Dosis de diálisis alcanzada en pacientes en hemodiálisis según el acceso vascular empleado
hombres 45-50 L); Kt bajo (mujeres<40L;
11.4.1 Regular Monitoring Flowering Phenology and Grazed Treatment Clippings Biomass
During the regular monitoring parameters taken for ‘plant fitness’ analysis, flowering phenology was also recorded from January when the first flower was produced. Parameters were; number of flowers per plant; and flowering period (Carter et al., 1997; Vuckovic et al., 2007).
In March 2012 the grazed treatment commenced, therefore a further parameter, (dry biomass of clippings taken) was also included. Monitoring dates as well as parameters measured are shown in Table 14.
Table 14. Regular Herbivore Requirement Parameters Taken First (main) batch Second batch Reference used in
results section
Parameters measured of each plant
3rd January 2012 17th January 2012 January 2012 Flower number 31st January 2012 14th February 2012 February 2012
29th Feb, 2nd & 5th March 2012
13th & 15th March 2012
March 2012 Flower number
Grazed treatment weights 27th March 2012 10th April 2012 April 2012 Flower number
Grazed treatment weights 24th April 2012 8th May 2012 May2012
22nd May 2012 5th June 2012 June 2012 19th June 2012 3rd July 2012 July 2012
15th July 2012 31st July 2012 August 2012 All Harvest measurements 23rd August 2012 6th September 2012 September 2012 Flower presence
25th April 2013 25th April 2013 April 2013 Flower number
Grazed treatment weights 25th May 2013 25th May 2013 May 2013 Flower number
Grazed treatment but clippings not weighed 27th June 2013 27th June 2013 June 2013
25th July 2014 25th July 2014 July 2013 27th August 2013 27th August 2013 August 2013
11.4.2 Harvest Flowers
At harvest time, flower number was again taken, flower aroma was also graded both during pre-harvest measurements and after harvest following methods described by Murrell et al. (1982) whereby “single umbels are sniffed and assigned a rank” of 0 (no odour) to 4 (strong odour) (Murrell et al.,1982). During harvest, flowers were placed in sealed plastic bags to allow
compounds to concentrate inside. The air sample inside the bag was then sampled near the end of the day and again ranked from 0 (no odour) to 5 (very strong odour).
After the flower scent test was complete all flowers were fresh weighed, these were then dried and reweighed for the dry biomass measurement (Figure 29).
Figure 29. Separating dry plant parts and weighing dry flower biomass.
11.4.3 Growth Habit and Hirsuteness
Additional observations were made immediately prior to harvest, of growth habit (prostrate, decumbent or erect) and hirsuteness (glabrous, very sparsely hairy, sparsely hairy or hirsute) (Smith et al., 2009).
11.4.4 Harvest Biomass
Plants were harvested by cutting to 5cm above soil level. Although a shorter cut than the grazed treatment, it was thought that this would still be
comparable to cattle grazing due to Natural England guidelines (2010)
advising grazing to a height of 5-7cm. With this height plants could still survive for further analysis yet a suitable amount of plant matter would be obtained. Fresh and dry shoot weights were calculated.
11.4.5 Plant Chemical Analysis
To establish the nutritional value and levels of defence against herbivores, plants were analysed to determine nitrogen and hydrogen cyanide (HCN) content of the leaves (Table 15). Originally tannin levels were to be tested, but due to limited amounts of suitable plant material, leaf-nitrogen and leaf- HCN analyses were prioritised.
Figure 30. Digestion of dried plant material with sulphuric acid and a Calgon tablet to transform organic nitrogen into ammonium for leaf-nitrogen analysis.
Table 15. Methods of plant chemical analysis
Chemical Solution/Method Reference
Leaf Nitrogen (N)
Determined by Kjeldahl
For each sample, approximately 0.5g of dry leaves were accurately weighed into a Kjeldahl digestion tube. Organic nitrogen was first transformed into ammonium by digesting the dried leaves with addition of 5mL sulphuric acid and a Calgon tablet as a catalyst (a blank is digested first) (Figure 30). The amount of ammonium produced was allowed to cool and 20mL of deionised water added. Sodium hydroxide was then added (25mL) to the solution to increase pH. The solution was steam distilled in the Kjeldahl apparatus, boric acid indicator was used in a 50mL collection flask, placed under the condenser tube. Sulphuric acid was then used to titrate the solution until it turned pink. The formula to determine percent leaf-nitrogen was then calculated:
%N = (T – B) x N x 1.401 g sample Key: T = mL of titrated sample B = mL of titrated blank N = acid normality Baker & Thompson1 992 Hydrogen cyanide (HCN)
Determined using the Dawson’s qualitative method. Sodium picrate papers were prepared beforehand by cutting chromatography paper into strips which were then soaked in a solution of sodium carbonate, picric acid and distilled water. A small leafy branch of approximately 0.1g (exact weights recorded) was randomly selected from the fresh plants. Plant material was placed in a boiling tube, mixed with five drops of toluene and crushed with a rod to aid cell breakdown. Picrate papers were then placed in the tube, suspended above the sample and stoppered (Figure 31). Tubes were left in a fume cupboard for two hours then picrate papers removed. In the presence of HCN, the yellow picrate paper would turn an orange/brown colour. Gebre- hiwot & Beuselinck2 001; Smith et al., 2005
Figure 31. Picrate papers suspended over crushed plant samples with toluene for leaf-HCN analysis.
11.5 Post-Harvest: Grading Leaf-HCN – The Eyedropper
Technique
For the qualitative leaf-HCN results obtained, picrate papers used in the leaf- HCN test were graded by eye according to the darkness of colouration from 0 (no colour change) to 4 (dark orange/brown) a method adapted from that of Egan et al. (1998). These eye-graded results were also used for ‘presence and absence’ tabulation.
To quantify these results more accurately for both statistical analysis and to eliminate human error or bias, a novel method was developed. All picrate papers were photographed on A4 paper using a Canon Powershot G12 camera (2736 pixels, 180dpi), at no flash, auto mode, held approximately 30cm above the papers in natural daylight conditions on the day of analysis. Two batches of picrate papers were made for the experiment which differed slightly in colour. To counteract this inconsistency each page of used picrate papers was photographed together with an unused standard picrate paper from that batch as a control (Figure 32).
Figure 32. Picrate papers image for The Eye-dropper Technique. The single paper on the right of the page is the standard.
Images were opened up in Adobe Illustrator CS version 11.0.0 (Adobe Systems Incorporated, 2003) and the eyedropper tool used to first select six random areas from top to bottom of the standard picrate paper. From the colour identified by the eyedropper tool the green hue number (degrees of colour) was recorded each time. It was found that the darker orange the picrate paper was, the lower the green hue would be and was the most reliable of the three colours to use (based on trials using both visual ranking and the eyedropper technique). Out of these six figures the minimum value was picked as the standard zero HCN for that batch. The eyedropper was then placed over the darkest area on the bottom of each picrate paper with the green hue figure recorded. Each green hue figure was subtracted from the standard and the difference used as the leaf-HCN amount. To ensure those with no leaf-HCN were recorded as such, the confidence interval from the six standard results was used, i.e. a confidence interval of seven would mean that a difference between the picrate paper and the standard, up to and including seven, would be replaced with a zero.