Diatom biomonitoring of streams: Reliability of reference sites and the response of metrics to environmental variations across temporal scales

Ecological Indicators 11 (2011) 1647–1657

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Diatom biomonitoring of streams: Reliability of reference sites and the response of metrics to environmental variations across temporal scales Nathan J. Smucker ∗ , Morgan L. Vis Department of Environmental and Plant Biology, Ohio University, Athens, OH, USA

a r t i c l e

i n f o

Article history: Received 7 September 2010 Received in revised form 10 April 2011 Accepted 13 April 2011 Keywords: Temporal variation Reference condition Nutrient Agriculture Acid mine drainage (AMD) Index

a b s t r a c t Benthic diatoms are widely used indicators of human impacts on stream ecosystems because they are very responsive to changing environmental conditions. However, little research has explicitly focused on their reliability with regards to temporal variation in assemblage structure and environmental conditions. We examined variability in diatom-environment relationships at bi-weekly, monthly, and yearly time scales from 7 reference, 7 agricultural, and 2 acid mine drainage (AMD)-impacted streams, and how nutrient and pH fluctuations may affect the interpretation of diatom metrics and the Diatom Model Affinity (DMA) index. Reference streams had less bi-weekly variability in NO3 -N concentrations than non-reference streams. The % eutraphentic diatoms and DMA scores were more strongly correlated with seasonal means of NO3 -N and PO4 -P concentrations than with same day concentrations. Most nutrient indicator metrics had strong correlations with watershed land use. All 14 non-AMD streams experienced substantial increases in NO3 -N and decreases in temperature from November to May, which were associated with high species turnover, substantial changes in community structure, reduced diversity and richness, increased relative abundances of high nutrient diatoms, and decreases in low nutrient diatoms and DMA scores. The % acidophilic diatoms and DMA scores were significantly correlated with increased pH associated with greater precipitation at AMD sites from December to April (r = −0.77, r = 0.62, respectively; P < 0.01). Yearly, DMA scores for all reference streams were consistently in the minimally impaired category, whereas scores for non-reference streams varied among impairment categories. Reference sites serve as reliable benchmarks for diatom ecological integrity during the summer. In this region, June to October is a recommended time period for diatom sampling in monitoring programs because subsequent shifts in hydrologic regimes, nutrients, and diatom assemblages occurred, affecting all sites and masking among stream differences attributable to agricultural land uses. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Diatoms are excellent indicators of impacts on the ecological integrity and water quality of streams (Jüttner et al., 1996; Taylor et al., 2007; Stevenson et al., 2008). They are particularly useful because they have high species diversity, a wide range of autecologies, and are present in most aquatic environments (Round et al., 1990; van Dam et al., 1994). Nutrients, pH, and ionic composition are especially important to the structure and diversity of diatom assemblages (DeNicola, 2000; Potapova and Charles, 2003; Potapova and Charles, 2007; Soininen, 2007), which make diatoms highly effective indicators of human activities affecting these parameters, such as urbanization (Sonneman et al., 2001;

∗ Corresponding author. Present address: U.S. EPA Atlantic Ecology Division, 27 Tarzwell Drive, Narragansett, RI 02882, USA. Tel.: +1 401 782 9624. E-mail addresses: [email protected], [email protected] (N.J. Smucker), [email protected] (M.L. Vis). 1470-160X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ecolind.2011.04.011

Newall and Walsh, 2005), deforestation (Naymik et al., 2005), agriculture (Leland and Porter, 2000; Fore and Grafe, 2002), and acid mine drainage (AMD) from abandoned coal mines (Smucker and Vis, 2009; Zalack et al., 2010). Numerous indices and metrics have been developed to assess the severity of anthropogenic impacts on streams and to communicate biomonitoring results to the public, watershed groups, government agencies, and policymakers more effectively than presenting complex species abundance data with multivariate relationships (Hill et al., 2000; Wang et al., 2005; Stoddard et al., 2008). Although metrics and indices provide meaningful scores that indicate responses to stressors, determining the severity of impacts and developing goals for remediation and conservation depend on reference stream conditions (Hughes et al., 1986; Hughes, 1995). Most biomonitoring methods rely on reference sites, which serve as benchmarks of stream water quality because they represent the best attainable condition in a region and how biological communities would exist in the presence of minimal human impacts (Karr and Chu, 1999; Stoddard et al., 2006; Herlihy et al., 2008).

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Reference sites are selected to meet criteria that often include nutrient concentrations, alkalinity, and land use in the upstream watershed at the least disturbed sites in a region (Hughes, 1995; Stoddard et al., 2006; Whittier et al., 2007). Although reference sites serve as the basis for most biomonitoring methods and they are acknowledged to have some variation over time, little research has investigated the extent to which temporal variability may affect monitoring and management decisions as a result of any changes in assemblage composition and environmental conditions. If temporal variation in reference sites is great, then attainment criteria may be inappropriately defined as a result of misguided expectations of condition, and the ability to detect impacts of stressors on biological condition would be reduced. Also, temporal variability in exposure to stressors at non-reference sites might muddle relationships of biological metrics with environmental conditions, potentially hindering their applications in biomonitoring programs. Comparing the variability of reference sites with that of non-reference sites would improve the ability to distinguish the effects of environmental stress from the natural range of variation. Chemistry and diatom communities, which are often only sampled once for assessments, could differ drastically depending on when they were collected (Lancaster et al., 1996; Winter and Duthie, 2000; Hamsher et al., 2004). While land use is fairly consistent and has longer time scales of variation, water chemistry can be more variable throughout a sampling season and among seasons as a result of precipitation events and climate regimes (Driscoll et al., 1987; Johnson et al., 1997; Fitzhugh et al., 1999). Diatoms have been typically considered to be responsive to short term environmental conditions when compared to other biomonitoring organisms, such as fish and macroinvertebrates (Lowe and Pan, 1996). This quicker response is partly due to their short generation time compared to the longer life histories of macroinvertebrates and fish (Williams, 1964; Banse, 1982). Further complicating monitoring programs, response times of diatoms to water chemistry change can be variable among streams with different severities of impairment (Lavoie et al., 2008). Sources of variability like these could have implications for management decisions, restoration plans, and conservation efforts. Few studies have investigated the response to and integration of change in chemistry by diatom assemblages, even fewer have focused on how temporal variability can affect biological assessments of human impacts using diatoms, and none, to our knowledge, have examined temporal variability and reliability of reference streams. Therefore, investigating temporal variability is imperative for determining how biomonitoring results may be affected by fluctuations in environmental conditions and assemblage composition. The leading causes of stream impairment in the Western Allegheny Plateau of southeast Ohio are agricultural activities, which increase nutrient loads as a result of animal waste and fertilizers, and acid mine drainage (AMD) from abandoned coal mines, which contributes high acidity and concentrations of metals (OEPA, 2000). The primary objectives of this study involved basic and applied ecological approaches: (1) to identify how the temporal variation of water chemistry, diatom communities, metrics, and diatom index scores affect assessments, (2) to characterize diatom relationships with fluctuations in nutrient concentrations, and (3) to determine the reliability of reference sites on which assessments are based. To achieve our objectives, we examined three temporal scales at 7 reference and 7 non-reference sites, which included bi-weekly variation during a summer biomonitoring sampling season, month to month variation for 1 year, and year to year variation for 3 years. We also sampled 2 AMD impacted streams to document their bi-weekly and monthly variability in relation to pH.

