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Chapter 4: Biodegradation processes of cellulosic wet wipes in aquatic…

- - - - Determined by:
        
        Initial tensile strength of control strips (wetted in tap water, dried [ 40.c], stored and processed identically to treatment strips)
        
        Tensile strength loss = [1 = Tensile Strength treatment strips / Tensile Strength reference strips ] x 100/ Incubation Time
        
        TS Treatment strips = maximum TS recorded for each strip incubated in the field
        
        TS Reference strips = mean tensile strength of control strips not incubated in field
        
        Incubation time = number of days strips were incubated in the field
        
        Tensile strength loss = % of initial tensile-strength lost per day of incubation (after Teigs et al. 2013)
        
        Statistical analyses
        
        Nested ANOVA to evaluate differences in mean % TSL per day among ecoregions and cotton materials (fixed effect) using streams nested within ecoregions as a random effect
        
        linear mixed model used to test variation of % TSL per degree-day along the acidification gradient using the fabric type and the acid neutralizing capacity, or aluminimum concentrations, as fixed effects and the streams as random effects
        
        Similarly, the relationships between **% TSL per day and nutrient concentrations were examined using linear mixed models** - nutrients conc. and fabric type (fixed effects) and ecoregions and streams nested within ecoregions (random effects)
        
        Variation in microbial abundance, phylotype richness and diversity among cotton fabric incubated along the acidification gradient - mixed ANOVA with cotton type (Fixed effect) and streams (random effect)
        
        All Mixed Models - REML and lmerTest
        
        Significance of fixed effects derived using Satterthwaite approximation for degrees of freedom as it produces acceptable Type 1 error rates for smaller sampes
        
        NMDS analyses of fungal and bacterial community profiles used to assess differences among sites and among cotton fabric using the function metaMDS in vegan
        
        Bray Curtis coefficient (quantify dissimilarity among sites and fabrics based on community profiles)
        
        Goodness of fit estimated with stress function (0-1)
        
        Axes values from NMDS correlated using SPearman rank correlation (cor.test) with ANC and aluminium concentration values to identify variables that corresponded to among-site difference in microbial community profiles
        
        Linear regression models performed to link % TSL per day of each cotton fabrics using the caret and mass packages
        
        For models, data divided into two parts - training data set 80% used to build the model and validation data set (i.e. 20% of the original data set) used to guagse the models performance - performance and accuracy estimated using root mean square error and Pearson correlation coefficient between observed and predicted values of validation data set
        
        Treatment strips placed in shallow trays of 70% ethanol, cleaned for 30s with light brush to remove adhering sediment/debris
        
        Tensiometer measurements - mark 10, motorized test stand, pulled at a rate of 2cm/min
        
        Accounting for differences in decomposition rate (i.e. temperature differences across acidification gradient): TS also expressed as % of initial TSL per degree-day by replacing time with degree-days in the Equation
        
        Degree days estimated by summing the mean daily temperatures at each stream, based on hourly readings greater than 0 degrees celsius
        
        Time to yield 50% TSL estimated by fitting the tensile loss to a function of incubation time according to a linear model (better AIC than exponential model) using the time series experiments performed in three reference streams
    - - Strips placed in shallow trays filled with distilled water (not ethanol like other strips) and cleaned with brush
        
        Pieces of cotton (1.5 x1.5cm) crushed in Eppendorf tubes containing 750 microlitres of PowerBead Tube solution from PowerSoil DNA Isolation Kit - total DNA extracted using PowerSoil DNA Isolation Kit
      - Abundance of fungi and bacteria estimated by qPCR using primer sets Fung5F/FF390R, 968F/1401R - target fungal 18S rRNA genes and the bacterial 16s rRNA genes