outline naomi

7/30/2019 Outline Naomi

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FORMULATION AND EVALUATION OF NUTRIENT SOLUTION DERIVED

FROM FERMENTED Sargassum muticum FOR HYDROPONICS LETTUCE

(Lactuca sativa L.) PRODUCTION1/

1/A thesis outline presented in partial fulfillment of the requirements for

graduation of the degree of Bachelor of Science in Chemistry, College of Arts andSciences, Visayas State University, Visca, Baybay City, Leyte. Prepared in the

Department of Pure And Applied Chemistry (DoPAC) under the guidance and

supervision ofDr. Felix M. Salas.

NAOMI LEEH LUZARESPOBADORA

CHAPTER I

INTRODUCTION

Nature and Importance of the Study

Nowadays, farmers are faced with the problem of where to plant their crops.

Some farmers do not have their own land. They are only adapting the tenancy system.

They till the soil plant the crops and let them grow but are only given a share of the fruit

of their labour. So those farmers who wish to commercialize the crops they grow cannot

do so. One way of resolving this problem is through hydroponics.

Hydroponics simply means growing plant without soil. All the essential nutrients

for the plants are called hydroponic nutrients which are dissolved in water and directly

absorbed by the plant. Thus, the farmers who do not have their own land can produce

crops for commercial purposes. The crops can be grown in their backyard or in places

where in-ground agriculture or gardening is not possible. In general, hydroponic gardens


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require only about 20% of the overall space required of soil gardens for the same

vegetable production (http://www.hydroponicvegetablegardening.com) . Moreover,

hydroponics provides less worry on the amount of water because the water stays in the

system and can be reused. Due to the controlled system, no nutrition pollution is released

into the environment. Another advantage of hydroponics gardening is that it preserves the

quality of the soil. It minimizes if not totally eliminate the damage done by soil

gardening. Hydroponics gardening involves a natural farming practice because it

promotes high productivity and high profitability with lower labour requirements and a

minimum negative impact on the environment. This type of gardening contributes to the

realization of sustainable agriculture for small farmers.

One of the foreseen problems in hydroponics is the expensive nutrient solution

available in the market which is not affordable to the farmers. One way of solving this

problem is the utilization of the seaweed, Sargassum muticum, as alternative nutrient

solution on hydroponic plants. Sargassum muticum is a seaweed species abundant in the

coastal areas of the Philippines. It usually grows near the shore lines at a rapid rate.

In this study, organic solution will be formulated and compared to the commercial

hydroponic solution. Fermented Sargassum muticum will be used as organic solution and

lettuce as hydroponic plant. Lettuce is a green leafy vegetable of high nutritional value.

Being high in nutritional value, it offers a number of health benefits. It is also a high

value crop plant which is relatively easy to grow.
http://www.hydroponicvegetablegardening.com/http://www.hydroponicvegetablegardening.com/


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Objectives of the study

This study will be conducted with the following objectives:

1. To determine the amount of nitrogen, phosphorus, potassium, sulfur, calcium,and magnesium in the fermented Sargassum muticum;

2. To formulate a nutrient solution derived from fermented Sargassum muticumbased on its nutrient analyses and in reference to the commercial hydroponic

solution; and

3. To evaluate the applicability of the formulated nutrient solution for lettuceproduction.

Scope and Limitation of the Study

This study will be limited to the analysis of nitrogen, phosphorus, potassium,

sulfur, calcium, and magnesium. The formulation of nutrient solution will be based on the

chemical analysis of the fermented Sargassum muticum and that of the commercial

nutrient solution (hydrosol) for hydroponics system. Appropriate inorganic salts will also

be added based on the difference between the nutrient analysis ofSargassum muticumand

that of the hydrosol.

Time and Place of Study

The chemical analyses will be conducted at the Department of Pure and Applied

Chemistry (DoPAC) of the Visayas State University Visca, Baybay City, Leyte. The

evaluation of the nutrient solution will be done at the Department of Horticulture of the

Visayas State University form June 1, 2012 to December 30, 2013.