2. Methods 2.1. Study sites During July to September 2005, 44 wadeable streams in the Western Allegheny Plateau (WAP) of southeast Ohio that historically met reference criteria set by the Ohio Environmental Protection Agency (OEPA) were sampled to design biomonitoring methods using diatoms (Smucker and Vis, 2009). Sites were selected randomly from an OEPA database of all sites historically sampled for fish, macroinvertebrates, or chemistry (Ed Rankin, personal communication). Of these 44 sites, 7 currently met reference site criteria set by the OEPA and the remaining 37 were considered non-reference and potentially impaired sites (OEPA, 1999; details in Smucker and Vis, 2009). Briefly, OEPA criteria were PO4 -P <0.06 mg l−1 , NO3 -N <0.47 mg l−1 , conductivity <532 ␮S cm−1 , Ca2+ <69, Mg2+ <18, Na+ <17, and Cl− <30 mg l−1 , and we added the requirements that >66% of their upstream watershed was forested and that no historical coal mining existed in the whole watershed. For this temporal study, we focused on the 7 sites meeting all reference criteria, and 7 sites that were selected randomly from the remaining 37 non-reference sites sampled in 2005. Reference and non-reference sites were distributed throughout the WAP of southeast Ohio and represented similar ranges in watershed area. The 14 sites were sampled bi-weekly from June 18 to August 27 in 2007 (i.e., 6 sampling dates separated by 2 weeks) to characterize shorter temporal dynamics, monthly from June 2007 to May 2008 to document seasonal patterns, and during August of 2005, 2006, and 2007 to investigate year to year variation. The date range for bi-weekly sampling was chosen because biomonitoring surveys are typically conducted in the summer and the OEPA sampling season is June 15–October 15. Baseflow conditions in streams exist during this time, which reduces variability associated with high flow events frequently occurring in spring. Two sites impacted by acid mine drainage (AMD) were sampled bi-weekly from June 15 to August 31, 2007 and monthly through April 2008. We were unable to sample some sites between December 2007 and March 2008 because of high-flow conditions. To aid in displaying trends when this occurred, means of water chemistry and metrics were calculated for the missing data points using data immediately prior to and subsequent to the missed sampling date. 2.2. Sampling and laboratory techniques Diatoms were sampled by collecting 10 rocks equidistantly spaced along a zigzag pattern from the downstream to upstream ends of riffles. The same riffles were sampled on each date to reduce any variability associated with spatial factors. Diatoms within a 7.1 cm2 rubber O-ring placed tightly on the upper surface of rocks were removed using a firm-bristled toothbrush. Composite samples of diatoms from each site were immediately placed on ice until return to the lab where they were homogenized by vigorously shaking for 30 s and preserved as 10-ml subsamples in CaCO3 buffered 2.5% glutaraldehyde. Samples were cleaned using boiling H2 O2 and HNO3 to remove organic matter and rinsed repeatedly until attaining a neutral pH. Samples were settled onto round cover slips in a chamber that ensured random and even distributions (Battarbee, 1973). Cover slips were mounted on slides using the mounting medium NAPHRAXTM (Brunel Microscopes Ltd., Hazelbrook, Wiltshire, UK) and 500 valves were identified to species along a transect at 1000× magnification with a light microscope (Olympus BX40TM , Center Valley, PA, USA). The keys used for species identification were primarily those of Kramer and LangeBertalot (1986, 1988, 1991a,b), but primary literature was also used and taxonomy was updated accordingly to account for recently erected genera, described species, and transferred species.

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At each site, handheld probes were used to measure conductivity, pH, and temperature (Waterproof ECTestr® and pHTestr 30® , Oakton, Vernon Hills, IL, USA). Filtered chemistry samples were collected from the water column at the upstream end of riffles and immediately placed on ice until returned to the lab for analysis (0.45 ␮m pores; Millipore® , Billerica, MA). PO4 -P was measured using the ascorbic acid method and NO3 -N using the cadmium reduction method (APHA, 1995). Landuse variables in the upstream watershed of each site were compiled using the National Land Cover Database (USGS, 1992), and included % forest, % pasture, and % row crops.

tion. A subsequent NMS set to three axes was conducted with 50 runs of real data, 50 runs of randomized data, and 250 maximum iterations with a random starting configuration, which had acceptable levels of stress (<0.20). DMA scores from each sampling year were plotted against each other to investigate major differences among years. Pearson correlations of index scores with 1-time measurement, 2-year, and 3-year means of nutrients were calculated to examine relationships of long-term year to year variation in nutrients with diatom communities.

