Characterization of natural biofilms in temperate inland waters

Journal of Great Lakes Research 38 (2012) 429–438

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Characterization of natural biofilms in temperate inland waters N. Kanavillil ⁎, M. Thorn, S. Kurissery Department of Interdisciplinary Studies, Lakehead University Orillia Campus, Orillia, ON, Canada L3V 0B9

a r t i c l e

i n f o

Article history: Received 29 August 2011 Accepted 20 June 2012 Available online 24 July 2012 Communicated by Hunter Carrick Keywords: Periphyton Inland waters Biofilm characterization Community dynamics Water quality Water quality indicators

a b s t r a c t Community dynamics of microalgae in natural biofilms grown on 10 × 3 cm glass slides were studied in three inland water systems in Central Ontario, Canada. The periphyton communities were analyzed for species composition, diversity, density and biofilm thickness. The usefulness of periphyton community dynamics and species diversity in water quality monitoring was tested. The density of microalgae varied from 2.4 × 10 7/cm 2 (Lake Couchiching) to 18 × 10 7/cm 2 (Lake Simcoe) with highest species diversity at Lake Couchiching. Lake Simcoe with its moderately high phosphorus and low organic carbon showed the highest density of microalgae while Lake Couchiching with lowest total phosphorus and highest organic carbon showed the lowest density of microalgae in biofilms. The results of analysis of variance showed significant variation in the number of genera, density, biofilm thickness and diversity of microalgae in the three sampling locations. The Mill Creek site with minimum anthropogenic disturbance, minimum light availability, lower water temperature and slow but steady flowing conditions recorded the lowest species diversity and number of genera. The dominant genera of diatoms were significantly different in the three sampling locations. This study thus showed the usefulness of periphyton community dynamics in the assessment of water quality in the inland water systems. © 2012 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved.

Introduction Periphyton grow in a variety of freshwater habitats and are considered integral to many ecological processes including primary production (Cecala et al., 2008; Liboriussen and Jeppesen, 2003), nutrient and biogeochemical cycling (Schelske, 1985; Schelske et al., 2006; Wetzel, 1993) and energy production for higher trophic levels (De Sousa et al., 2008). Periphyton are ecologically significant and are responsive to various environmental factors such as light availability, temperature, nutrient concentrations, water flow, and substrate type (Barranguet et al., 2004, 2005; Besemer et al., 2007; Danilov and Eklund, 2001; Ludwig et al., 2008; McGowan et al., 2005; Nandakumar et al., 2003; Veraart et al., 2008). Because of the sensitivity of periphyton, specifically diatoms, to various ecological changes, they have been used as potential biological indicators of aquatic ecosystems such as large lakes and river basins (Falasco et al., 2009; Kelly and Whitton, 1995; Schelske et al., 2006; Sladecek, 1986; Wiklund et al., 2010). Periphytic diatom communities generally reside in the shallow regions of aquatic ecosystems and can also receive terrestrially derived nutrients and detritus through land drainage or river run-off (Lambert et al., 2008). This places periphyton in a position for an early detection of water quality changes due to erosion and land run-off and/or anthropogenic input of effluents (Lambert et al., 2008; Rosenberger et al., ⁎ Corresponding author. Tel.: +1 705 330 4008x2633; fax: +1 705 329 4648. E-mail address: [email protected] (N. Kanavillil).

2008). The use and application of periphytic diatoms as indicator of water quality within small stream ecosystems in Canada has been demonstrated (Winter and Duthie, 2000a,b,c; Winter et al., 2003). However, there have been fewer studies regarding the use of periphytic diatoms as water quality indicators of large inland water ecosystems such as lakes and streams (Christie and Smol, 1993). Many inland water ecosystems have been impacted by the excessive anthropogenic activities (Crouzet et al., 1999; MacIsaac, 1996; Vitousek et al., 1997). The ecological disturbances can be caused by nutrient enrichment from agriculture run-off, change in land-use patterns resulting in increased erosion and turbidity, input of treated and untreated effluents from wastewater treatment plants or industries, physical and chemical disturbances caused by fishing or other water-sport activities, cottage development, establishment of invasive species etc. Recently, Lake Simco, a large lake in Central Ontario has been reported to have lost its cold water fishery and deteriorated water quality mainly due to the excess phosphorus loading occurred in tandem with the growth of human population in the watershed (Winter et al., 2007). The water from Lake Simcoe flows through Atherley Narrows to Lake Couchiching, a much smaller lake forming a part of the Trent Severn Waterway. Lake Couchiching is also exposed to intense anthropogenic activities. This includes fishing, a marina with hundreds of boaters, and water sports. Additionally, the frequent beach closures at Lake Couchiching demonstrate the intense anthropogenic activities. On the contrary, Mill Creek, a quiet lotic water system discharging into Lake Simcoe at Smith's Bay is exposed very little to anthropogenic activities because it meanders through a protected area of City of Orillia, ON. Unfortunately, data on

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N. Kanavillil et al. / Journal of Great Lakes Research 38 (2012) 429–438

Table 1 Mean values and standard deviations of limnological parameters and relative anthropogenic activities at the three study localities. ORP is oxidation reduction potential, SpCond is specific conductivity, DO is dissolved oxygen, TP is total phosphorus, RSi is reactive silicates, DOC is dissolved organic carbon. Detection limit for a method is indicated by an asterisk “*”. Lake Simcoe

Temp [°C] pH ORP [mV] SpCond [μS/cm] DO [mg/L] Turbidity [NTU] Chlorophyll [μg/L] TP [mg/L] Nitrate [mg/L] Nitrite [mg/L] RSi [mg/L] DOC [mg/L] Anthropogenic activities Fishing Boaters Water sports Other land drainages

