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ISSN 2094-1749

Volume: 3, Issue: 2, 2011 April – June 2011

Table of Contents

9. Morphometric Analysis of Third order River Basins using High

Resolution Satellite Imagery and GIS Technology: Special Reference to Natural Hazard Vulnerability Assessment

………Pradeep K. Rawat, P.C. Tiwari and Charu C. Pant

…70

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..........Tapas K. Mandal, Nragish Parvin and Mitali Saha

..101

12. Biofertilizers in Action: Contributions of BNF in Sustainable Agricultural Ecosystems

………..A.M., Ellafi, Gadalla, , A. and Galal, Y.G.M.

..108

13. Successional Changes in Herb Vegetation Community in an Age Series of Restored Mined Land- A Case Study of Uttarakhand India

............Shikha Uniyal Gairola, Prafulla Soni

..117

14. Short-term dynamics of the active and passive soil organic carbon pools in a volcanic soil treated with fresh organic matter

………Wilfredo A. Dumale, Jr., Tsuyoshi Miyazaki, Taku Nishimura and Katsutoshi Seki

..128

15. Rainwater Harvesting, Quality Assessment and Utilization in Region I

........Adriano T. Esguerra, Antonio E. Madrid, Rodolfo G. Nillo

..145

E-International Scientific Research Journal

ISSN: 2094-1749 Volume: 3 Issue: 2, 2011

70

Morphometric Analysis of Third order River Basins using High Resolution Satellite Imagery and GIS Technology:

Special Reference to Natural Hazard Vulnerability Assessment

Pradeep K. Rawat*, P.C. Tiwari* and Charu C. Pant**

*Department of Geography Kumaun University, Nainital, India ** Department of Geology Kumaun University, Nainital, India

Email: [email protected]

Abstract

The main objective of the study was to analysis the morphometric parameters of third order sub basins (TOSBs) special reference to natural hazard vulnerability assessment through integrated GIS database modeling on geo-informatics and morphometry-informatics modules. The Dabka River Basin (DRB) constitutes a part of the Kosi Basin in the Lesser Himalaya, India in district Nainital has been selected for the case illustration. Geo-informatics module consists of GIS mapping for location map, drainage map, drainage order map, lineament map, structural map, geological map etc. Morphometric module consists of morphometric analysis for several drainage basin parameters include drainage pattern, stream order, stream number, stream length, mean stream length, drainage pattern, drainage density, stream frequency, stream length ratio, relief ratio, elongation ratio, bifurcation ratio, form factor, circularity ratio and sinuosity index. Consequently the morphometric results integrated with geo-informatics parameters to assess the natural hazard vulnerability in all third order sub basins (TOSBs) and the final integrated results concluded that out of total 23 sub basins maximum 17 sub basins are highly vulnerable for several natural hazards whereas only 4 sub basins and 2 sub basins have respectively moderate and low natural hazards vulnerability.

Keywords: GI-Science, Geo-informatics, Morphometry-informatics, Natural Hazards

Introduction

Dwarfing all other mountains of the world in sheer height, Himalaya is the youngest mountain system, which is still undergoing tectonic movement due to prevailing geological conditions. Though each and every part of the world is more or less susceptible to natural calamities, the Himalaya due to its complex geological structures, dynamic geomorphology, and seasonality in hydro-meteorological conditions experience natural disasters very frequently, especially water-induced hazards (Bisht, 1991; Bora and Lodhiyal, 2010; Rawat et. al., 2011). Although the Himalaya is highly vulnerable for all type of hazards such as erosion, land slide, flood in



71

monsoon period and drought in non-monsoon period (as drying up of natural water springs and streams). In the mountain regions, such as, Himalaya, the problems of earthquake and landslides or hillslope instability are very common particularly in the geodynamically sensitive belts, i.e. zones of boundary thrusts and transverse faults (Valdiya, 1980). The presence of Main Boundary Thrust (MBT) and a number of other major and minor faults the study area is tectonically active which makes highly vulnerable the area for natural hazards whereas several morphometric parameters of river basin accelerating this vulnerability. In order to that present study highlights on these morphometric parameters through GIS (Geographical Information Science) database on geo-informatics and morphometry-informatics modules. Throughout the study area third order sub basins found highly vulnerable for several types of natural hazards and also responsible to accelerate the vulnerability for down order river basins. Therefore the study concentrated on third order sub basins (TOSBs) morphometric analysis. The watershed lies between the latitude 29°24'09"– 29°30'19"N and longitude 79°17'53"-79°25'38"E in the north-west of Nainital town along the tectonically active Main Boundary Thrust (MBT) of Himalaya, India. The region encompasses a geographical area of 69.06 km2 between 700 m and 2623 m altitude above mean sea level (Fig.1).