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CHAPTER II

REVIEW OF LITERATURE

Hydroponics Vegetable Production

Hydroponics is the art and science of growing crops in soilless medium. Instead

of soil, water enables the transport of nutrients necessary for the development of the

crops because the nutrients are concentrated in water solutions\, the crops are provided

with the best environmental conditions. The plants receive maximum feeding of nutrient

elements in a well-balanced range and in turn produce crops of excellent quality in terms

of flavour and palatability of fruits.

The introduction of hydroponics dates back to the mid-1930. Since then there has

been many advances made in soil less cultivation. The technique has been applied to a

number of crops including wheat, potato, tomato, cabbage, pechay, lettuce and many

others. In Japan, hydroponics was introduced by the US Army after the World War II and

upon increase in vegetable injury from microorganisms in the soil

(http://www.mayhillpress.com/ideal.html)

Hydroponic lettuce has become popular since consumers are becoming more

concerned about where these crops are grown. Field grown lettuce pose problems to the

food service industry because of the high labor cost in removing dirt with grit in the

plant. By growing lettuce in NFT (Nutrient Film Technique) hydroponic system, these

problems are eliminated (http://www.cropking .com/NFT_Lettuce_Herbs.shtml).

Compared to NFT, SNAP hydroponics system is simpler and more budget-friendly. This
http://www.mayhillpress.com/ideal.htmlhttp://www.mayhillpress.com/ideal.html


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type of hydroponics system is commonly used in small scale production for household

consumption. This only uses discarded household items.

Lettuce production often makes use of grow enhancers for bigger yields and better

crops. These enhancers provide plants with essential B vitamins which stimulate plant

growth. Grow enhancers are also often packed with seaweed or kelp extract which

contain plant hormones that encourages plant growth

(http://hydroponiclettuce.blogspot.com/) .

Applicability ofSargassum muticum as Nutrient Solution

Sargassum is a species of brown seaweeds growing abundantly in coastal areas

(see Figure 1). This species is considered a nuisance because it clogs the intake pipes of

boats, produce offensive smells when rotten and cause loss in amenity and recreational

use of water areas. Dense growth of Sargassum also affects species diversity of

indigenous marine fauna and flora in shallow subtidal regions

(http://www.issg.org/database/species/ecology.asp?si=727&fr=1&sts) . However, this

otherwise considered nuisance offers a number of uses. In Japan, it is added to soups and

fermented with other soy sauce ingredients to create specific flavour. It is also home to

some sea creatures and serves as their source of food. In some regions, it is collected as

fertilizer (http://www.wisegeek.com/what-is-sargassum.htm)
http://hydroponiclettuce.blogspot.com/http://www.issg.org/database/species/ecology.asp?si=727&fr=1&stshttp://www.issg.org/database/species/ecology.asp?si=727&fr=1&stshttp://hydroponiclettuce.blogspot.com/


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Figure 1. Sargassum muticum(http://www.google.com.ph/imgres?imgurl=http://www.seaweed.ie/_images/)

History dates the use of seaweeds, specially the large brown seaweeds, by coastal

people to fertilize their lands. In Brittany (France), farmers regularly collect brown

seaweeds to fertilize their fields. In Cornwall (United Kingdom), they mix the seaweed

with sand, let it rot and then dig it in. In Puerto Madry (Argentina), green seaweeds are

composted and used in trials for growing tomato plants. Because brown seaweeds are

most readily available in large quantities, they are usually used as basis in seaweed meal

drying. Seaweed meals provide approximately equivalent amount of nitrogen, less

phosphorus but more potassium, total salts and readily available micronutrients compared

to most animal manures (http://www.soeagra.com/abr/vol2/5.pdf) . A company in Ireland

that produces milled seaweed for the alginate industry is developing applications for

seaweed meal in Mediterranean fruits and vegetable cultivation. Species of Ascophyllum,

Ecklonia, and Fucus are commonly used as soil additives. Because of their large amounts

of insoluble carbohydrates and the trace amounts of elements they contain, they function
http://www.soeagra.com/abr/vol2/5.pdfhttp://www.soeagra.com/abr/vol2/5.pdf


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as both fertilizer and soil conditioner. An example of a commercially available dried

seaweed sold as a fertilizer is Afrikelp. It is based on the brown seaweed Eckloni maxima

(http://www.fao.org/docrep/006/y4765e/y4765e0c.htm) .