2.3. Diatom and statistical analysis

3.1. Bi-weekly dynamics

Species richness and Shannon diversity in 500 valves were calculated for each sample, and changes in assemblage composition over time and their relationship with changes in water chemistry were examined. We calculated scores for the Diatom Model Affinity (DMA) index developed in Smucker and Vis (2009) and nutrient metric scores using the relative abundance of species indicating high P, low P, high N, and low N conditions from Potapova and Charles (2007) and % eutraphentic diatom genera from Hill et al. (2000). Using relative abundances of genera, the DMA scores stream sites based on percent similarity to a model community from reference sites, with low similarity indicative of impairment (Passy and Bode, 2004). Briefly, the model community comprised 50% Achnanthidium + Cymbella, 20% Nitzschia, 15% Navicula, 10% Amphora + Cocconeis, and 5% Fragilaria + Gomphonema (Smucker and Vis, 2009). DMA scores were categorized as minimally (>66), moderately (55–66), or severely impaired (<55) as defined in Smucker and Vis (2009). High P and low P diatoms were defined as being typical of TP >0.10 mg l−1 and <0.01 mg l−1 , respectively, and high N and low N diatoms were defined as being typical of TN >3 mg l−1 and <0.20 mg l−1 , respectively (Potapova and Charles, 2007). The relative abundance of acidophilic diatoms in AMD impacted streams was also calculated (Hill et al., 2000). For all parametric statistics, data were either log10 or power transformed when needed to meet normality and homogeneity of variance assumptions. Pearson correlations were used to examine relationships of index and metric scores with nutrients at nonAMD impacted sites and with pH at AMD impacted sites using Number Cruncher Statistical Systems (NCSS 2004, Kaysville, UT, USA). For bi-weekly sampling data, correlations were calculated for same day water chemistry measurements and for the seasonal means to examine if diatoms had stronger relationships with same day measurements or seasonal means that integrate variability in nutrient resources. Repeated measures analysis of variance (rmANOVA) using NCSS 2004 was conducted on the bi-weekly and yearly datasets to explore if index and metric scores were significantly different among sampling dates and if variation differed between stream types. Repeated measures ANOVAs were not conducted on the monthly dataset because of missing data in the winter when some streams were flooded and diatoms could not be sampled. To examine bi-weekly and month to month changes in assemblage structure, Bray–Curtis similarity, which is useful for comparing the similarity of samples using abundance-based data (Bray and Curtis, 1957; Legendre and Legendre, 1998), was calculated between sampling dates for each sample. Jaccard similarity, which uses the presence–absence of species (Legendre and Legendre, 1998), was calculated to determine changes in species composition among sampling dates. Nonmetric multidimensional scaling (NMS) was conducted using the Bray–Curtis coefficient to explore seasonal changes in diatom communities using PC-ORD v. 5.0 (MjM Software, Gleneden Beach, OR, USA). Using the quick and dirty autopilot settings, NMS recommended a 3-dimensional solu-

The maximum concentrations of PO4 -P and NO3 -N during the summer were positively correlated with the % crops in the upstream watershed (r = 0.58, 0.54, respectively; P < 0.05). The concentrations of PO4 were similar between both types of sites, but the 7 reference sites had lower NO3 -N concentrations, conductivity, and temperatures than non-reference sites throughout the summer (Table 1, Fig. 1). Nutrient concentrations decreased toward the end of the summer sampling period at non-reference sites. All sites had variable nutrient concentrations, but differences in nutrient concentrations from one sampling occasion to the next were on average smaller at reference sites (mean ± 1 SE; PO4 -P = 0.02 ± 0.007, NO3 -N = 0.01 ± 0.004) than at non-reference streams (mean ± 1 SE; PO4 -P = 0.04 ± 0.009, NO3 -N = 0.03 ± 0.014). Conductivity and temperature at all sites increased slightly throughout the summer, possibly as a result of observably decreased flow and hotter days. At the two AMD sites, pH remained consistently 3.6–3.8 throughout the summer and conductivity ranged from 1440 to 1750 ␮S cm−1 . Regardless of impairment, Bray–Curtis similarity from month to month remained fairly high for all sites during the summer and was consistently ∼60% (Fig. 2A). Jaccard similarity was consistently around 45% from month to month, and was likely much lower than Bray–Curtis similarity because of the large number of rare species encountered (Fig. 2B). Shannon diversity was slightly greater in non-reference sites than in reference sites while species richness was similar, and Shannon diversity and species richness were extremely low in AMD sites (Fig. 2C and D). DMA and most metrics were not significantly different among sampling dates despite some fluctuations in DMA and metric scores throughout the bi-weekly sampling (rmANOVA, P > 0.05; Fig. 3A–F). The % high P diatoms were significantly greater and the % low N diatoms were significantly less on the last sampling date than the first (rmANOVA, P < 0.01). There were no significant interactions between stream type and dates, which indicated that variability in metric scores was similar between reference and non-reference streams. Reference sites had lower relative abundances of high N, high P, and eutraphentic diatoms and greater DMA scores and relative abundances of low N diatoms than non-reference sites (Fig. 3). The relative abundance of high P and eutraphentic diatoms at non-reference sites increased throughout the summer. High N diatoms at all sites had consistent relative abundances throughout the summer (June–September), and low N and low P diatoms at all sites decreased approximately 10% from the beginning to the end of summer (Fig. 3B and D). DMA scores for reference sites consistently remained in the least impaired category (>66), and non-reference sites were consistently in the moderate impairment category throughout the summer (Fig. 3F). In AMD streams, % acidophilic diatoms remained high while DMA scores were low throughout the summer with no significant differences among dates (rmANOVA, P > 0.05; Fig. 4). DMA and metric scores showed consistent relationships with land use throughout the summer period, although not always sig-

3. Results

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Table 1 Overall means and ranges of water chemistry from June 18 to August 31, 2007 and land use for reference, non-reference, and acid mine drainage (AMD) impacted sites. Sites

PO4 -P, mg l−1

NO3 -N, mg l−1

EC, ␮S cm−1

pH

% forest

% pasture

% crops

Reference Non-reference AMD

0.12 (0.06–0.14) 0.14 (0.09–0.20) 0.21 (0.04–0.33)

0.03 (0.01–0.05) 0.08 (0.01–0.21) 0.08 (0.01–0.21)

334 (311–349) 392 (343–430) 1670 (1440–1750)

7.7 (7.5–7.8) 7.9 (7.7–8.0) 3.6 (3.6–3.8)

83.9 (67.9–95.8) 65.8 (34.7–81.7)

12.2 (3.1–25.5) 23.4 (13.6–39.5)

3.3 (0.5–11.1) 10.4 (1.5–25.4)

EC = electrical conductivity.