Overall classification a b

Lake Couchiching

Mill Creek

Mean

SD

Mean

SD

Mean

SD

20.1 9.0 −693.6 226.3 14.1 5.4 2.7 0.0063 0.009* 0.006* 2.44 3.27

2.9 0.8 110.3 175.2 4.4 5.5 1.6 0.0023

21.1 8.8 −684.0 237.0 12.2 4.5 1.7 0.005* 0.009* 0.006* 2.39 4.27

2.7 0.8 107.4 24.1 3.7 4.7 0.6

17.4 8.5 −680.8 341.4 12.6 2.2 4.0 0.005* 0.015 0.006* 11.11 4.77

2.9 0.8 119.2 219.1 4.3 1.4 0.7

0.44 0.49

0.25 0.35

Lowa Low Low Mill Creek

Highb Very High Very High Land drainage

Nil Nil Nil Land drainage

Wastewater treatment plant Moderately high

High

Nil

0.0079 1.92 1.16

Very few instance of activity. Frequent instances of activity.

the periphyton from these important but contrasting ecosystems are sparse. The main objective of this study was to characterize the periphytic microalgal community mainly diatoms that develop on experimental surfaces suspended in three inland water ecosystems with varying exposure to anthropogenic activities. Thus, the hypothesis tested in this study is whether varying degrees of physical conditions such as lentic and lotic systems coupled with anthropogenic activities have a strong influence on the species composition of periphyton community and therefore the variation of the species composition can act as useful biological determinants of water quality in these ecosystems. The study involved microalgal species identification, observation on species succession and monitoring the community variation in these three different aquatic ecosystems. The three inland ecosystems studied included Lake Simcoe, Lake Couchiching and Mill Creek. Depending on the human influence at the sampling locations these ecosystems are broadly classified as least exposed (Mill Creek), moderate highly exposed (Lake Simcoe) and highly exposed to human activities (Lake Couchiching). Materials and methods Sampling sites The study was conducted in three ecologically distinct freshwater habitats located in Lake Couchiching (44°36′35.29′′N and 79°23′36.69′′ W), Lake Simcoe (N 44°35′21.7′′ and W 79°24′4.5′′), and Mill Creek (44°34′59.06′′N and 79°27′1.48′′W). All the sampling sites were located in Orillia, central Ontario, Canada. Lake Couchiching is a small lake with an approximate area of 80 km2 with an average depth of 6 m. Lake Couchiching serves as a connecting link between Lake Ontario and Georgian Bay via inland Trent-Severn waterway. The sampling point in Lake Couchiching is located close to a marina with hundreds of boaters and beach (with frequent closures because of microbial contamination) where there are active water sports and fishing activities occurring throughout summer. This is considered as the most disturbed habitat among the three studied (Table 1). The next sampling point is located

at the north-western part of Lake Simcoe. Lake Simcoe is a dimitic, hard-water lake (Winter et al., 2007) with an area of 722 km2 and has been identified as disturbed inland water ecosystem due to its nutrient enrichment and declining cold water fishery (LSEMS, 2003; Winter et al., 2007). Therefore, it has become the target of nutrient reduction strategies as part of the Lake Simcoe Management Plan. The Lake Simcoe Protection Act has been recently enacted as a means to lower nutrient loadings especially phosphorus. Between 1990 and 2003 the average total phosphorus loading of Lake Simcoe has decreased from 114 t/year to 67 t/year (Winter et al., 2007). Though the total phosphorus concentrations have declined, the end-of-summer dissolved oxygen concentration was still too low for the revival of cold water fishery (Eimers et al., 2005; Winter et al., 2007). The phytoplankton community in the Lake Simcoe has been continuously monitored since 1980 and the data showed a decline in phytoplankton density consistent with the phosphorus reduction (Evans et al., 1996; Nicholls, 1995). The average depth of Lake Simcoe is 17 m with 5% of the lake area having >30 m depth (Campbell and McCrimmon, 1970). The sampling location on the shore of this lake represents a moderately disturbed area as it is located about 1 km away from the lakeside cottages and the point of effluent discharge from City of Orillia's water treatment plant (Table 1). In contrast, Mill Creek is a small spring fed creek that drains into Smith's Bay, Lake Simcoe. The sampling location in the Mill Creek is located at the Scout Valley, a forested area managed and protected by the City of Orillia. The maximum depth of this stream at the sampling location is about 1 m and the width is about 2 m. This is considered as the least disturbed location among the three studied (Table 1). Glass slides, 3×10 cm, were used as the substrata for the growth of natural biofilms. The glass slides were detergent washed, rinsed with tap water followed by distilled water. They were air dried before using for the experiment. Altogether 24 slides were suspended initially at each sampling locations. The glass slides were suspended with the help of a periphytometer, which consisted of a wooden frame with clips to attach glass slides. The periphytometers at Lake Couchiching and Lake Simcoe were suspended at 50 cm below the water surface, whereas the periphytometer in Mill Creek was suspended 25 cm below the water surface.

N. Kanavillil et al. / Journal of Great Lakes Research 38 (2012) 429–438

Experimental procedure The colonization and succession of microalgal biofilms was studied between May and August 2009. The samples were collected at fixed time intervals; 1, 3, 5, 7, 10, 15, 20, 25, and 30 days after slide installation. These sampling intervals were decided after running trial experiments that tested various time intervals to monitor the periphyton growth on glass slides at these study sites. These intervals provided more frequent observation of the community development during the early phase while less frequent sampling during the later part of community development owing to the relatively stable nature of the community usually observed by 20th day after slide exposure. Because no appreciable difference was observed in the community build up between the slow flowing freshwater stream (Mill Creek) and the lakes, the same sampling intervals were employed to study at all three sampling sites. Duplicate slides were removed randomly from the periphytometer at all intervals except for 10, 20, and 30 days when quadruplicate slides were retrieved (Stevenson and Bahls, 1999). After collection, the slides were immediately transported to the laboratory for analysis in an immersed condition in water collected from the respective sampling locations. This study was repeated three times. The microalgal biofilm samples were prepared for observation by wiping one side of the slides with a clean cotton plug. The side observed always faced the water flow. Each slide was examined at nine fixed locations under a compound light microscope (Ken-a-vision) at 200× magnification. The biofilms were kept in moist condition during observation by adding water drops through a Pasteur pipette. The biofilm samples were analyzed for genera composition of microalgae, microalgal density (number/cm2) and biofilm thickness (μm). A higher magnification was used (400× and 1000×) for identification purpose. The microalgal genera were identified by using Round et al. (1990) and Prescott (1978). Biofilm thickness was determined according to Bakke and Olsson (1986) where the vertical displacement of the microscope stage measured by the attached microguage required to focus between the biofilm-liquid interface and the biofilm-substratum interface. This is recorded as the biofilm thickness. The mean wet biofilm thickness was determined by averaging biofilm thickness readings at nine locations spread over the slide surface. The algal density on each slide was measured at a magnification of 200× at nine fixed locations and expressed as an average number of organisms per cm−2. Limnological parameters The limnological parameters measured were temperature (°C), pH, dissolved oxygen (mg/L), oxidation reduction potential (ORP, mV), turbidity (NTU), specific conductivity (μS/cm), and chlorophyll a (μg/L). All limnological parameters were measured by using a Hydrolab Datasonde (Series 5, Campbell Scientific Canada Corp., Alberta) by immersing to the desired depth of the water (50 cm below water level at lakes and at 25 cm below at Mill Creek). The system is connected to a computer which recorded the parameters instantly. In addition, water samples were collected and analyzed for nutrients such as nitrate, nitrite, phosphate, total phosphorus, reactive silicates and organic carbon using standard analytical protocol (APHA, 2005). Data analysis The biofilm algal density, thickness, and genera composition were compared between and with limnological parameters by using linear regression analysis and repeated measures ANOVA. This analysis was chosen because the biofilm parameters monitored are considered not completely independent of each other even though different slides were observed for different intervals. This view was strengthened by the fact that a good correlation was observed between biofilms parameters and

