U and SR
(Table 1), which resulted in the significant interaction
term. Across all 20092010 sample dates, SR had a sig-
nificantly greater mean chlorophyll a concentration than
both sites T and U. Comparison of fall 2005 chlorophyll
a concentrations with fall 2009 concentrations for LR, T
and U, found a significant site by year interaction, driven
primarily by the difference at the LR between the 2 years.
However, there were no obvious differences in the mea-
sured environmental parameters between the 2 years at LR
(Tables 1, 2).
There was a significant site effect for the 2005 samples
in CR, NCP and GPP, respectively. Site LR had signifi-
cantly higher NCP and GPP than the other sites.
Fig. 2 Mean chlorophyll concentrations (?1 SE) for epilithic
communities at the untreated (U), treated (T), small reference (SR)
and large reference (LR) sample sites on the five sample dates. Sites
that are significantly different based on posteriori, pair-wise tests
(FPLSD P \ 0.05) are indicated by different letters. Sites with the
same letter are not significantly different. For the 20092010 samples,
these comparisons are indicated on the spring 2009 graph. nd no data
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Environmental Management (2012) 50:10351046
Community respiration was more similar among sites. NCP
was significantly lower at U than T. NCP was negative at
site U, indicating a heterotrophic community (Fig. 3).
There were significant differences among the four sites
for the 20092010 samples in epilithic CR, NCP and GPP,
with significant site by date interactions for CR and GPP
(Fig. 3). Across all dates, the three types of metabolic rates
were all substantially higher, and significantly different, at
LR than the other sites. The interaction with date resulted
from differences from LR being lower in summer 2009 and
in spring 2010. This corresponded to water temperatures
being relatively higher on these dates at SR and U
(Table 1). The NCP and GPP rates were significantly lower
at U than T and SR. As in the 2005 samples, NCP was
negative at site U, indicating a heterotrophic community.
Values for CR were more similar among these three sites,
and not significantly different. Date had a significant main-
factor effect only for CR, which was significantly highest
(more negative change in dissolved oxygen) in spring 2010
compared to all other dates. Comparison of fall 2005
metabolic rates with fall 2009 rates for sites LR, T and U,
found only GPP was significantly different between the
2 years. While the trends among sites were similar for the
2 years, GPP was slightly higher overall in 2009 than 2005
(Fig. 3). This corresponded to slightly higher pH and
alkalinity in fall 2009 at most of the sites (Table 1).
The chlorophyll a/phaeophytin ratio for the summer
2009-spring 2010 samples was significantly affected by site
and date with a nonsignificant interaction term. Across
dates, both reference sites had significantly higher ratios
than T and U (Fig. 4). On two of the three dates, the ratio at
U was higher than for site T. Across all sites, the ratio was
significantly lower in summer 2009 than spring 2010.
Discussion
Comparison of Metabolism Rates to Other Studies
A wide variety of methods have been used to measure
primary productivity in epilithic communities in streams,
which can influence measurement values, and make com-
parisons among studies difficult (Bott and others 1985).
Given that, the NCP values in this study cover the range
reported for streams in deciduous biome studies, with site
LR being comparable to productive, open-canopy streams.
Site SR was at the higher end for small-order, heavily
shaded streams, and U was near the lowest of all reported
values (Allan 1995). The range of values of GPP reported
for mountain streams affected by hardrock AMD (ca.
50300 mg O2/m2/h) are generally higher than for those
found in this study (Crossey and La Point 1988; Hill and
others 1997; Niyogi and others 2002). Higher values in
Environmental Management (2012) 50:10351046 1041

Fig. 3 Mean rates for epilithic

community respiration (CR), net
community productivity (NCP)
and gross primary productivity
(GPP) (?1 SE) at sites U, T, SR
and LR on the five sample dates.
Sites that are significantly
different based on posteriori,
pair-wise tests (FPLSD
P \ 0.05) for each of the three
metabolism measurements,
respectively, are indicated by
different letters. For the
20092010 samples, these
comparisons are indicated on
the spring 2010 graph.