0.35

0.25 0.20 0.15 0.10

0.5 0.4 0.3 0.2 0.1

0.00

0 J

A S O N D

J

J

F M A M 30

900

C

800

Temperature °C

Conductivity µS cm-1

0.6

0.05

J

B

0.7

NO3-N mg l-1

0.30

PO4-P mg l-1

0.8

A

700 600 500 400 300 200

J A S O N D

J

F M A M

D

25 20 15 10

100 0

5 0

J

J

A S O N D

J

F M A M

J

J

A

S O N D

J

F M A M

Fig. 1. Means ± 1 SE of chemistry parameters from June 2006 to May 2007. Black lines = reference sites, gray lines = non-reference sites. Vertical dashed lines indicate the end of bi-weekly sampling.

Table 2 Pearson correlations (r) of landuse variables with scores and means of Diatom Model Affinity (DMA) index scores, % low nitrogen (LN), % low phosphorus (LP), % eutraphentic (eut), % high nitrogen (HN), and % high phosphorus (HP) diatoms throughout the bi-weekly sampling period (n = 14). Indicators with negative response to increased impairment Metric

DMA

% LN

% LP

Indicators with positive response to increased impairment

Date

% forest

% pasture

% crops

6/18 7/2 7/16 7/30 8/13 8/27 Mean 6/18 7/2 7/16 7/30 8/13 8/27 Mean 6/18 7/2 7/16 7/30 8/13 8/27 Mean

−0.10 0.37 0.38 0.13 0.28 0.24 0.26 0.48 0.61 * 0.43 0.46 0.47 0.65 * 0.58 * 0.24 0.39 0.22 0.24 0.08 0.31 0.25

0.03 −0.35 −0.36 −0.13 −0.20 −0.15 −0.23 −0.47 −0.55* −0.34 −0.41 −0.41 −0.59* −0.52 −0.17 −0.25 −0.06 −0.12 0.05 −0.18 −0.12

0.09 −0.37 −0.48 −0.13 −0.40 −0.39 −0.29 −0.53* −0.58* −0.47 −0.42 −0.46 −0.66* −0.64* −0.27 −0.35 −0.26 −0.14 −0.06 −0.31 −0.41

Bold terms significant at P < 0.10, asterisks denote P < 0.05.

Metric

% eut

% HN

% HP

Date

% forest

% pasture

% crops

6/18 7/2 7/16 7/30 8/13 8/27 Mean 6/18 7/2 7/16 7/30 8/13 8/27 Mean 6/18 7/2 7/16 7/30 8/13 8/27 Mean

−0.24 −0.34 −0.11 −0.27 −0.18 −0.18 −0.26 −0.56* −0.58* −0.59* −0.28 −0.34 −0.58* −0.51 −0.56* −0.51* −0.51* −0.35 −0.27 −0.52* −0.51

0.21 0.27 0.02 0.20 0.14 0.13 0.20 0.50 0.47 0.48 0.19 0.23 0.49 0.41 0.44 0.38 0.35 0.25 0.28 0.40 0.38

0.29 0.44 0.24 0.40 0.37 0.35 0.36 0.55* 0.62* 0.60* 0.29 0.30 0.54* 0.63* 0.47 0.58* 0.55* 0.37 0.36 0.63* 0.66*

N.J. Smucker, M.L. Vis / Ecological Indicators 11 (2011) 1647–1657

0.7

A

0.9 0.8

B

0.6

% Jaccard similarity

% Bray-Curtis similarity

1.0

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

0.5 0.4 0.3 0.2 0.1 0

J

A S O N D

J

J

F M A M

4.0

A

S O N D

J

F M A M

80

C

3.5 3.0 2.5 2.0 1.5 1.0

60 50 40 30 20

0.5

10

0.0

0 J

D

70

Species richness

Shannon diversity

1651

J A S O N D J

J

F M A M

J A S O N D J F M A M

Fig. 2. Means ± 1 SE of diatom community composition at reference sites (black lines), non-reference sites (gray lines), and AMD sites (dotted gray lines). Bray–Curtis (A) and Jaccard (B) similarities comparing the similarity of each month with the prior month, beginning with comparison of July with June 2006 and ending with comparing May with April 2007. Bi-weekly and monthly changes in Shannon diversity (C) and species richness (D).

nificant at P < 0.05 (Table 2). DMA scores, % low P, and % low N diatoms were positively correlated with % forest in the upstream watershed while being negatively correlated with % pasture and % crops in the upstream watershed; the % eutraphentic, high P, and high N metrics showed the opposite pattern (Table 2). With the exception of % low P diatoms, species-based metrics

tended to have stronger correlations with land use variables than the genus-based DMA and % eutraphentic diatoms. DMA, % eutraphentic, and % high N diatoms had the strongest correlations with PO4 -P and NO3 -N, and these correlations tended to be stronger with seasonal means than with same day measurements (Table 3).