431

increasing intervals of time (age). However, to get a better understanding between the sampling locations, a pair-wise analysis between two sampling locations was carried out using one-way ANOVA. Before the analysis of variance indicated distributions were not normal, the data were log transformed. The statistical analyses were performed using SPSS statistical software (IBM SPSS Statistics, ver 19, SPSS Inc.). Regression analyses were performed between algal density, thickness, number of genera, and the limnological parameters to determine the interactions between these data sets. In addition, the species diversity was calculated by using the Shannon–Wiener Index (Ricklefs, 2001). The results of the ANOVA showed no significant variation for algal density (F2,26 =1.5; p>0.05), biofilm thickness (F2,26 =1.56; p>0.05), and the number of genera (F2,26 =2.5; p>0.05) between the three sampling periods (repeated studies) at the three study locations and therefore the data for the three sampling periods of each location were pooled for further interpretation and analyses. Specific growth rates in terms of algal density, biofilm thickness and number of genera of microalgae have been estimated according to the following formula: G ¼ ðB−AÞ=A Where G is the specific growth rate (in terms of algal density or biofilm thickness or number of microalgal genera), A and B are initial and final values of a parameter between two sampling intervals. Results Limnological parameters The water temperature and chlorophyll a concentrations between the three sampling locations showed significant variation (one way ANOVA F2,52 = 24.9; p b 0.05 and F2,52 = 7.4; p b 0.05 for chlorophyll a and water temperature, respectively). The highest chlorophyll a concentration was recorded at the Mill Creek, which was the least disturbed area among the three studied, also received the lowest sunlight. Here, the water temperature was found to be the lowest. The concentrations of reactive silicate and total organic carbon were highest at Mill Creek. The total phosphorus concentration showed higher values at Lake Simcoe sampling location than the other two sites. The organic carbon concentration showed higher values in Lake Couchiching and Mill Creek compared to Lake Simcoe (Table 1). The parameters pH, dissolved oxygen, turbidity, conductivity, oxidation reduction potential, nitrate, nitrite, reactive silicate and total phosphorus did not show any significant variations between these locations (Table 1; one way ANOVA F2,52 = b1.14; p > 0.05 for all parameters). Biofilm characteristics: microalgal density, biofilm thickness and number of genera The variation in microalgal density in the three study locations is plotted in Figs. 1A, B, and C. The general trend in density variation showed an initial period of increase, a climax at around day 25 and a decrease thereafter. The maximum microalgal density was recorded in Lake Simcoe (1.8 × 10 8 cells/cm 2 on day 25 in the month of May, 2009) while the minimum (1.2 × 10 7 cells/cm 2 in the month of May, 2009) was recorded in Lake Couchiching, the most disturbed ecosystems among the three studied. The variation in algal density did have an impact on the biofilm thickness in the three ecosystems. The variation in biofilm thickness roughly followed the same pattern as that of algal density i.e. a gradual increase from day 1 and reaching maximum on day 20 or day 25 followed by a decrease (Figs. 1A, B and C). The algal density and biofilm thickness showed a positive correlation in all the three sampling locations (r 2 = 0.7193, p b 0.05; r 2 = 0.31, p > 0.05; and

432

A

N. Kanavillil et al. / Journal of Great Lakes Research 38 (2012) 429–438

Biofilm species composition, succession, and diversity

200

Density x 106/cm2

Lake Simcoe 150

Mill Creek Lake Couchiching

100

50

0 Day 1

Day 3

Day 5

Day 7 Day 10 Day 15 Day 20 Day 25 Day 30

Duration of slide exposure

Biofilm thickness (µm)

B 100.00 Lake Simcoe

80.00

Mill Creek Lake Couchiching

60.00

40.00

20.00

0.00 Day 1

Day 3

Day 5

Day 7 Day 10 Day 15 Day 20 Day 25 Day 30

Duration of slide exposure

C

35.00 Lake Simcoe

30.00

No. of algal genera

Lake Couchiching The microalgal genera composition of biofilms at Lake Couchiching is shown in Appendix 1a. The species composition varied with time. The initial colonizers included Achnanthes, Cymbella, Fragilaria, and Navicula, and they dominated the initial biofilm community. As the community developed, Achnanthes became the dominant diatom genera which increased its percentage composition from 50% to 67% from days 1 to 10, respectively. The percentage composition of Achnanthes remained relatively constant thereafter until the end of the study. Overall composition of the microalgae showed equal representation of diatoms and cyanobacteria during the initial phase of biofilm development, however, over the time period a gradual dominance of diatoms (97% and 99% by days 5 and 25, respectively) is observed as compared to other types of algae such as green algae, chrysophyta and cyanobacteria. The variation in the dominance of algal genera is reflected in the Shannon Diversity Index which showed a consistent decline from day 1 to day 30 indicating the loss of species diversity with the dominance of Achnanthes (Fig. 3).