Chemical Analysis ofSargassum

Besides water, plants obtain inorganic substances from the soil essential for

metabolism. These substances exist as ions and are absorbed by plants. Salts of

phosphorus (P), potassium (K), nitrogen (N), sulfur (S), calcium (Ca), iron (Fe),

Magnesium (Mg), together with carbon (C), hydrogen (H), and oxygen (O) are the 10

essential nutrients required by plants for maximum growth. Other elements such as boron

(B), copper (Cu), manganese (Mn), molybdenum (Mo), zinc (Zn), and sodium (Na) are

also needed by plants but only in minute amounts. Hence, they are called trace elements

or microelements (Hill, et al., 1967)

In the elemental analysis of Sargassum species conducted by Marin, et al. (2001)

at La Paz, Baja California Sur, Mexico, sargassum was analysed for nitrogen to calculate

protein content. It was found to have 6.3 0.04 % crude protein. Other analyses for

essential plant elements with their corresponding values are listed as follows: K-15.9

0.06 mg/g, P-2.7 0.07 mg/g, Mg-7.54 0.07 mg/g, Ca-6.41 0.094 mg/g, Fe-263

9.65 ppm, Zn-1195.41 ppm, Cu-14 0.29 ppm (http://www.ots.ac.cr/

tropiweb.attachments/volumes/vol574/30-Marin-Sargassum.pdf) . The details of the

nutrients analyzed are shown in Table 1.
http://www.fao.org/docrep/006/y4765e/y4765e0c.htmhttp://www.ots.ac.cr/%20tropiweb.attachments/volumes/vol574/30-Marin-Sargassum.pdfhttp://www.ots.ac.cr/%20tropiweb.attachments/volumes/vol574/30-Marin-Sargassum.pdfhttp://www.ots.ac.cr/%20tropiweb.attachments/volumes/vol574/30-Marin-Sargassum.pdfhttp://www.ots.ac.cr/%20tropiweb.attachments/volumes/vol574/30-Marin-Sargassum.pdfhttp://www.fao.org/docrep/006/y4765e/y4765e0c.htm


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Table 1. Analysis ofSargassum spp.

(http://www.ots.ac.cr/tropiweb.attachments/volumes/vol574/30-Marin-

Sargassum.pdf)

nutrient concentrationmoisture 7.70.06 %

crudeprotein 6.30.04 %ether extract 0.450.03 %crude fiber 6.40.08 %

Ash 33.30.11 %GE 2.130.02 kcal/g

NDF 47.10.42 %

ADF 44.50.35 %

cellulose 6.20.37 %Mg 7.540.07 mg/g

K 15.90.06 mg/g

Na 28.70.16 mg/g

Ca 6.410.094 mg/g

P 2.70.07 mg/g

Fe 2639.65 ppm

Zn 1195.41 ppm

Cu 1195.41 ppm

Pb 180.02 ppm

Se 850.13 ppbHg 1810.26 ppb

tannic acid 220.53 mg/g
http://www.ots.ac.cr/tropiweb.attachments/volumes/vol574/30-Marin-Sargassum.pdfhttp://www.ots.ac.cr/tropiweb.attachments/volumes/vol574/30-Marin-Sargassum.pdfhttp://www.ots.ac.cr/tropiweb.attachments/volumes/vol574/30-Marin-Sargassum.pdfhttp://www.ots.ac.cr/tropiweb.attachments/volumes/vol574/30-Marin-Sargassum.pdf


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CHAPTER III

MATERIALS AND METHODS

Collection and Preparation of Sample

Sargassum muticum will be collected from the shores of Baranggay Libjo,

Merida, Leyte. The collected samples will be washed with tap water. After washing, one

kilogram sample will be added with three liters of water and will be boiled for three

minutes. Samples will be placed in a clean container after boiling and will be added with

one kilogram mascovado to provide a good source of nutrients for the bacteria that will

aide in the fermentation process. The container will be covered with cheese cloth to allow

air to pass through while preventing the entry of insects. The samples will then be

fermented for a month.