Table 3 Pearson correlations of Diatom Model Affinity (DMA) index scores, % low nitrogen (LN), % low phosphorus (LP), % eutraphentic (eut), % high nitrogen (HN), and % high phosphorus (HP) diatoms from all non-AMD impacted sites (n = 14) with one-time measurements and means of NO3 -N and PO4 -P during summer 2007. Indicators with negative response to increased impairment

Indicators with positive response to increased impairment

Metric

Date

NO3 -N

PO4 -P

Mean NO3 -N

Mean PO4 -P

DMA

6/18 7/2 7/16 7/30 8/13 8/27 Mean

0.12 −0.39 −0.52 −0.62* −0.16 −0.26 –

−0.27 −0.20 −0.36 −0.62* 0.28 0.37 –

−0.36 −0.59* −0.78** −0.74* −0.79** −0.76** −0.78**

−0.14 −0.03 −0.29 −0.10 −0.35 −0.35 −0.25

% LN

6/18 7/2 7/16 7/30 8/13 8/27 Mean

−0.20 0.05 −0.21 0.32 −0.29 0.07 –

−0.47 0.18 −0.59* −0.31 0.12 0.10 –

−0.41 −0.08 −0.50 −0.04 0.08 0.11 −0.13

−0.30 −0.14 −0.02 −0.04 −0.21 −0.22 −0.17

% LP

6/18 7/2 7/16 7/30 8/13 8/27 Mean

0.01 0.31 0.12 0.34 −0.47 0.33 –

−0.63* 0.42 −0.16 −0.31 0.22 −0.11 –

0.08 0.11 −0.12 0.20 0.37 0.44 0.21

−0.43 −0.27 −0.25 −0.13 −0.19 −0.35 −0.27

Bold terms P < 0.10, *P < 0.01, **P < 0.001.

Metric

Date

NO3 -N

PO4 -P

Mean NO3 -N

Mean PO4 -P

% eut

6/18 7/2 7/16 7/30 8/13 8/27 Mean

0.08 0.51 0.36 0.61* 0.11 −0.07 –

0.75** 0.32 0.40 0.83** 0.15 −0.39 –

0.33 0.69** 0.66* 0.56* 0.66** 0.72** 0.69**

0.62* 0.76** 0.53 0.38 0.70** 0.38 0.65*

% HN

6/18 7/2 7/16 7/30 8/13 8/27 Mean

0.05 0.08 −0.35 −0.42 0.34 −0.17 –

0.73** −0.13 0.48 0.09 −0.26 −0.12 –

0.19 0.19 0.28 0.05 0.12 0.16 0.18

0.66** 0.58* 0.57* 0.51 0.36 0.76** 0.58*

% HP

6/18 7/2 7/16 7/30 8/13 8/27 Mean

0.34 −0.27 −0.38 −0.37 0.30 −0.05 –

0.54* −0.30 0.50 0.19 −0.32 −0.21 –

−0.03 −0.01 0.07 −0.04 0.04 0.07 0.02

0.27 0.37 0.31 0.29 0.24 0.52 0.36

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50

50

A

B 40 % low P

% high P

40 30 20

30 20

10

10

0

0

40 35 30 25 20 15 10 5 0

J J A S O N D J F M A M 50

C

D

40 % low N

% high N

J J A S O N D J F M A M

30 20 10 0

J J A S O N D J F M A M

J J A S O N D J F M A M

100 85

F

75

60

DMA

% eutraphentic

E 80

40

65 55 45

20

35

0

25 J J A S O N D J F M A M

J J A S O N D J F M A M

Fig. 3. Means ± 1 SE of bi-weekly and monthly changes in % high P (A), % low P (B), % high N (C), % low N (D), % eutraphentic diatoms (E), and Diatom Model Affinity index scores (F) at reference sites (black lines) and non-reference sites (gray lines). Vertical dashed lines indicate the end of bi-weekly sampling.

% acidophilic (r = -0.77, P < 0.01)

80

8

60

6

pH

DMA or % acidophilic

100

pH

40

4 2

20 DMA (r = 0.62, P = 0.01)

0 J

J

A

S

O

N

D

J

F

M

A

Fig. 4. Means ± 1 SE of pH (gray line), % acidophilic diatoms (dotted black line), and Diatom Model Affinity (DMA) index scores (black line). Pearson correlations of % acidophilic diatoms and DMA with pH are given. Vertical dashed line indicates the end of bi-weekly sampling.

3.2. Monthly dynamics Nutrient concentrations had great seasonal variability and fairly consistent patterns between the two types of streams (Fig. 1).

Changes in NO3 -N concentrations were substantially greater than in PO4 -P concentrations during the fall to spring months with the greatest peaks in NO3 -N occurring from January to April. At nonreference sites, NO3 -N decreased from concentrations >0.18 mg l−1 in June to <0.10 mg l−1 from August to September, and had peaks ∼0.40 mg l−l from January to February and again in April. At reference sites, NO3 -N concentrations steadily increased after October to a maximum of ∼0.23 mg l−1 in February before decreasing until May. Temperatures decreased steeply from September to November, and remained ∼5 ◦ C through March before increasing in April and May. Conductivity increased slightly until October, peaked in November at non-reference sites, and decreased to below summer levels until May. AMD sites had pH values around 3.8 until September, after which pH increased until reaching the highest values of 6.0–6.8 in March and April (Fig. 4). Bray–Curtis similarity between monthly samples at all sites decreased greatly from October to January, which indicated major changes in community composition during this time period (Fig. 2A). This variability coincided with the large changes in PO4 P and NO3 -N concentrations occurring at the same time (Fig. 1). Bray–Curtis similarity increased to approximately 50% from February to April, before decreasing again in May (Fig. 2A). Jaccard similarity between samples at all sites remained fairly consistent for all months (45–50%) except January and March, indicating great species turnover during these months (Fig. 2B). In the NMS

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Table 4 Mean ± 1 SE nutrient concentrations among the three sampling years for reference sites and non-reference sites. Sites NO3 -N, mg l−1 Reference Non-reference PO4 -P, mg l−1 Reference Non-reference