Mill Creek Lake Couchiching

25.00 20.00

Lake Simcoe The microalgal genera composition during the study period is shown in Appendix 1b. The initial colonizers in Lake Simcoe biofilm were the same as in Lake Couchiching. The dominant genera included Achnanthes, Diatoma, Fragilaria, Cymbella, and Navicula. As observed in Lake Couchiching, Achnanthes dominated the biofilm algal community. The percentage composition of this genus increased from 47% to 87% from day 1 to day 15. Here, the diatoms dominated the microalgal community throughout the study period (96% to 99% on day 1 and from day 7, respectively). The Shannon Diversity Index values showed a consistent decline from day 1 to day 30 which has a direct relationship with the dominance of Achnanthes (Fig. 3).

15.00 10.00 5.00 0.00 Day 1

Day 3

Day 5

Day 7 Day 10 Day 15 Day 20 Day 25 Day 30

Duration of slide exposure Fig. 1. Variation of average microalgal density (A), biofilm thickness (B) and number of microalgal genera (C) over the period of slide exposure at three sampling locations. Each data point represents average of three repeated studies and error bars indicate SD.

r 2 = 0.51, p b 0.05, for Lake Couchiching, Lake Simcoe and Mill Creek, respectively; Fig. 2). Even though this is an expected relationship, it may not be always true as seen in Lake Simcoe where the significance level was much lower than the other two ecosystems. The pattern of variation in the number of microalgal genera in the periphyton community in the three ecosystems was dissimilar. The highest number of genera in Lake Couchiching and Mill Creek was recorded on day 20 while in Lake Simcoe the maximum number of genera was recorded on day 10. The minimum number of genera in Lake Couchiching and Lake Simcoe was recorded on day 1 while that in Mill Creek it was recorded on day 10. These differences could be indicative of the variation in the environmental stresses present in these three ecosystems. During the study period, Lake Couchiching recorded the maximum number of genera while the minimum number of genera was observed in Mill Creek. The correlation between the number of genera, the algal density and biofilm thickness resulted in a positive relationship, while none of these relationships were statistically significant (Fig. 2).

Mills Creek The generic composition of biofilm microalgae in Mill Creek is shown in Appendix 1c. The initial dominant genera to colonize the biofilms were Achnanthes, Cocconeis, and Navicula. As the biofilm developed, both Achnanthes and Cocconeis increased their percentage composition and remained as the dominant genera. Unlike the other two sampling locations, Cocconeis became the most dominant genera in Mill Creek and its percentage composition increased from 4% to 66% from day 1 to day 10 and reached the highest value of 95% on day 30. In Mill Creek the composition of microalgae showed considerable presence of green algae in the initial phase of biofilm development (12% and 6% on days 1 and 3, respectively) which was gradually dominated by the diatoms towards the end of the study. The dominance of Cocconeis reduced the genera diversity, which is reflected in the Shannon Diversity Index values (Fig. 3). Specific growth rates Algal density The specific growth rates showed an initial increase (day 3) followed by a decrease (day 5) and then (from day 7) the growth rate fluctuated over time (Fig. 4A). The only exception observed was at Lake Simcoe where the growth rate in terms of algal density fell negative after day 15. This has been noticed in the diversity index where there was a sudden decrease in diversity at Lake Simcoe more or less at the same time period (between days 10 and 15). Number of genera The number of microalgal genera also showed an initial increase (day 3) followed by a decrease (days 5–7) during the early stages of biofilm growth followed by a gradual decrease (Fig. 4B). The growth in number of genera fluctuated widely except in Lake Simcoe during

N. Kanavillil et al. / Journal of Great Lakes Research 38 (2012) 429–438

Mill Creek 9

= 0.5148

8 7 6 5 4 3

R2

100

= 0.3314

8

Thickness (µm)

R2

Density (log10) No./cm2

9

Density (log10) No./cm2

433

7 6 5

50

80 60 40 20

4

0

3 0

R2 = 0.6018

100

0

10

20

30

0

No. of genus

Thickness (µm)

10

20

30

No. of genus

Lake simcoe 9

R = 0.7193 8 7 6 5 4 3

100

R2 = 0.2894 8

Thickness (µm)

2

Density (log10) No./cm2

Density (log10) No./cm2

9

7 6 5 4

50

100

0

10

20

0

8 7 6 5 4 3

100

R2 = 0.0807

8 7 6 5 4

20

30

R2 = 0.2246

80 60 40 20 0

3 100

10

No. of genus

Thickness (µm)

Density (log10) No./cm2

Density (log10) No./cm2

R = 0.425

50

20

30

Lake Couchiching 9

2

0

40

No. of genus

Thickness (µm)

9

60

0

3 0

R2 = 0.3093

80

0

10

20

No. of genus

Thickness (µm)

30

0

10

20

30

No. of genus

Fig. 2. Results of regression analysis between microalgal density, biofilm thickness and number of microalgal genera during the study period at three sampling locations.

the initial phase and exhibited a negative growth rate in Lake Simcoe towards the later part of the biofilm development.

rate fluctuated widely indicating an increase and decrease in number of genera over the same time period. The relationship between biofilm characteristics

Biofilm thickness The rate of growth of biofilm thickness also exhibited an initial increase (day 3) followed by a decrease (days 5–7; Fig. 4C). The growth was rapid during the initial phase of biofilm development; however, the growth rate was not sustained during later part of biofilm development in all the three sampling locations. In Mill Creek, the growth 2

Lake Couchiching Lake Simcoe Mill Creek

Diversity index

1.6

1.2

0.8

0.4

0 Day 1

Day 3

Day 5

The results of the repeated measure ANOVA showed highly significant variation in density (F1,2 = 380.28, p b 0.05), diversity (F1,2 = 156.28, p b 0.05), thickness (F1,2 = 186.9, p b 0.05) and the number of microalgal genera between the three study locations (F1,2 = 15.93, p = 0.05). The pair-wise analysis of the biofilm parameters such as the thickness, density and diversity with the number of days (duration) showed significant difference between early period of biofilm development (e.g. thickness during 1, 3, 5 and 7 days) with later stages of community development (e.g. thickness during 10, 15, 20, 25 and 30 days). However, this was not clearly demonstrated in case of the number of genera which showed a significant difference only between those observed on days 1 and 3 with that observed on 15 and 20 days. The ANOVA results between pair-wise locations showed significant variation between all three pair-wise comparisons in the number of genera while the thickness and density showed significant variation only between Lake Simcoe and Mill Creek (Table 2).