Posteriori, pair-wise tests for
dates indicated CR in spring
2010 was higher than all other
dates (FPLSD P \ 0.05). nd no
data

many of those streams probably resulted from little to no
canopy cover, and a high biomass of filamentous chloro-
phytes (see below). In study sites more comparable to ours,
Bauers (2003) used whole-stream methods to measure
productivity in small-order, forested streams in Ohio
impacted by coal-mining AMD. GPP rates at her most
impacted sites were similar to rates at U in our study (ca.
26 mg O2/m2/h), and rates at her most productive refer-
ence stream were similar to the values for SR and T in our
study (ca. 40 mg O2/m2/h).
Factors not necessarily associated with photosynthesis
and respiration may affect measurements of metabolism
based on changes in dissolved oxygen in communities
impacted by AMD. For example, both chemical oxygen
demand, and Fe-oxidizing bacteria associated with mi-
crozones of reduced Fe, may increase oxygen consumption
in communities with high concentrations of Fe (Sode
1983). Additionally, photoreduction of metal oxides may
occur at high light intensities (Hrncir and McKnight 1998).
The degree to which these may affect the accuracy of
metabolic measurements based on changes in dissolved
oxygen concentrations needs to be assessed.
Effects of AMD and Treatment on Biomass
and Metabolism
Most of the AMD sources in the watershed have a pH of
four or less, but in some cases, such as at U, the pH of the
source can be higher, probably because the AMD discharge
contacts a geologic formation containing limestone (PA
DEP 1998). As a result, the major impact at U was metal
precipitates on the substrata (see below). Benthic algal
biomass and epilithic productivity were lower at U than all
our other sites. The negative NCP values at U on every
sample date indicate it was a heterotrophic community,
where the energy fixed by autotrophs did not meet the
respiratory demands. Crossey and La Point (1988) found
the GPP to CR ratio decreased with increasing AMD stress,
which can ultimately lead to a heterotrophic community.
Our study suggests AMD stress may have a greater effect
on photosynthetic processes than respiratory processes, as
CR was not significantly different among SR, T and U.
Similarly, Tlili and others (2011) found photosynthetic
efficiency was more affected by metal contamination stress
than respiration of heterotrophs in a stream biofilm. A

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Fig. 4 The chlorophyll a/pheophytin ratio (?1 SE) for epilithic
communities at U, T, SR and LR for three sample dates. Sites that are
significantly different based on posteriori, pair-wise tests (FPLSD
P \ 0.05) are indicated by different letters on the spring 2010 graph.
Posteriori, pair-wise tests for dates indicated the ratio was signifi-
cantly lower in summer 2009 than spring 2010 (FPLSD P \ 0.05)
potential explanation is that metal precipitate associated
with AMD physical stress reduces light availability for
photosynthesis, whereas organic matter energy sources for
heterotrophic bacterial and fungal respiration in the com-
munity may not be as affected. However, heterotrophic
microbial respiration was found to decline with metal
precipitate in stream affected by hard rock AMD (Niyogi
and others 2001).
Chlorophyll a, NCP and GPP in the epilithon were lower
on every date at U than at SR, which is similar in size and
canopy cover. Although herbivore densities were lower at
U compared to SR (Table 1), less herbivory did not com-
pensate for the negative effects of AMD on the epilithon at
U. High AMD stress can exacerbate nutrient limitation of
algae because Fe and Al compounds can bind P (Smucker
and Vis 2009). In addition, dissolved inorganic carbon may
be lowered in heavily impacted AMD streams (Hogsden
and Harding 2012a). However, alkalinity, pH, acidity, and
TP were all similar for sites U and SR. While total Fe, Mn
and Al concentrations at the U and T sites can exceed EPA
criteria for aquatic life at times (USEPA 2005), other
studies have shown that periphyton biomass is not neces-
sarily impacted at similar conductivities and concentrations
of metals from coal mine drainage when the community is
not buried by thick precipitates (Verb and Vis 2000;
Hamsher and others 2004; Bray and others 2008). In those
studies, pH at untreated sites was usually much lower than
at U, with most of the metals presumably in a dissolved
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Environmental Management (2012) 50:10351046
form, and thus there was high chemical but not physical
stress. In addition, affects of dissolved Fe and Mn on
periphyton community structure occur at relatively high
concentrations (Wellnitz and Sheldon 1995; Guasch and
others 2012), near or below our mean total, aqueous con-
centrations. The pH at U was always above 6.0 during the
study period, and therefore under oxidizing conditions in
the stream water, more of the Fe and Mn existed as pre-
cipitates based on equilibria of the solid phase, rather than
in dissolved form (Stumm and Morgan 1996). There was a
substantial coating of orange precipitate on the substrata
(ca. 23 mm) at U and it is likely that resuspension of
precipitate is what raised total Fe and Mn in the water
column at this site. This suggests the primary AMD impact
on epilithic metabolism at site U in our study was physical.