The fermented sample will be filtered with cheese cloth prior to chemical

analysis.

Chemical Analyses

Nitrogen Analysis

A modified Kjeldahl method will be used to determine the total nitrogen

consisting of organic and ammonium forms. Wet digestion method will be applied.

Sulfuric acid will be utilized to digest organic matter of the fermented Sargassum

muticum. The sample will be digested for three hours. Details of the digestion process are

presented in Appendix 1.Sulfuric acid will convert organic matter to ammonium sulfate

in the process. The solution will be made basic prior to distillation of ammonia. The


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distilled ammonium sulfate will be received in boric acid and will be titrated with a

standard acid (Black, 1965). The details of the procedure and calculation are shown in

Appendix 2.

Phosphorus Analysis

Appendix 1 lists the details of the digestion of the sample with aqua regia prior to

analysis of phosphorus using UV-VIS spectrophotometer. The clear solution from the

digest will be determined according to Murphy and Rileys (1962) method. Details of the

procedure are presented in Appendix 3.

Sulfur Analysis

Sulfur as a sulfate ion will be determined using the turbidimetric method. The

sample will first be digested with nitric acid (as in Appendix 1) to convert the organic

sulfur to sulfate ion. Sulfate ion will be reacted with barium ion to form a turbid

suspension of insoluble barium sulfate. The scattering of light caused by this suspension

will indicate the presence of sulfate ion in the sample. Procedure will be based on

Chauddry and Cornfield (1966) as shown in Appendix 4.

Calcium, Potassium, and Magnesium Analysis

Calcium, potassium and magnesium will be determined using Atomic Absorption

Spectrometer (AAS). The sample will be digested with aqua regia as shown in Appendix

1. The clear solution that will be obtained will be subjected to Atomic Absorption

Spectrometric analysis according to Murphy and Rileys (1962) method shown in

Appendix 5.


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Formulation and Application of Nutrient Solution

The nutrient solution will be formulated by adding inorganic salts of pre-

determined amounts to the filtrate that will be obtained from the fermented Sargassum

muticum sample solution. The calculated amount of inorganic salts will be added to

obtain a nutrient solution chemically of the same nutrient composition as the standard

nutrient solution for hydroponics as shown in Table 2.

Table 2. Chemical Analysis of Commercial Hydrosol nutrient Solution (Poliquit, 2010)

nutrient concentration

nitrogen 215.00

phosphorus 37.00

potassium 218.00

calcium 152.00

sulfur 54.00

magnesium 42.00

iron 4.08

manganese 0.96

zinc 0.48copper 0.36

boron 0.036

molybdenum 0.012

total 723.93

The inorganic salts to be added will be computed using the formula

() ()

X in the equation represents the required element. The weight of inorganic salt will be

calculated from the difference obtained. This will be done using the formula:


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Figure 2. SNAP hydroponics setup(http://www.google.com.ph/imgres?q=SNAP+hydroponics+setup)

The styropor cups (6 ounces) will hold the lettuce seedlings in place. Holes will

be provided at the bottom of the cups and a screen net will be used to cover the holes.

The styropor cups will be half-filled with coco coir dust (Figure 3).

Figure 3. Seedling plug(http://www.google.com.ph/imgres?q=seedling+plugs)

The bottom of the cups will be immersed in the nutrient solution while the roots

have not yet developed. Upon development of the roots, the solution will be maintained

at 2-4 cm between the bottom of the cups and the top of the solution. Nutrient delivery

will be maintained at about 9 litres of the solution as prepared and will be poured onto the


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styropor fruit boxes. This may vary, however, depending on the size of the styropor fruit

boxes and the number of plants grown. Four litres of nutrient solution will be replenished

every two weeks depending on the availability of nutrient solution.

Horticultural Characteristics of Hydroponically Grown Lettuce

The following horticultural data will be gathered:

1. Plant height (cm). This will be determined by measuring the height of theplant from the base up to the tip of the longest leaf.