Day 7 Day 10 Day 15 Day 20 Day 25 Day 30

Duration of slide exposure Fig. 3. Variation of Shannon Diversity Index of microalgal genera observed on biofilms developed on the glass slides at the three sampling locations during the study. Each data point represents average of three repeated study at each sampling location and error bars indicate SD.

Between sampling durations The ANOVA test on the microalgal density with the duration of slide exposure showed a significant variation (F7,23 = 5.15, p = 0.003) while thickness only showed a moderate variation (F7,23 = 2.488, p = 0.06). However, the number of microalgal genera with the duration of slide

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N. Kanavillil et al. / Journal of Great Lakes Research 38 (2012) 429–438

A

Discussion

0.8

Growth rate in algal density

Lake Couchiching Lake Simcoe

0.6

Mill Creek 0.4 0.2 0 Day 1

Day 3

Day 5

-0.4

Growth rate in No. of genera

B

Day 7 Day 10 Day 15 Day 20 Day 25 Day 30

-0.2

Duration of slide exposure Lake Couchiching

0.8

Lake Simcoe Mill Creek

0.6 0.4 0.2 0 Day 1

Day 3

Day 5

Day 7 Day 10 Day 15 Day 20 Day 25 Day 30

-0.2 -0.4 -0.6

C

Duration of slide exposure

20

Lake Couchiching

Growth rate in biofilm thickness

Lake Simcoe 15

Mill Creek

10 5 0 Day 1

Day 3

Day 5

Day 7 Day 10 Day 15 Day 20 Day 25 Day 30

-5 -10

Duration of slide exposure

Fig. 4. Average specific growth rates of biofilms (average of three repeated studies at each sampling locations) with the duration of slide exposure at the three sampling locations calculated with respect to the growth in microalgal density (A), number of genera (B) and biofilm thickness (C). See methodology for details of calculation of specific growth rates (error bars indicate SD).

exposure did not show a significant variation (F7,23 =0.72, p = 0.66). This further confirms the pair-wise comparison results as mentioned above. Table 2 Variations of cell density, biofilm thickness and the number of microalgal genera between three study locations. The test used is one way ANOVA. The data were log transformed before running the analysis. Groups Density Couchiching vs Simcoe Simcoe vs Mill Creek Mill Creek vs Couchiching Thickness Couchiching vs Simcoe Simcoe vs Mill Creek Mill Creek vs Couchiching Number of genera Couchiching vs Simcoe Simcoe vs Mill Creek Mill Creek vs Couchiching

df

F

p

1.16 1.16 1.16

3.65 10.84 2.77

0.07 0.004 0.11

1.16 1.16 1.16

1.13 5.75 2.03

0.30 0.02 0.17

1.16 1.16 1.16

22.06 16.87 7.73

b0.001 b0.001 0.01

The substrate used for studying biofilms can have a significant impact on the growth and composition of biofilms. The major surface properties that are known to affect biofilm formation include surface roughness, surface pH, surface tension, surface wettability, presence of bacterial biofilm and grazers (Becker, 1996; Marszalek et al., 1979; Sekar et al., 2004). Glass slides used in this study are inert in nature and have been used to sample natural biofilm communities by many workers (Aloi, 1990; Cattaneo and Amireault, 1992; Danilov and Eklund, 2001; Marszalek et al., 1979). Danilov and Eklund (2001) who compared various substrates for biofilm studies have recommended glass slides as the suitable substratum for natural biofilm studies. The variation of percentage composition of microalgae (diatoms and other forms of microalgae) over the study period showed a slight variation over the one that has been previously reported (Sekar et al., 2004). In the present study, Lake Couchiching showed equal dominance of diatoms and other forms of microalgae during the initial phase of community development which over the period of time shifted to a community dominated by diatoms (more than 50% of the total cell density and the number of genera). Sekar et al. (2004) observed the periphyton community in the fresh water reservoir initially co-dominated by Chlorella vulgaris and Cocconeis scultellum with other forms of diatoms. This trend was slightly different to the one observed by Korte and Blinn (1983) who observed diatoms such as Achnanthes minutissima and Cocconeis placentula dominating the community during the initial phase of community development. In the study from the shallow lakes in Denmark by Liboriussen and Jeppesen (2006), the community initially dominated by diatoms and cyanobacteria was shifted to chlorophyta over time. They observed this shift was related to the variation of concentration of total phosphorus in the water column. In the present study, Lake Simcoe and Mill Creek periphyton community showed the dominance of diatoms throughout the study period. This is not the trend observed previously by Sekar et al. (2004) or Korte and Blinn (1983) or Liboriussen and Jeppesen (2006). Sekar et al. (2004) have noticed an initial dominance or a co-dominance of green algae and diatoms made way to the dominance of Bacillariophyceae which over the time paved way to a community dominated by Cyanobacteria. The nature of study site might have influenced this succession pattern. For example, the study carried out by Sekar et al. (2004) was in a protected freshwater reservoir in a tropical area where the disturbances such as the variation of limnological parameters, water flow patterns and influence of anthropogenic activities are minimal and highly predictable. Many authors have reported on the influence of nutrient concentrations, sunlight availability, depth and or flow rate on the species composition and succession of periphyton community (Cattaneo, 1987; Horner et al., 1990; Lambert et al., 2008; Liboriussen and Jeppesen, 2006; O'Reilly, 2006; Uehlinger et al., 2010). The biofilm community composition of Lake Simcoe and Lake Couchiching showed similarities. The ANOVA results showed no significant variation in microalgal density and biofilm thickness between these two stations however the number of genera showed significant difference (Table 2). Diatoms of the genus Achnanthes were the strong pioneer species and the dominant species throughout the entire sampling period for both the study sites. Achnanthes sp. has been reported as a common pioneer species in a variety of freshwater aquatic habitats such as reservoirs (Sekar et al., 2004), lake systems (Liboriussen and Jeppesen, 2003; Poulickova et al., 2004), and river systems (Veraart, et al., 2008; Winter et al., 2003; Winter and Duthie, 2000a, b). Prostrate diatoms such as Achnanthes sp. and Cocconeis sp. are strong initial colonizers of biofilms because they closely adhere to substrates and have the ability to produce mucilage that facilitates their attachment (Sekar et al., 2004). Also, small sized diatoms (e.g., Achnanthes minutissima) are fast reproducers and can compete