Metal precipitates have been found to greatly lower
benthic algal biomass in several studies (McKnight and
Feder 1984; Sode 1983; Wellnitz and Sheldon 1995;
Smucker and Vis 2011). While metal oxides can bury cells
and reduce light, Niyogi and others (1999) and Niyogi and
others (2002) found some filamentous chlorophytes were
able to proliferate in iron-oxide dominated deposition, and
thus algal biomass and productivity remained high
(although productivity declined at very high metal depo-
sition rates). High concentrations of dissolved metals from
hard rock mining greatly reduced grazer populations in
these streams, further stimulating algal productivity.
However, in areas with aluminum oxide deposition, algal
biomass and productivity were always low in these studies
(and in Smucker and Vis 2011), presumably because Al is
more toxic to algae than Fe (Genter 1996). In a previous
study, we found the concentration of Fe in the precipitate at
site U to be about an order of magnitude higher than Al
(DeNicola and Stapleton 2002b). In 12 years of monitor-
ing, we have found that the epilithic community at U was
dominated by a few cyanobacteria and acidobiontic diatom
species, and have never found high algal biomass at this
site (DeNicola and Stapleton 1999). Blooms of filamentous
chlorophytes greatly increase in abundance following
canopy removal (Hansmann and Phinney 1972) and may be
restricted to environments with high irradiance (Lowe and
others 1986). Additionally, some taxa are genetically tol-
erant of metals (Reed and Gadd 1989). The much more
open canopy of the high elevation, moderately AMD-
impacted streams in Niyogi and others (1999), along with
reduced herbivory, favored extensive growth of tolerant
filamentous chlorophytes, even with moderate Fe oxide
precipitate. The relatively greater canopy cover at the
untreated AMD site in this study may have prevented such
growth, and thus epilithic biomass and productivity were
very low, despite a near absence of herbivores. Iron pre-
cipitate on the substrata also may have contributed to
lowering light at U, but its rate and depth were low
Environmental Management (2012) 50:10351046
compared to some high productively sites in Niyogi and
others (2002) (DeNicola and Stapleton 2002b), suggesting
that it played less of a role than canopy cover in limiting
epilithic biomass and productivity.
Epilithic biomass and metabolic rates were much greater
at our large reference site than all other sites. Given that there
were no higher order stream sites that were not affected by
AMD in the headwaters, LR was located on a tributary fur-
ther downstream within the Slippery Rock Creek watershed.
This stream is substantially different than the other streams
of the headwaters in terms of geology and landuse. The high
alkalinity and pH at LR results from the streambed inter-
secting a limestone formation, which stratigraphically lies
below the streams in the headwaters area, and does not
influence their water chemistry. In addition, much of the
landuse in the LR drainage is agriculture, which results in
substantially higher nutrient concentrations compared to the
other sample sites. The high nutrient concentrations and a
relatively open canopy at this site resulted in a very pro-
ductive epilithic community, characterized by a thick mat of
diatoms with an overstory of filamentous chlorophytes.
Although the AMD treated site, T, was similar in canopy
cover to LR, epilithic biomass and productivity were much
lower. As mentioned above, much higher nutrient concen-
trations at LR drove the high productivity at that site.
However, biomass and productivity at T were also lower
than at SR, which had similar nutrient concentrations but a
much more closed canopy, as well as substantially higher
macroinvertebrate densities. While there was no visible
metal oxide precipitate on the substrata at T, the total Fe and
Mn concentrations were slightly higher at T than that of SR.