2. Number of leaves. This will be determined by counting the number of leavesper plant every week.

3. Leaf length (cm). This will be determined by getting the average length of theleaves of the lettuce plant from the base up to its apex.

4. Leaf width (cm). This will be determined by taking the average width of theleaves of the lettuce plant.

5. Leaf area index (cm2). The area of the leaf will be determined using theformula;

Leaf area index = length x width x 0.75 (correction factor)

6. Yield (g/plant). This will be determined by multiplying the leaves and stemparts of the plant using a digital weighing scale.

Experimental Design and Statistical Analysis

A Completely Randomized Design (CRD) will be used for this study. Analysis of

variance (ANOVA) will help compare the significance among treatments. Each treatment


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LITERATURE CITED

BLACK, C.A. (ed). 1965. Methods of Soil Analysis. Part 2. Chemical and

Microbiological Properties. American Society of Agronomy Monograph No. 9.Madison, Winconsin. 1572pp.

CHAUDDRY, I.A. and A.H. CORNFIELD.1966. The Determination of Total Sulfur in

Soil and Plant Material. Analyst. 91:528-530.

HILL, J. B. et al.,1967. Botany A Textbook for Colleges. McGraw-Hill, Inc. pp. 136-137

MURPHY, J. and J.P. RILEY. 1962. A Modified Single Solution for the Determination

of Phosphate ion in the Natural Water. Analytical Chemistry. Ad. 27:31-36.

POLIQUIT, P.M. 2010. Formulation and Evaluation of Nutrient Solution Derived fromFermented Fish Entrails for Hydroponics Lettuce Production. Unpublished

Undergraduate Thesis. pp

ROBIN, N.A. 2008. Formulation and Evaluation of Nutrient Solution Derived from

Fermented Tomato for Hydroponics Pechay Production. Unpublished

Undergraduate Thesis. pp14-15.

http://www.cropking .com/NFT_Lettuce_Herbs.shtml

http://www.fao.org/docrep/006/y4765e/y4765e0c.htm http://www.hydroponiclettuce.blogspot.com/http://www.hydroponicvegetablegardening.com http://www.issg.org/database/species/ecology.asp?si=727&fr=1&sts http://www.mayhillpress.com/ideal.html http://www.ots.ac.cr/tropiweb.attachments/volumes/vol574/30-Marin-Sargassum.pdf http://www.soeagra.com/abr/vol2/5.pdfhttp://www.wisegeek.com/what-is-sargassum.htm
http://www.fao.org/docrep/006/y4765e/y4765e0c.htmhttp://www.fao.org/docrep/006/y4765e/y4765e0c.htmhttp://hydroponiclettuce.blogspot.com/http://hydroponiclettuce.blogspot.com/http://www.hydroponicvegetablegardening.com/http://www.hydroponicvegetablegardening.com/http://www.issg.org/database/species/ecology.asp?si=727&fr=1&stshttp://www.issg.org/database/species/ecology.asp?si=727&fr=1&stshttp://www.mayhillpress.com/ideal.htmlhttp://www.mayhillpress.com/ideal.htmlhttp://www.ots.ac.cr/tropiweb.attachments/volumes/vol574/30-Marin-Sargassum.pdfhttp://www.ots.ac.cr/tropiweb.attachments/volumes/vol574/30-Marin-Sargassum.pdfhttp://www.soeagra.com/abr/vol2/5.pdfhttp://www.soeagra.com/abr/vol2/5.pdfhttp://www.wisegeek.com/what-is-sargassum.htmhttp://www.wisegeek.com/what-is-sargassum.htmhttp://www.soeagra.com/abr/vol2/5.pdfhttp://www.ots.ac.cr/tropiweb.attachments/volumes/vol574/30-Marin-Sargassum.pdfhttp://www.mayhillpress.com/ideal.htmlhttp://www.issg.org/database/species/ecology.asp?si=727&fr=1&stshttp://www.hydroponicvegetablegardening.com/http://hydroponiclettuce.blogspot.com/http://www.fao.org/docrep/006/y4765e/y4765e0c.htm