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better for nutrients. This allows them to colonize substrates faster than the rest of the microalgal genera (Sommer, 1981). This is in support of the current study where Achnanthes was observed as the dominant genus. The diversity of Lake Couchiching and Lake Simcoe biofilms was influenced by the substantial dominance of Achnanthes sp. The biofilm diversity was highest during the initial phase of the biofilm development which then steadily declined with age (Fig. 3). As the density of Achnanthes sp. increased, the species richness decreased and the population distribution became skewed. The consistencies between Lake Couchiching and Lake Simcoe biofilms is a reflection of the similar study site conditions including light penetration, substrate, water depth, pH, and temperature. This also showed the influence of human developmental activities on the periphyton species composition and growth (Porter-Goff, 2010; Taylor et al., 2004). The degree of anthropogenic activities at the three sampling locations is different (Table 1). Anthropogenic activities may lead to eutrophic conditions such as have been observed in Lake Simcoe (Winter et al., 2007). On the contrary, some pollutants may act as environmental stressors (e.g. pharmaceuticals, heavy metals, caffeine, etc.) by reducing productivity and diversity (Kolpin et al., 2002). Thus, the extent and type of human activities and the input of pollutants could influence the health of an aquatic ecosystem. The present study provided an opportunity to test the hypothesis that varying degrees of anthropogenic activities in combination with the other environmental factors influence the periphyton community composition and therefore reflects the water quality. Mill Creek, with no exposure to anthropogenic activities recorded lowest density and diversity of periphyton, even though this could be influenced to a certain extent by the existing physical parameters such as availability of sunlight and water flow. The other two sampling sites, Lake Simcoe and Lake Couchiching, being exposed to varying degrees of anthropogenic activities (Table 1) showed greater diversity, higher density of microalgae, and biofilm thickness. Lake Simcoe is known for the higher concentration of total phosphorus than the normal lentic systems for the past several decades (Eimers et al., 2005). This higher concentration has been attributed to the effluent discharge, both industrial as well as agricultural, originating from the surrounding watersheds. Even though the management strategies could bring the phosphorus loading down to a great extent (Winter et al., 2007), the present study also recorded a higher concentration of total phosphorus in Lake Simcoe compared to the other two study locations where total phosphorus was generally below detection by our methods (Table 1). The possible source of this higher nutrient load is the wastewater treatment plant located close to (b1 km from this sampling location) this sampling site as well as agriculture run-off. Thus, these human activities have influenced the microalgal density and diversity at Lake Simcoe sampling location. There are reports available on the varying relationships between the nutrient concentration in water and the microalgal productivity in the freshwater ecosystems (Bowman et al., 2005; Ludwig et al., 2008). Often a good relationship between water nutrient concentration and phytoplankton composition and productivity (Jeppensen et al., 2000; Schindler, 1978; Tilman, 1982) is observed, however, this is not always true with periphyton (Cattaneo, 1987; Dodds et al., 2002; Hansson, 1988). This failure or weak relationship between the water nutrient concentration and growth of periphyton community is because of the poor reflection of water nutrient concentration in the periphyton community (Cattaneo, 1987; Lambert et al., 2008). Additionally, there are various factors other than the nutrient concentration that determine the growth of periphyton community, especially the diatom component, in the periphyton as described by Cattaneo (1987), Tison et al. (2007), Lambert et al. (2008). The circumstances in Lake Couchiching sampling location are slightly different. This location is prone to very high water sports activities as well as boating and fishing activities though out the

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summer. Operation of motor boats and water sports result in the introduction of organic wastes into the water column. This can happen by the direct emission of fossil fuels from the motor boats as well as the re-suspension of organic matter from the sediment. This sampling location showed a high concentration of dissolved organic carbon than Lake Simcoe. There are reports demonstrating a direct relationship between the dissolved organic carbon concentration and the primary productivity/growth of microalgae in the water column (Córdoba et al., 2008; Köster et al., 2005). This higher concentration of the dissolved organic carbon coupled with a slightly higher value of water temperature has resulted in a higher periphyton diversity in this sampling location. This higher growth of periphyton (both in terms of diversity and density) is quite similar to the one observed in Lake Simcoe which has been supported by the ANOVA results (no significant variation between these two sampling locations in density and biofilm thickness, Table 2). Thus, the variations observed in the biofilm parameters could be largely explained by the water quality which is influenced by the anthropogenic activities. Thus, the results are in agreement with the general hypothesis tested during this study. The biofilm composition of Mill Creek was different from that of Lake Couchiching and Lake Simcoe. Cocconeis sp. was the dominant diatom genera of Mill Creek biofilms, which is different from Achnanthes sp. of Lake Couchiching and Lake Simcoe. Achnanthes sp. did have a strong presence in Mill Creek biofilms along with Fragilaria sp. Achnanthes sp. initially dominated the Mill Creek biofilm as a pioneer species, but was quickly surpassed by Cocconeis sp. in density by day 5. The diversity index followed the same pattern as the other study sites (Fig. 3). The diversity steadily decreased as Cocconeis sp. increased in dominance within the biofilm. Veraart et al. (2008) when studying a freshwater stream system found that biofilms were dominated by Cocconeis plancentula and Achnanthes minutissima. Winter and Duthie (2000a, 2000c) found that in a Southern Ontario stream system A. minutissima was the dominant diatom in stream epipelic and epilithic diatom communities. The biofilm community of Mill Creek is similar to that of biofilms studied in other freshwater stream systems. The dominance of Achnanthes sp. and Cocconeis sp. in Mill Creek biofilms is a reflection of the ability of these adnate taxa to tightly adhere to the substrate surface resisting the continuous flowing conditions, even though the flow rate varies with time (0.13± 0.18 m 3/ sec). Flowing environments such as Mill Creek favours low drag, compact, and prostrate forms such as Cocconeis sp. (Larned, 2010). Furthermore, Cocconeis sp. has a rapid doubling rate allowing it to quickly colonize and subsequently dominate the diatoms (Sekar et al., 2004) as observed in the Mill Creek biofilm community. These factors, in addition to the anthropogenic activities contributed to the difference in species composition between sampling locations. It has been reported that flowing conditions do have a strong influence on the periphyton growth and community development (Uehlinger et al., 2010). Horner et al. (1990) when studying the periphyton community composition and growth in the laboratory reported that moderate flowing conditions of the range 10–60 cm/sec supported the highest periphyton biomass build-up. Thus the comparison between the lakes and between lakes and stream demonstrates the influence of variation of physical and chemical composition of the ecosystem on the periphyton community even within a same watershed. Lake Simcoe biofilm samples had the highest biofilm microalgal densities relative to Lake Couchiching and Mill Creek. High nutrient loadings have been documented in Lake Simcoe since the early 1900s, which would contribute to higher primary productivity of diatom biofilms leading to increased cell densities (LSEMS, 2003). Lake Simcoe has also experienced a substantial growth of invasive zebra mussels (Dreissena polymorpha), which has been implicated in water-clarifying effects (Cecala et al., 2008). Cecala et al. (2008) found that the establishment of zebra mussels has lead to a 4% increase in whole-lake epipelic and epilithic algal primary productivity and has been attributed to the deeper