Given the eH and pH, the solubility of Fe and Mn would
have been low, which suggests there may have been a small
amount of metal precipitate that reduced algal productivity
at the treated site relative to SR. Although water chemistry
did not vary much at site T on different sample dates in this
study, other studies have shown more chemical variability in
streams receiving passive treatment for AMD (Petty and
Barker 2004; Verb and Vis 2000). In 11 years of monitoring
seven metals at these sites, we have found mean concen-
trations were below EPA criteria for aquatic life (DeNicola
and Stapleton 1999), but some, especially Zn, can exceed
levels that affect periphyton community structure (Tlili and
others 2011). As a result, there also may have been episodic
events of AMD contamination associated with storm events,
which along with low nutrients, limited epilithic biomass
and productivity at the treated site. Productivity at T was
significantly higher than at U, however, indicating passive
treatment reduced AMD impacts. The major change at site T
since treatment began has been mainly an increase in pH and
alkalinity. There was no visible precipitate at the initiation
of, or following treatment at this site, but total aqueous Fe
and Mn have decreased slightly since treatment began
1043
(DeNicola and Stapleton, personal communication). It is
likely the combination of less chemical and physical AMD
stress, and a more open canopy, resulted in the higher pro-
ductivity at T than U.
Chlorophyll a/Pheophytin Ratios
The chlorophyll a/pheophytin ratio was significantly lower
at U and T than for the two reference sites, indicating AMD
stress increased algal senescence in the epilithic commu-
nity. Sode (1983) found Fe(OH3) precipitation can result in
more degradation product. Since pheophytin increases in
algal cells when light quantity decreases (Guasch and Sa-
bater 1998), burial by metal precipitates should lower the
chlorophyll/pheophytin ratio. As a result, we expected the
ratio to be lowest at site U because it had the most pre-
cipitate. However, the ratio was higher at U than T in the
fall and spring, when the canopy at U was more open. It
could be the very low biomass at U in combination with
seasonally, relatively high irradiance, decreased self-shad-
ing, and shading associated with Fe precipitates, within the
algal biofilm at this site. The use of this ratio to assess
AMD requires further exploration. It appears as if the
chlorophyll/pheophytin ratio may be sensitive to physical
AMD stress (burial by precipitates reduces light), but its
relationship to chemical AMD stress remains unclear.
Structural Versus Functional Measures of AMD Stress
Stream ecologists have long recognized that both structural
and functional aspects of stream ecosystems can be used as
indicators of environmental impacts, and to assess recovery
(Warren 1971). Measures of community structure are much
more widely used in biomonitoring programs and some
have argued they are more sensitive because species
replacements within the community due to environmental
changes may not always correspond to functional changes
(Odum 1985; Niyogi and others 2002). Others argue that
functional attributes are more useful indicators of change
because they are not constrained by regional biota, and
have less spatial and temporal variation (Hill and others
1997; Brooks and others 2002). Corcoll and others (2012)
compared both structural and functional measures of
periphyton in response to metal contaminants. They found
initial exposure mostly affected function, but structural
metrics were more sensitive indicators of chronic impacts.
In streams affected by AMD, changes in algal community
structure have been shown to be good indicators of impacts
and recovery due to treatment (e.g., Verb and Vis 2005;
Zalack and others 2010), although knowledge of species
autecology is key to interpreting effects on community
structure. The objective of our current study was to
determine whether functional aspects of the epilithic
123
1044
community also could be used as indicators of AMD stress
and of recovery associated with passive treatment.
We found biomass and community metabolism were
negatively affected by AMD stress. In general, these metrics
were highest in reference sites, lowest at the untreated site,
and intermediate at the site below passive treatment, and
thus were good indicators of the amount of stress. Spatial
differences among sites were generally much greater than
temporal differences. Significant site by date interactions for
chlorophyll a, CR and GPP showed that the magnitude of the
differences among sites differed temporally, although the
overall rank among sites was consistent. Water chemistry
did not vary much within each site for the different sample
dates, and significant interactions between site and date for
epilithic biomass and metabolism were more related to
seasonal changes in canopy cover. In particular, epilithic
biomass and metabolism were generally higher in spring
samples at small-order sites when the canopy was relatively
more open, even though water temperatures were colder.
