2.0.20.0, http://www.granitebaysoftware.com).
After an acclimatisation period of 24 h to allow macrofaunal establishment within the aquaria, luminophores (Partrac Tracer 2290 pink, size 125–355 μm, 20 g aquaria−1) were evenly distributed across the sediment surface immediately prior to the start of each time lapse sequence (1 image 15 min−1 for 96 h, i.e. 384 images sequence−1). Images were saved with colour JPEG (Joint Photographic Experts Group) compression. Bioirrigation activity was estimated from changes in water column concentrations of an inert tracer, (Sodium bromide, NaBr, dissolved in seawater [Br−] = 800 ppm, 5 mM, stirred into the overlying seawater) for RG7204 manufacturer 8 h on day 8 of each experimental run, during which time the aquaria were isolated from the seawater supply. Water samples (5 ml) were taken at 0, 1, 2, 4, and 8 h (following Forster et al., 1999 and Mermillod-Blondin et al., 2004) and immediately filtered (47 mm ∅ GF/F filter) and frozen (−18 °C). [Br−] was analysed using colorimetric analysis using a FIAstar 5000 flow injection analyzer (FOSS Tecator, Höganäs, Sweden). Additional water samples (50 ml, 47 mm ∅ GF/F filter) were taken at 0 and 8 h to determine any changes in nutrient concentrations (NH4–N, NOx–N, PO4–P and SiO2–Si) of the
overlying water column and analysed using a nutrient autoanalyser (Branne and Luebbe, AAIII). The distribution of luminophore particles within the sediment profile was quantified, following Solan et al. (2004b), using a Dinaciclib molecular weight custom made semi-automated macro in ImageJ (v. 1.44), a public domain Java based programme (http://rsbweb.nih.gov/ij/download.html). The macro sequentially opens each image and splits it into three separate colour (RGB) channels. The user traces the sediment–water interface (=upper region of interest) using the segmented line tool in the green channel. Identification Phospholipase D1 of luminophores below the sediment–water interface is achieved in the red channel using an appropriate
threshold level that distinguishes the luminophore particles from the background sediment. The threshold image is converted to a bitmap (0 = background sediment, 1 = luminophore pixels), allowing the total number of luminophore pixels in each row to be summed for each depth row. In addition, the mean (lummean), median (lummed) and coefficient of variation (lumCV = standard deviation/mean) of the vertical distribution of luminophores recovered from the final image in each sequence were calculated. A process-based, spatially explicit simulation model (Schiffers et al., 2011) was applied to the timelapse sequence data (1 image 30 min−1 for 72 h, i.e. 145 images sequence−1).
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