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penetration of sunlight due to water clearance by zebra mussel. The present study also showed a slightly higher values of total phosphorus concentration and lowest concentration of dissolved organic carbon in Lake Simcoe as compared to other two localities (Table 1) and these factors might have contributed to the higher biofilm algal density. Contradictory reports on the relationship between the nutrient concentration and periphyton composition and growth are available. While Masseret et al. (1998) have shown positive influence of nutrients on the species composition and growth of periphyton other studies such as Bowman et al. (2005) and Ludwig et al. (2008) did not support this idea. Nevertheless, nutrient availability showed a direct influence on the growth of phytoplankton, if not on periphyton (Tomasky et al., 1999). The factors contributing to the higher productivity of Lake Simcoe biofilms requires further investigation in order to provide better insight into the relationship between the environmental parameters and microalgal biofilm growth. Mill Creek on average had the lowest biofilm densities and thicknesses, whereas Lake Couchiching has the highest biofilm thickness and Lake Simcoe has the highest density. The low density and thickness values of Mill Creek can be attributed to differences in a variety of factors including high canopy cover (low light availability), flow rate (0.13 ± 0.18 m 3/sec), nutrient concentration (Table 1), lower depth, and lower temperature (Table 1). The Lake Couchiching and Lake Simcoe sampling sites had very low canopy cover and therefore received more sunlight, whereas the Mill Creek sampling site had a thick canopy hindering light penetration. A decrease in light availability has a direct negative impact on algal primary productivity and has been found to be one of the most significant factors determining periphyton growth (Veraart et al., 2008; Von Schiller et al., 2007). The canopy cover has been related to developmental activities such as a low canopy cover in areas with high developmental activities as reported by Lhotka and Loewenstein (2006). Mill Creek biofilm samples were also exposed to increased hydraulic forces due to their continuous exposure to flowing water even though the flow rate varies with time. Flow events can disrupt biofilm formation through the removal of biomass once the structural threshold is exceeded or through increased sediment abrasion (Larned, 2010). Hydraulic forces also disrupt diatom immigration, biomass accumulation, species composition, and structural development (Larned, 2010). Similar observation was reported by Burkholder et al. (1990) when studying the influence of water turbulence on the biofilm thickness in the boundary layer between water and the study substrata. The water turbulence or microcurrents on the substratum surface was reported to influence variation in the biofilm thickness on the slide surfaces (Neu and Lawrence, 2006). In their study on bacterial biofilms conducted in a bioreactor, the flow patterns have shown to result in population heterogeneity over space and time. Similar observation was made in a natural microalgal biofilm by Sekar et al. (1998) who observed colonization of stalked diatoms towards the edges of the slides while non-stalked and adnate diatoms were found largely colonizing towards the center. They have also attributed the variation in colonization to the microcurrents at the surface of the slides. On a different note, Christensen and Characklis (1990) noted that the biofilm thickness and biomass were the function of the age and the thickness, which was influenced by the species diversity in the biofilm. In the present study too, this was found to be true. Lake Simcoe showed the most diversified generic composition (Fig. 3) while the biofilm thickness was not the highest (Fig. 1). On the contrary, Lake Couchiching with highest species diversity produced highest biofilm thickness (Fig. 1). These all translate into the species specificity in the biofilms and their dominance in regulating the biofilm characteristics such as the biofilm thickness. The low microalgal density and biofilm thickness in the Mill Creek therefore could be the result of the combined effects of low light availability, hydraulic forces (increased shear forces) and other physical conditions therefore form a great topic of future study. On the other hand, the higher organic