Location MapDabka Watershed

I N I AD

1:25000

250 10500 1/2m Km

Location MapDabka Watershed

Index

Meteorological station

Drainage

Third order River Basins

Km0 200100

Figure 1: Location Map



72

Although in this technology era we are using various digital techniques with the help of indigenous software for morphometric analysis but the morphometric studies on river basins were first introduction by Horton, 1932 and the idea was later developed in detail by Miller 1953, Schumm 1956, Melton 1958, Smith 1958, Morisawa 1962, Strahler 1964. In order to that a number of other studies have been carried out as a traditional morphometric analysis without any scientific application of the morphometric results (Khan, 1998, Nag, 1998; Biswas et al., 1999; Shrimali et al., 2000; Srinivasa et al., 2004; Chopra et al., 2005 and Nookaratnam et al., 2005). Whereas the present morphometric analysis advocating a scientific application of the results for natural hazard vulnerability assessment which is a major environmental problem of the Himlaya because natural hazards in the region cause great loss to life and property and poses serious threat to the process of development with have far-reaching economic and social consequences. In view of this the proposed work will fill up this highly realized gap and thus will have great scientific relevance in the field of natural hazard and risk management in Himalaya and other mountainous parts of the world.

Methodology

The study comprises mainly two components, (a) lab/desk study and (b) field investigations. Geo-structural maps were prepared during field study and details were verified and modified with other maps prepared during the lab/desk study. The procedure adopted for morphometric analysis and GIS mapping has been outlined in Fig. 2 and describing as below:

GIS Mapping The necessary base maps for morphometric analysis carried out through GIS Mapping using Indian Remote Sensing Satellite (IRS-1C) LISS III and PAN merged data of 2010 and SOI Topographical Sheets (56 O/7NE and 56 O/7NW) of the area at scale 1:25000 (Fig. 2). These required GIS maps are location map, drainage map, drainage order map, lineament map, structural map, geological map etc. The satellite images of the study area were registered geometrically using SOI Topographical Sheets (56 O/7NE and 56 O/7NW) of the area at scale 1:25000. For carrying out this important exercise uniformly distributed common Ground Control Points (GCPs) were selected and marked with root mean square (rms) error of one pixel and the images used were resampled by cubic convolution method. Both the data sets were then co-registered for further analysis initially, the LISS and PAN data were co-registered with root mean square (rms) error of 0.3 pixel and the output FCC was transformed into Intensity, Hue and Saturation (IHS) colour space images. The reverse transformation from IHS to RBG was performed substituting the original high-resolution image for the intensity component, along with the hue and saturation components from the original RBG images. This merge data product obtained through the fusion of IRS –1C LISS – III and PAN was used for the generation of GIS mapping through digital image processing techniques supported by intensive ground truth surveys were used for the interpretation of the remote sensing data. In order to enhance the interpretability of the remote sensing data for digital analysis several image enhancement techniques, such as, PCA, NDVI etc. were employed (Fig. 2).

Morphometric Analysis: The morphometric parameters are calculated based on the formula suggested by (Horton, 1945), (Strahler, 1964), (Schumm, 1956), (Nookaratnam et al., 2005) and



73

(Miller, 1953) given in result section of the article. Morphometric parameters like stream order, stream length, bifurcation ratio, drainage density, drainage frequency, relief ratio, elongation ratio, circularity ratio and compactness constant are calculated. Data Integration and Natural Hazard Assessment: GIS base maps and the morphometric results have been integrated and superimposed to identify vulnerability for erosion, landslide and flash flood hazards following scalogram modeling approach (Fig. 2).