2005

2006

2007

0.10 ± 0.04 0.23 ± 0.03

0.08 ± 0.02 0.10 ± 0.03

0.01 ± 0.004 0.07 ± 0.05

0.03 ± 0.01 0.06 ± 0.03

0.11 ± 0.03 0.16 ± 0.05

0.06 ± 0.01 0.11 ± 0.02

correlated with % row crops (n = 14, r = −0.55, P < 0.05), and reference sites had more positive axis 3 scores than non-reference sites (Fig. 5). Shannon diversity and species richness remained consistent at all sites from June to November, before decreasing to minimums in March or April, which coincided with a large increase in relative abundances of Gomphonema olivaceum (Hornemann) Brébisson and Gomphonema micropus Kützing from November to March (Fig. 2C and D). At AMD sites, species richness increased from March to May in concert with greater pH, but assemblages still had low Shannon diversity because they were dominated by Achnanthidium minutissimum (Kützing) Czarnecki, rather than Eunotia exigua (Brébisson) Rabenhorst and Frustulia krammeri Lange-Bertalot and Meltzeltin. With the exception of % eutraphentic diatoms, all metrics and DMA scores decreased substantially from November to March. This decline was likely because G. olivaceum and G. micropus, which are considered eutraphentic (Hill et al., 2000), did not have defined nutrient optima and dominated assemblages at almost all sites during these months. Excessive relative abundances of Gomphonema species have negative effects on DMA scores. Seasonal means of metrics only had significant correlations with landuse variables in the summer. In AMD streams, the % acidophilic taxa decreased and DMA scores increased as pH increased, but DMA scores were always in the category of severe impairment (Fig. 4). The % acidophilic diatoms and DMA were strongly correlated with same day pH measurements (r = −0.77, 0.62, respectively; P ≤ 0.01; Fig. 4). 3.3. Yearly dynamics

Fig. 5. Nonmetric multidimensional scaling ordination of reference (䊉) and non-reference sites () on axes 1 vs. 2 (A), 1 vs. 3 (B), and 2 vs. 3 (C) using the Bray–Curtis coefficient (stress = 0.19). Orange = June–October samples, white = November–February samples, green = March–May samples. Large symbols = seasonal means for reference sites (䊉) and non-reference sites () with 1 standard error bars.

ordination, axis 1, axis 2, and axis 3 explained 34.9%, 20.3%, and 17.1% of the variation, respectively (72.2% total). The NMS ordination showed strong seasonal changes in diatom communities regardless of being reference or non-reference sites (Fig. 5). Mean axis 1 scores were significantly different among the three seasons (rmANOVA, P < 0.001). Mean axis 3 scores for summer were

Reference sites had the lowest PO4 -P concentrations among years, ranging from 0.03 to 0.11 mg l−1 , and non-reference sites ranged from 0.06 to 0.16 mg l−1 (Table 4). Mean NO3 -N concentrations ranged from 0.01 to 0.10 mg l−1 and were consistently lowest at reference sites and ranged from 0.07 to 0.23 mg l−1 at nonreference sites. DMA scores for reference sites were consistently in the minimal impairment category each year (Fig. 6). Non-reference sites had more variable DMA scores among years, with 4 sites classified in the same category two of three years, 2 never being classified in the same category, and 1 being in the same category for all 3 years. Despite the variability in DMA and metric scores among years, no year had significantly different scores than other years (rmANOVA, P > 0.05). However, PO4 -P concentrations were greater in 2006 than in 2005 (rmANOVA, P = 0.01; Table 5). NO3 -N concentrations were greater in 2005 and 2006 than in 2007 (rmANOVA, P < 0.01). Although variability existed in DMA scores at non-reference sites, strong relationships existed between DMA scores and the concentrations of PO4 -P and NO3 -N each year (Table 5). Correlations of DMA scores with 2- and 3-year means of NO3 -N were stronger than with same year measurements. Correlations of DMA scores with 2- and 3-year means of PO4 -P were similar to same year measurements. Eutraphentic diatoms were strongly correlated with nutrients in 2007 when using same year measurements and 2- and 3-year means (Table 5). Species-based metrics did not

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Table 5 Pearson correlations of DMA and % eutraphentic diatoms (eut) with nutrient measurements determined as the year of sampling, the mean of the year prior to and year of sampling, and the mean of 2 years prior to and year of sampling (n = 14). Same year

DMA 2005 DMA 2006 DMA 2007 % eut 2005 % eut 2006 % eut 2007

2-year mean

3-year mean

PO4 -P

NO3 -N

PO4 -P

NO3 -N

−0.47 −0.48 −0.62* NS NS 0.83*

−0.42 −0.53* −0.52 NS 0.57* 0.62*

– −0.60* −0.48 – NS 0.79*

– −0.57* −0.64* – 0.53* 0.66*

PO4 -P – – −0.56* – – 0.77*

NO3 -N – – −0.57* – – 0.60*

Bold *P < 0.05, all other given terms P < 0.1, NS = not significant.

have significant correlations with 1 year or yearly means of nutrients.

4. Discussion These results offer insight into our three objectives for this study: (1) in general, all streams had similar variability in diatom assemblages during the summer, but non-reference streams with more agricultural impacts in their watersheds had much greater variations in NO3 -N concentrations, which may complicate the ability to discern relationships with nutrient concentrations, (2) species-based metrics had stronger relationships with land use variables than the genus-based DMA and % eutraphentic diatoms, but DMA and % eutraphentic diatoms had stronger relationships with nutrient concentrations, which were even stronger when using seasonal means, and (3) despite seasonal variability of all streams, reference sites served as reliable benchmarks during the summer and among years, whereas non-reference streams showed greater variability among years.