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BUDGETARY REQUIREMENTS

Prepared by:

NAOMI LEEH L. POBADORA

Items Quantity Unit Cost (Php) Cost(Php)

Conc. HCL 500 mL 750/1L 375.00

Conc.HNO3 100 mL 1250/1L 125.00

Boric Acid 200 g 729/250g 685.00

Bromcresol green 1 g 925/5g 6.50

Methyl red 1 g 475/25g 46.72

Ethanol 200 mL 1250/500mL 250.00

NaOH 500 g 1872/1kg 650.00

K2SO4 100 g 860/250g 410.00

Anhydrous CuSO4 10 g 475/100g 9.50

Conc. H2SO4 500 mL 815/1L 200.00

NH4-molybdate 20 mL 3800/1L 76.00

Ascorbic acid

soln50 mL 1078/450 mL 120.00

KSb-Tartrate 10 mL 1408/500 mL 28.16

Air pump 1pc. 330/pc. 330.00

Styrofoam box 6 pcs. 20/pc. 120.00

Miscellaneous 2000.00

TOTAL 6196.36


18/27

Appendix 1: Wet acid digestion of the fermented Sargassum muticum

Reagents

Concentrated hydrochloric acid

Concentrated nitric acid

Procedure

1. Measure 1 ml of fermented Sargassum muticum sample and place in a 100 mlbeaker.

2. Add 10 ml concentrated hydrochloric acid.3. Heat gently over hot plate for about 30 minutes.4. Add 5 ml concentrated nitric acid.5. Cool the solution when 5 to 10 ml remains in the beaker.6. Transfer the solution to 100 ml volumetric flask and dilute to the mark with

deionized water.

7. Collect filtrate.8. Store in appropriate container.9. Label properly.


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Appendix 2: Determination of total nitrogen (Black, 1965)

Reagents

1. Boric acid indicator solution. Place 160 g of pure boric acid in a 5-L bottlemarked to indicate a volume of 4-L. Add about 3800 ml of water and swirl the

flask until the boric acid id dissolved. Add 80 ml of mixed indicator solution

which can be prepared by dissolving 0.099 g of bromcresol and 0.066 g of

methyl red in 100 ml ethanol. Then add 0.1g sodium hydroxide continuously

until the solution assumes a reddish purple tint (pH ca. 5.0) and make the

solution 4 L by adding water. Mix the solution thoroughly before use. This is

a 4% boric acid solution.

2. Salt mixture. Mix thoroughly 100 g potassium sulfate, 10g anhydrous coppersulfate and 1 g salt mixture.

3.

Weigh 400 g of 40% technical sodium hydroxide (4 N) and dissolve to 1 L

distilled water in 1 L volumetric flask.

4. Concentrated sulfuric acid.5. Sulfuric acid or hydrochloric acid, standard, 0.1 N.

Procedure

A.

Digestion

1. Measure 1 ml of fermented Sargassum muticum solution.2. Add 1 g of salt mixture.3. Add 5 ml of 6 M sulfuric acid.


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4. Digest the sample. Regulate the heating so that the sulfuric acid condensesabout one third of the way up the neck of the flask. The flask is rotated at

intervals to facilitate the digestion of the sample. When the sample no longer

contains carbonaceous materials as indicated by the disappearance of the

blackish color, stop the digestion.

5. Allow the flask to cool.B. Distillation1. To determine the ammonium-N liberated by digestion, place a 250 ml

Erlenmeyer flak containing 25 ml of the 4% boric acid indicator solution

under the condenser of the distillation set-up so that the end of the condenser

is below the surface of the boric acid solution.

2. old the distillation flas at a 45 angle and pour 125 ml of 10 N sodiumhydroxide down the neck so that the alkali reaches the bottom of the flask

without mixing the digest.

3. Attach the flask as quickly as possible to the distillation set-up, mix thecontents thoroughly by swirling and immediately start the distillation.

Regulate the heating to prevent suc-bac of boric acid and to minimie

frothing or bumping during distillation. Chec if the flow of cold water

through the condenser is sufficient to eep the temperature of the distillation

about 35 C.