load in the water may have resulted in a lower algal density, but higher diversity as seen in Lake Couchiching. A higher total phosphorus concentration and lower organic load might have resulted in a higher algal density in biofilms as seen in Lake Simcoe. The specific growth rates (Fig. 4A, B, C) and regression (Fig. 2) for the density, thickness, and number of algal genera revealed a limited interaction between these variables in all the study sites. The specific growth rates of algal density and thickness did not closely reflect one another indicating a poor association between the two variables. The specific growth rates for the number of algal genera were highly variable and did not demonstrate the growth patterns of either density or thickness (Figs. 1, 4). The two lentic ecosystems showed similarity in periphyton species diversity and its dynamics over time, even though the human activities in these ecosystems were different. The species diversity was high during the initial phase of periphyton community development which over the period of time lowered owing to the dominance of certain algal genera and by the disappearance of certain others (Appendices 1a, b, c). This was true in case of lotic system (Mill Creek) as well except for the dominant microalgal genera, which were different from the lentic systems. Even though the variations can be influenced by the changes in physical conditions at the sampling locality such as the water flow, light penetration, nutrient concentration etc. influence of human activities is working as the main driving force of periphyton community development. As demonstrated in the foregoing sections, the colonization of substratum surfaces is also influenced by the physical attributes of the sampling locality as some adnate algae prefers to colonize substratum surface exposed to flowing conditions while prostrate algae colonize the surface in lentic systems with minimal water currents (Sekar et al., 1998). Microalgal biofilms are sensitive to the environmental parameters of an aquatic system and thus can act as an indicator of ecosystem perturbations. Both periphyton and phytoplankton have been used as biological indicators of trophic status in aquatic ecosystems (Almeida, 2001; Kelly and Whitton, 1995; Lambert et al., 2008; Winter and Duthie, 2000a; Wu and Kow, 2002). Lambert et al. (2008) found that periphyton on all substrata were more responsive to increased recreational activities than phytoplankton due to their position close to the land-water interface. A variety of trophic indices based on periphyton and diatoms have been developed by various workers. Some examples are: Specific Pollutosensitivity Index (Coste, 1986), European Economic Community Index (Descy and Coste, 1991), Generic Diatom Index (Rumeau and Coste, 1988), Sladecek's Index (Sladecek, 1986), and the trophic diatom index (Kelly and Whitton, 1995). In the present study the variation in species diversity and algal density with respect to the water quality parameters such as the organic carbon content and total phosphorus concentration driven by the anthropogenic activities provided evidence that the periphyton community composition and dynamics can be used as indicators of the health of inland water ecosystems. The variations observed in terms of species diversity, species composition, microalgal density and colonization patterns of periphyton reveal the influence of both anthropogenic activities and the physical conditions of the environment. This strengthens the hypothesis tested; however, there are inconsistencies with respect to the variations. For example, the species diversity or colonization pattern did not produce a clear difference between the two lentic systems even though it showed variations in biofilm thickness as well as number of microalgal genera. However, the lentic systems showed variations with the lotic system especially in species composition and colonization pattern. Thus, more research is needed to get a thorough understanding of microalgal biofilm community composition and dynamics with respect to ecosystem perturbations and variations in physical conditions before suggesting a system of biological factors to indicate the health of these ecosystems.

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Conclusions The study showed periphyton community is dominated by diatoms rather than other forms of microalgae throughout the study period. Lake Couchiching exposed to the highest degree of human activities resulted in highest species diversity and the number of genera of microalgae. The relationship between the human activities and periphyton community composition has been documented earlier by Porter-Goff (2010). Lake Simcoe with highest phosphorus concentration among the three localities showed the highest microalgal density. Mill Creek with the lowest sunlight availability, continuous water flow and lowest influence of human activities showed lowest species diversity and biofilm thickness. While Achnanthes sp. was the dominant genus in lentic systems, Cocconeis sp. was found to be the dominant genus in the lotic system. The ANOVA results showed significant variation in number of genera of microalgae between the three study locations while the other two biofilm characteristics did show significant variation. The significant variation in microalgal genera between the three sampling locations indicates the potential to use this aspect as an indicator of water quality in the lentic and lotic ecosystems. The relationship between the biofilm characteristics such as the biofilm thickness, microalgal density and number of microalgal genera showed mixed results in the three sampling locations. The results showed density of microalgae or number of genera may not decide the thickness of biofilms, but, it depends on the species present and the size of the microalgae. Acknowledgements Lakehead University Senate Research Committee Research Fund is acknowledged for the financial support. Christopher Vaillant is acknowledged for his help during the field and laboratory studies. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jglr.2012.06.014. References Almeida, S.F.P., 2001. Use of diatoms for freshwater quality evaluation in Portugal. Limnetica 20 (2), 205–213. Aloi, J.E., 1990. A critical review of recent freshwater periphyton field methods. Can. J. Fish. Aquat. Sci. 47, 656–670. American Public Health Association, 2005. In: Eaton, A.D., Clesceri, L.S., Rice, E.W., Greenberg, A.E., Fransen, M.A.H. (Eds.), Standard methods for the examination of water and wastewater. Centennial Edition. Washington, DC. Bakke, R., Olsson, P.Q., 1986. Biofilm thickness measurements by light microscopy. J. Microbiol. Methods 5, 93–98. Barranguet, C., Van Beusekom, S.A.M., Veuger, B., Neu, T.R., Manders, E.M.M., Sinke, J.J., Admiraal, W., 2004. Studying undisturbed autotrophic biofilms: still a technical challenge. Aquat. Microb. Ecol. 34, 1–9. Barranguet, C., Veuger, B., Van Beusekom, S.A.M., Marvan, P., Sinke, J.J., Admiraal, W., 2005. Divergent composition of algal-bacterial biofilms developing under various external factors. Eur. J. Phycol. 40, 1–8. Becker, K., 1996. Exopolysaccharide production and attachment strength of bacteria and diatoms on substrates with different surface tensions. Microb. Ecol. 32, 23–33. Besemer, K., Singer, G., Limberger, R., Chlup, A., Hochedlinger, G., Hodl, I., Baranyi, C., Battin, T.J., 2007. Biophysical controls on community succession in stream biofilms. Appl. Environ. Microbiol. 73 (15), 4966–4974. Bowman, M.F., Chambers, P.A., Schindler, D.W., 2005. Epilithic algal abundance in relation to anthropogenic changes in phosphorus bioavailability and limitation in mountain rivers. Can. J. Fish. Aquat. Sci. 62, 174–184. Burkholder, J.M., Wetzel, R.G., Klomparens, K.L., 1990. Direct comparison of phosphate uptake by adnate and loosely attached microalgae within an intact biofilm matrix. Appl. Environ. Microbiol. 56, 2882–2890. Campbell, J.S., McCrimmon, H.R., 1970. Biology of the emerald shiner Notropis antherinoides Rafinesque in Lake Simcoe, Canada. J. Fish Biol. 2, 259–273. Cattaneo, A., 1987. Periphyton in lakes of different trophy. Can. J. Fish. Aquat. Sci. 44, 296–303. Cattaneo, A., Amireault, M.C., 1992. How artificial are artificial substrata for periphyton. J. North Am. Benthological Soc. 11 (2), 244–256.

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