4.1. Seasonal variability Outside of the typical sampling season of June to October, PO4 -P and NO3 -N concentrations showed very similar seasonal trends in reference and non-reference streams. Stream flows were noticeably higher at all sites from November to March than during the summer, and nutrient concentrations also increased during this time. Increased NO3 -N concentrations in streams resulting from upstream N losses in watersheds are strongly associated with increased discharge, which often shows distinct temporal trends comparable to our results (Johnson et al., 1969; Murdoch and Stoddard, 1993; Mitchell et al., 1996). In addition, terrestrial biotic processes occurring in summer can help retain and consume NO3 N, which minimizes N losses to streams during this time period, whereas greater N loss occurs during the dormant season (Vitousek and Melillo, 1979; Fitzhugh et al., 1999; Mitchell, 2001). A major snowstorm, which occurred along the western edge of the WAP in the beginning of March 2008, may have contributed to a second peak in NO3 -N loads to some streams following snowmelt and flooding, which has been previously observed elsewhere (Rascher et al., 1987; Wigington et al., 1996). The similar increases in nutrients at all sites could also be influenced in part by historical land use (Harding et al., 1998), because much of southeastern Ohio was deforested for agriculture and industry during the mid-1800s to early 1900s. Regardless, the dramatic changes in environmental conditions were associated with simplification of the diatom community as indicated by large decreases in diatom diversity and richness from November to April. High rates of species turnover and changes in diatom assemblage structure also occurred during this time as indicated by low Jaccard and Bray–Curtis similarities, respectively.

Decreases in the scores of DMA and diatom metrics associated with low nutrients coincided with increased NO3 -N from fall to spring while % eutraphentic diatoms increased. The large increase in Gomphonema species, which tend to be typical of high nutrient conditions (Hill et al., 2000), at all sites was likely one of the most important factors affecting metrics and DMA scores in the monthly study. In addition to much greater nutrient availability, this increase in Gomphonema abundances also coincided with colder temperatures and greater light availability associated with natural seasonal changes. High N diatoms did not increase with the observed increase in NO3 -N because the two dominant species, G. olivaceum and G. micropus, present in the current study were not classified as high N diatoms for the region in Potapova and Charles (2007), but G. olivaceum was associated with greater nutrients in other regions. When these two species were included as high N diatoms, this metric increased as NO3 -N increased. Temporal variability of pH in AMD streams coincided with noticeably greater flow from January to April 2008 because of mixing with more alkaline surface waters and a dilution of the acidity. Conductivity ranged from 1320 to 2000 ␮S cm−1 in the summer, but was less than 700 ␮S cm−1 from January to April. Diatom communities responded quickly to the increase in pH, which correlated with greater DMA scores and lower % acidophilic diatoms. Species richness increased with pH, but Shannon diversity was still low because A. minutissimum dominance replaced the previously dominant acidophilic species, E. exigua and F. krammeri. A. minutissimum is a species that is often abundant following disturbances, can quickly colonize scoured substrata, and can tolerate moderately acidic pH (Stevenson and Bahls, 1999; Verb and Vis, 2000; Smucker and Vis, 2009). The dilution effects in the current study were contrary to other studies in which pH decreased as a result of greater discharge from abandoned mines and infiltration of AMD contaminated groundwater with minimal dilution effects from runoff or increased discharges from non-AMD impacted streams with greater alkalinity (Fitzhugh et al., 1999; Verb and Vis, 2000). Although AMD impacts can vary greatly from site to site, AMD contaminated groundwater intrusion was likely not a factor at the sites in the present study, and any increased contaminants associated with greater AMD discharge were offset by greater surface flow from non-AMD sources, as has been reported in other situations (Johnson and Thornton, 1987).

4.2. Biomonitoring implications The DMA index and metrics were excellent indicators of nutrient concentrations and land use across temporal scales during summer months, ranging from bi-weekly to monthly and to yearly sampling, which added further support for their effectiveness in bioassessments. Changes in pH at AMD sites were also signaled by DMA scores and % acidophilic diatoms throughout the year. The genus-based DMA and % eutraphentic metric had stronger relationships with nutrient concentrations, especially with NO3 -N,

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Fig. 6. Scatter plots of DMA scores for each year sampled. Minimal impairment category includes scores ≥ 67, moderate impairment includes scores between 54 and 66, and severe impairment category includes scores ≤53. Black circles = reference sites, white circles = non-reference sites.

than species-based metrics, but species-based metrics were more strongly correlated with land use variables in the watershed than % eutraphentic diatoms or DMA. Although DMA and metric scores were not always significantly correlated with land use, these metrics had strong relationships with land use variables in a larger dataset, with the exception of % eutraphentic diatoms (Smucker

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and Vis, 2009). This indicates that certain diatom metrics can be strong indicators of watershed alterations. The correlations of DMA and % eutraphentic diatoms with same day nutrient concentrations were rarely significant, but they were more often significant and consistently stronger when using means of NO3 -N and PO4 -P, which indicated they were likely good integrators of the variability in nutrient concentrations throughout the summer. However, species-based metrics were not as strongly correlated with nutrient concentrations. This pattern was the opposite of what was observed in a larger study with more sites in the WAP, in which species-based nutrient metrics had more significant and stronger correlations with nutrients, other chemistry parameters, and land use than the genus-based metric (Smucker and Vis, 2009). This finding supports the notion that greater amounts of ecological information for discerning impacts can be gleaned from species taxonomy than from genus taxonomy (Hill et al., 2001). Genus-based metrics tend to be more conservative estimates of diatom responses to environmental conditions, especially nutrients, because they lump varying responses to environmental conditions of species within genera, potentially becoming less informative with greater sample representation and larger spatial scales (Hill et al., 2001). Another possible explanation is that genera are likely less susceptible to variation associated with dispersal and regional distributions, which can explain a significant amount of variation in species composition (Smucker and Vis, in press). With only 14 sites sampled throughout the region, the effects of species dispersal and natural variation may have been prominent factors that confounded and reduced correlations of species metrics with nutrients. Greater coverage of regional conditions and longer environmental gradients in the larger study were likely explanations of why species metrics had stronger relationships with water chemistry parameters than the genus metric. Regardless of strong or non-existent correlations with nutrients at 14 sites, an important finding of our study was that DMA and metric scores remained consistent throughout the summer, with the exception of % high P and % low N being greater and less on the last sampling date than the first, respectively. The reliability of reference sites and knowledge of natural variations is important to management decisions, as in other ecosystems (Landres et al., 1999). Mid-June to mid-October would be an acceptable sampling season for diatoms in this region because DMA and metric scores were not significantly different among dates and community similarities were consistently high from month to month (e.g., Bray–Curtis). For comparison purposes, a >60% threshold in Bray–Curtis similarity among replicate counts of the same slides has been used to define acceptable variation in quality assurance protocols (Kelly, 2001), and likewise for examining spatial variability among stream riffles (Hollingsworth and Vis, 2010). In addition, diatom assemblages in winter and spring were significantly different from summer assemblages in the NMS ordination. The summer time period has typically been the accepted sampling season for fish and macroinvertebrates in bioassessments largely because of fairly consistent base flow conditions, but it had not been previously examined for diatom bioassessments. Changes in water chemistry and diatom assemblages at all sites were very prominent after October, indicating that this would be an appropriate end to the sampling season. The observed natural temporal variability among seasons that affected all streams in a similar manner could mask among stream differences attributable to agricultural land uses. Defining this sampling period reduces temporal variability that may be related to flood regimes or seasonal shifts that can naturally affect all sites in a region (Hawkins et al., 2010), as was observed in the current study. In addition, streams in winter and spring were often either difficult or impossible to sample simply because of dangerously high flow events, which add further complications for interpreting biomonitoring results because of