4. When about 50 ml of distillate have been collected, lower the receiver flask sothat the end of the condenser is above the surface of the distillate.


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22/27

Appendix 3. Determination of Total Phosphorus (Murphy and Rileys, 1962)

Mixed reagent

Successively add with a graduated cylinder to a 500 ml pyrex bottle and

homogenize after each condition:

-50 mL of 2.5 M sufuric acid

-15 mL of NH4- molybdate solution

-30 mL of ascorbic acid solution

-5 mL of KSb-tartrate solution

-100 mL water

Procedure

1. Digestion of sample as in appendix 1.2. Pipette into separate test tubes 3 mL of sample extracts and standard solution.3. Add 3 mL of mixed reagent and homogenize.4. Allow the solution to stand for at least one hour for the blue color to develop

its maximum.

5. Measure the absorbance on spectrophotometer.


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Appendix 5. Determination of Sulfate (Chauddry and Cornfield, 1966)

Reagents

Ammonium chloride, calcium hydroxide, barium acetate, acetic acid,

hydrochloric acid.

Standards

1. Stock solution:a.

100-ppm sulfate: use certified NIST traceable standard.

2. Instrument calibration standards:a. Pipette the designated amounts of 100-ppm sulfate stock solution into 100

mL volumetric flask. Add 2.674 g ammonium chloride. Dissolve and

dilute to the mark with deionized water and mix well.

Working solution

1. Precipitating solution:a. Weigh 255g barium acetate and dissolve in 500mL deionized water in 1 L

volumetric flask.

b. Add 100 mL acetic acid.c. Dilute to the mark with deionized water and mix well.

2.

Seed solution:

a. Add 210mL of 100mgL-1 sulfate stock solution to a 500mL volumetricflask.

b. Add 2mL hydrochloric acid.


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c. Add 13.87g ammonium chloride.d. Dilute to the mark with deionized water and mix well.

Extracting solution

1. 0.5N ammonium chloridea. Weigh 26.74g ammonium chloride into a 1L volumetric flask containing

700mL of deionized water.

b. Add 0.06g calcium hydroxide.c. Dissolve, dilute to the mark with deionized water and mix well.

Procedure

1. Pipette 5mL of fermented Sargassum muticum sample into glass test tubes.2. Add 1mL seed solution.3. Add 1mL precipitating solution and mix by slow inversion. This step is

critical. Each sample should be mixed in exactly the same manner.

4. Pour the samples into the glass cuvettes and allow to stand for 10 minutes.5. Analyse the sample on the spectrophotometer at 550 nm.6. Treat the six calibration standards in the same manner.7. Prepare one extraction blank for each set of samples.

Calculation

1. Construct a graph of sulfate concentration versus absorbance.2. Determine mgL-1 sulfate in the sample from the graph.3. Calculate:


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mg/L SO4 = (mgL-1 SO4 in samplemgL

-1 SO4 in sample blank) x dilution factor

4. Final results will be reported in mg/kg dry weight (mgL-1) calculated as follows:

Appendix 5: Determination of Calcium, Potassium, and Magnesium (Murphy and

Riley, 1962)

Reagents

Strontium chloride

Cesium chloride

Procedure

1. Digestion of sample as in Appendix 1.2. An aliquot of digested Sargassum muticum solution, 1mL, will be mixed with

2.5mL 10000 mgL-1

strontium chloride for Ca and Mg and 1mL of 50000

mgL-1 cesium chloride for K and dilute to 25 mL with deionized water.

3. Samples will be read for the total calcium, total magnesium, and totalpotassium using atomic absorption spectrometry at a wavelength of 239.9,

202.6, and 769.9 nm, respectively.


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4. Readings will be converted into elemental concentration.Calculation

where:

c = concentration of the metal being analysed (mg/kg sample)

R = AAS reading (mgL-1

)

V = volume of the solution (mL)

W = weight of the sample used (g)

5. Instrument readings are recorded in mg/L solution concentration. Final resultswill be reported in mg/kg dry weight (ppm) calculated as:


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