An overview over the frequency of parameters that were measured, sampling performed for laboratory analysis with additional instruments and methods that were used is given in table 1.
Table 1: The frequency of sampling and analysis, instruments and methods that were applied.
Days of sampling pr. week Parameters Instrument/Methods
3 pH YSI ProPlus Multiprobe
3 Temperature (oC) YSI ProPlus Multiprobe
3 Dissolved oxygen (%) YSI ProPlus Multiprobe
3 Conductivity (µS/cm) YSI ProPlus Multiprobe
3 Turbidity (NTU) Turbidity Meter 2100 P
1 Nitrogen (mg/L) APHA (2005) 4500
1 Phycoerythrin (g/m2) Bennett & Bogorad (1973)
3 Photographs Olympus Though TG-850
3.3.1 Nutrient manipulation
In order to maintain even nutrient levels across all experimental groups and to prevent dilution caused by continuous water addition, the water was spiked with nitrate (NO3-) and phosphate (PO43-). The nutrient concentrations were based on the optimal concentrations for Phormidium growth and proliferations at 0.01 mg phosphorus/L (Wood et al., 2015) and a modified concentration of 0.5 mg nitrate/L
Stock nitrate solution: 1.39 g sodium nitrate (NaNO3) was dissolved in 1 L Milli-Q water, making 1000 mg/L nitrate stock solution.
Stock phosphorus solution: 1.83 g dipotassium hydrogen phosphate (K2HPO4) was dissolved in 1 L Milli-Q water, making 1000 mg/L phosphate stock solution.
Estimated volume of stock solution used for 70 L water (C1V1 = C2V2) was 35 mL/70 L for NO3-
and 0.7mL/70 Lfor PO43-.
The first day of the experiment each header tank was spiked with 105 mL NO3- and 2.1 mL PO43, which is a three-folded volume of the estimated concentration of nutrients for 70 L conducive to enhance growth development at the experimental start-up. When the outflow tanks were topped up with river water, the water added was spiked with an estimated nutrient concentration depending on the volume of water added. In addition, each header tank was spiked with 105 mL NO3- and 2.1 mL PO43- after conducting nutrient sampling once a week.
3.3.2 Nutrients
Water samples (150 mL) from each header tank were collected in a glass bottle for nitrate/nitrite, ammonium nitrogen and dissolved reactive phosphorus nutrient analysis. The water samples were stored on ice until processed (within 2 hrs.). Approximately 45 mL water sub-sample was filtered through GF/C filters (Whatman, UK) directly into 50 mL falcon tubes.
All samples for nutrients were stored frozen (-20˚C) until analysis. The nutrient sampling was conducted once a week, once before nutrient spike manipulation and after manipulation.
3.3.3 Physio-chemical water characteristics
Using a handheld YSI ProPlus multiprobe temperature (oC), pH, dissolved oxygen (%) and conductivity (µS/cm) were measured on Mondays, Wednesdays and Fridays throughout the five-week sampling period, in both header tanks and outflow tanks. Continuous water temperature was measured at five minute intervals using temperature loggers (Hobo Pendant, Onset) attached to a cobble in the outflow tank. Turbidity (NTU) was measured using the Turbidity Meter 2100 P on Mondays, Wednesdays and Fridays throughout the five-week sampling period. Three samples from each header tank were measured and an average value calculated.
3.3.4 Biomass
Samples were collected for chlorophyll-a (µg/L), phycoerythrin (PE g/m2), ash-free dry weight (AFDM g/m2) and Phormidium biovolume (BV mm3/m2). The first assessment took place on day five, then continuously every seventh day throughout the experimental period. Eight rocks
from each channel set were chosen for biomass monitoring every week, resulting in a total of 32 cobbles sampled per week. For every cobble selected for sampling, growth was documented by taking a digital photo for visual comparison and analysis of changes in mat size.
Milli-Q water (100 mL) was added into a plastic tray in which the selected cobble was placed.
Biomass material was removed by thoroughly brushing the surface area of the cobble with a plastic brush. The biomass of each cobble was transferred into 100 mL bottles and put on ice for further analysis preparation. For analysing surface area, each cobble was measured by covering it with tin foil and removing the edges that were not within the surface area (see chapter 3.3.5). After scrubbing and measuring surface area, the sampled cobbles were returned to the channels upside down to prevent them from being analysed repeatedly.
By using an ultra turrex (Ultra Turrex T8.01 Netzgerät. IKA-WERKE) the 100 mL biomass samples were homogenized at full speed for maximum one minute. For cell counting and Phormidium biovolume, 1.5 mL of the homogenized sample was pipetted into a 1.7 mL Eppendorf tube and preserved with two drops of Lugol’s solution. The sub-sample was stored in the dark at room temperature until further analysis.
A 30 mL sub-sample was pipetted into a 50 mL Falcon tube for AFDM analysis and stored frozen (-20˚C). Two sub-samples (30 mL) were filtered (GF/C filters Whatman, UK) for chlorophyll-a and phycoerythrin. The filtered water was discarded and the filters were folded, inserted to 1.7 mL Eppendorf tubes and stored frozen (-20˚C).
3.3.5 Surface area
For determining the surface area of each cobble, a standard curve was developed. Triplicated square pieces of tin foil measuring 1 cm2, 25 cm2, 50 cm2, 100 cm2 and 225 cm2 were weighed, and the relationship between weight and areas established using linear regression (Fig. 14).
The tin foil for each rock was then weighed, and equation 1 was used to determine the surface area:
Equation 1:
𝑥 = 𝑌 3.3661
x: the rock surface area in cm2
Y: the tin foil weight (mg) of tin foil covering each surface area
Figure 11: Linear regression between weight (mg) and area (cm2) of tinfoil standards used to determine surface area of each rock replicate.
3.3.6 Changes in size of Phormidium mats
Throughout the whole experiment 48 selected cobbles where photographed three times a week to compare the growth (cm2)of the mats. A plastic object of a known size was used in each photograph for scale. The photographs were taken at the experimental site, and the Phormidium-mat surface area in the images were further analysed using the image processing software, e.g. Image J as described in Annex 3.
y = 3.3661x R² = 0.9951
0 100 200 300 400 500 600 700 800
0 50 100 150 200 250
Foil weighth (mg)
Area of squares of tin foil (cm2)