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the potentially confounding influence of flow variability on diatom assemblage structure. Year to year variation was lowest in reference sites, which were consistently classified as minimally impaired, but greater variation existed at non-reference sites. Despite this variability, DMA scores consistently had strong relationships with PO4 -P and NO3 -N. Relationships were even stronger when yearly means of nutrients were calculated. These stronger relationships of DMA with mean nutrients potentially indicated that although nutrient concentrations varied somewhat from 1 year to the next, DMA was a good indicator of average conditions across years, which was also similar to the finding that DMA was more strongly correlated with mean nutrient concentrations than one-time measurements in the biweekly dataset. Species-based nutrient metrics did not have strong relationships with nutrient concentrations among years possibly because of reasons previously discussed in the bi-weekly dataset. Regardless, reference sites serve as temporally reliable benchmarks to set restoration and conservation goals. Acknowledgments Funding for this research was provided by U.S. EPA STAR grant R831365 and Ohio University (Clippinger Fellowship, Ohio Center for Ecology and Evolutionary Studies, Student Enhancement Award, Graduate Student Senate Grant for Original Research). Sam Drerup, Denise House, Jason Zalack, and Emily Hollingsworth are gratefully thanked for sampling assistance. Dina Lopez, Prosper Gbolo, Sam Drerup, Molly Semones, and Alex VandenBroek assisted with water chemistry analysis. We greatly appreciate Emily Johnston, Patty Contreras, and Alex VandenBroek for assisting with data entry. Suggestions for manuscript improvement by Jared DeForest, Kelly Johnson, Brian McCarthy, and two anonymous reviewers are also appreciated. References APHA (American Public Health Association), 1995. Standard Methods for the Examination of Water and Wastewater, 19th edition. American Public Health Association, American Water Works Association, and Water Environment Federation, Washington, DC. Banse, K., 1982. Cell volumes, maximal growth rates of unicellular algae and ciliates, and the role of ciliates in the marine pelagial. Limnology and Oceanography 27, 1059–1071. Battarbee, R.W., 1973. A new method for estimating absolute microfossil numbers with special reference to diatoms. Limnology and Oceanography 18, 647–653. Bray, J.R., Curtis, J.T., 1957. An ordination of the upland forest communities of southern Wisconsin. Ecological Monographs 26, 325–349. DeNicola, D.M., 2000. A review of diatoms found in highly acidic environments. Hydrobiologia 433, 111–122. Driscoll, C.T., Yatsko, C.P., Unangst, F.J., 1987. Longitudinal and temporal trends in the water chemistry of the North Branch of the Moose River. Biogeochemistry 3, 37–61. Fitzhugh, R.D., Furman, T., Webb, J.R., Cosby, B.J., Driscoll, C.T., 1999. Longitudinal and seasonal patterns of stream acidity in a headwater catchment on the Appalachian Plateau, West Virginia, U. S. A. Biogeochemistry 47, 39–62. Fore, L.S., Grafe, C., 2002. Using diatoms to assess the biological condition of large rivers in Idaho (USA). Freshwater Biology 47, 2015–2037. Hamsher, S.E., Verb, R.G., Vis, M.L., 2004. Analysis of acid mine drainage impacted streams using a periphyton index. Journal of Freshwater Ecology 19, 313–324. Harding, J.S., Benfield, E.F., Bolstad, P.V., Helfman, G.S., Jones III, E.B.D., 1998. Stream biodiversity: the ghost of land use past. Proceedings of the National Academy of Sciences of the United States of America 95, 14843–14847. Hawkins, C.P., Olson, J.R., Hill, R.A., 2010. The reference condition: predicting benchmarks for ecological and water-quality assessments. Journal of the North American Benthological Society 29, 312–343. Herlihy, A.T., Paulsen, S.G., Van Sickle, J., Stoddard, J.L., Hawkins, C.P., 2008. Striving for consistency in a national assessment: the challenges of applying a reference-condition approach at a continental scale. Journal of the North American Benthological Society 27, 860–877. Hill, B.H., Herlihy, A.T., Kaufman, P.R., Stevenson, R.J., McCormick, F.H., Johnson, C.B., 2000. Use of periphyton assemblage data as an index of biotic integrity. Journal of the North American Benthological Society 14, 451–457. Hill, B.H., Stevenson, R.J., Pan, Y., Herlihy, A.T., Kaufmann, P.R., Johnson, C.B., 2001. Comparison of correlations between environmental characteristics and stream

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