Such effects of canopy cover would be a confounding factor
that needs to be considered when attempting to use benthic
productivity or biomass to assess AMD.
Other studies similarly have found a direct, negative
correspondence between AMD stress and biomass (Sode
1983; Vinyard 1996; Hill and others 2000; this study).
Many, however, have found either increases in biomass with
AMD impacts (Crossey and La Point 1988; Niyogi and
others 2002; Bray and others 2008), a nonlinear response
(Smucker and Vis 2011), or no differences among untreated,
reference and treated sites (Verb and Vis 2000; Simmons
and others 2005; Verb and Vis 2005). Though there are
fewer studies examining benthic algal metabolism in AMD
stressed streams, similar conflicting results are apparent
(Bauers 2003; Crossey and La Point 1988; Hill and others
1997; Niyogi and others 2002; Sode 1983; Tease and Coler
1984; this study). Such conflicting responses of productivity
to stress are more likely to be created by toxic substances
(such as AMD effects) than those related to increased
nutrients (e.g., eutrophication) (Warren 1971). With eutro-
phication, increases in resources to the community can
result in a few species becoming exceedingly abundant;
therefore, both structural and functional changes usually
corresponded uniformly to increases in nutrient loading.
However in communities affected by toxic substances, the
relationship between changes in community structure and
ecosystem productivity depends on whether or not thresh-
olds for the elimination of sensitive trophic levels are
exceeded. For example, the elimination of sensitive herbi-
vores by toxic substances can permit the proliferation of
resistant algal taxa (Warren 1971), and this seems to be
the case in some AMD studies. Chemical AMD stress that
is high enough to decrease herbivores but not affect toler-
ant algal taxa, can increase benthic algal biomass and
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Environmental Management (2012) 50:10351046
productivity under conditions of high irradiance and slightly
elevated nutrients, even with moderate Fe precipitate
(Niyogi and others 2002). In contrast, this study and Bauers
(2003) indicate that in conditions where light, and nutrients
are more limiting, dominance by a few algal taxa resulting
from low to moderate AMD stress, corresponded with a
decrease in biomass and productivity. Thus light and
nutrients can act as a constraint on benthic algal productivity
when herbivores are lost.
While the AMD chemistry of sites in this study was fairly
typical of other AMD-impacted and AMD-treated Appala-
chian streams, it is apparent that even within the same
watershed, differences among sites in other environmental
variables made it difficult to determine how specific AMD
stressors influenced periphyton productivity. An under-
standing of AMD stress that encompasses both hard rock
and coal mining impacts is more difficult because AMD
chemistry varies greatly depending on the mineral geology
of the site. In addition, the many interacting external (e.g.,
light, DOC, nutrients, current) and internal (e.g., community
physiognomy, exposure history) factors that ultimately
affect toxicity are more pronounced. Partitioning variance
among those factors has been shown to better resolve
periphyton responses to specific AMD stressors (Tlili and
others 2011), but AMD stress often simplifies food webs
(Hogsden and Harding 2012b). Therefore, more specific
knowledge of the autecologies of taxa is required to connect
structure with function. Experimental studies are needed to
provide a better understanding of how specific chemical and
physical AMD-stress thresholds interact with other envi-
ronmental factors to affect community structure, and func-
tional aspects, of the benthic community in streams.
Ultimately, this information may lead to the development of
better treatment systems. Our problem is to understand the
functioning of the parts as they lead to the behavior of
wholes (Warren 1971).
Acknowledgments We are grateful to Margaret Dunn, The Slippery
Rock Creek Watershed Coalition, Stream Restoration Inc. and the Knox
Office of the Pennsylvania DEP who were responsible for building most
of the treatment systems in the watershed. We thank Michael Stapleton
for assistance with water chemistry analysis. Comments by three anon-
ymous reviewers improved the manuscript. This study was supported by
funds from the Department of Biology at Slippery Rock University, and a
Scholarly Endeavor Grant from Slippery Rock University.
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