Figure 2: Procedure Adopted for Study

Selection of Data sources

GIS Database Management (DBMS)

Geo-informatics Modeling

Morphometry-informatics Modeling

Field Survey for Data Sources

Verification

Desk/Lab study Field study

Acquisition of geo-coded data (Liss+Pan)

1:25000

Acquisition of Topographic Maps

1:25000

Data Integration and Superimposition To Assess Natural Hazards Vulnerability

Ground truth survey on

Preliminary Preliminary

GIS Mapping

Preliminary GIS Mapping

Ground truth Survey on Final

Results for Verification and

Final Morphometric Results

Final GIS Mapping

Morphometric Analysis and Natural Hazard Vulnerability Assessment



74

In scalogram modelling approach (Cruz, 1992), an arithmetic operation was combined with the corresponding numerical weights for the vulnerable factors. To assess the combined vulnerability level a multiple hazard vulnerability map has been carried out through integration and overlaying of all existing natural hazards vulnerability with in third order subbasins (TOSBs).

Result and Discussion

Geo-informatics Geo-informatics module consists of GIS mapping for location map, drainage map, drainage order map, lineament map, structural map, geological map etc. a brief discussion is given as below: Drainage Pattern: Drainage network is a significant indicator of the process of landform development in a geographical unit. Horton (1932) advocated, a drainage basin is an ideal unit for understanding the geo-morphological and hydrological processes and for evaluating the runoff pattern of the streams. The geological settings of the area as portrayed by the main steams and their tributaries generally control the drainage of the watershed. Generally the rectangular drainage pattern has developed at many places in the watershed. The drainage pattern of the Lesser Himalayan Ranges is quite different from that of Siwalik Hills falling in watershed. This difference in the drainage pattern is mainly due to the presence of active Main Boundary Thrust (MBT) in the watershed that separates the Lesser Himalaya from Siwaliks. Dabak river is a Sixth order stream includes as many as 495 first, 105 second, 22 third, 5 fourth and 2 fifth order streams (Fig. 3 and Table 1). Third order Sub-Basins (TOSBs): As discussed introduction and methodological section that through out the study area third order sub basins found highly vulnerable for several types of natural hazards and also responsible to accelerate the vulnerability for down order river basins. Therefore the study concentrated on third order sub basins (TOSBs) morphometric analysis. Fig. 4 and Table 1 showing that there are total twenty three third order sub basins in the study area which all have been selected for comprehensive morphometric analysis.

Table 1: Number of Streams in different stream orders

Stream order No. Of Stream 1st 495 2nd 105 3rd 23 4th 5 5th 2

6th 1



75

I. Order Streams

II. Order Streams

III. Order Streams

IV. Order Streams

V. Order Streams

VI. Order Streams

Dabka

R.

East Dabka R.West Dabka R.

Km0 1 2

1:25000

0.5

Drainage OrderDabka Watershed

Index

Figure 3: Drainage map of the area of present investigation

Lineament and Structural Setting: A lineament is a linear feature in a landscape which is an expression of an underlying geological structure such as a fault. Typically a lineament will comprise a fault-aligned valley, a series of fault or fold-aligned hills, a straight coastline or indeed a combination of these features. Fracture zones, shear zones and igneous intrusions such as dykes can also give rise to lineaments. Lineament orientations are dominantly found in NE to SW and NW to SE orientations in the study area (Fig. 4).

Geology and Structural Setting: Geologically the study area is located in the southeastern extremity of the Krol belt forming outer part of Lesser Himalaya in Kumaun (Auden 1934). The watershed encloses rocks of the Blaini-Krol-Tal succession which are thrust over the autochthonous Siwalik Group along the Main Boundary Thrust (MBT) of Himalaya. The rocks of the area are divisible into Blaini and Krol groups (Rawat and Pant 2007). The Blaini Group has been further sub divided into Bhumiadhar, Lariakantha, Pangot and Kailakhan formations in an ascending order of succession (Fig. 4) The oldest rocks exposed in the watershed comprise quartzwacke, quartzarenite, diamictite, siltstone and shale (Bhumiadhar Formation) followed upward by predominantly arenaceous Lariakantha



76

Formation, which intern is followed by the diamictites, purple grey slates, siltstone and lenticular pink siliceous dolomitic limestone of the Pangot Formation. The upper most Kailakhan Formation comprises dark grey carbonaceous pyritous slate and siltstone. The Blaini Group transitionally grades into the Krol Group. The lower most formation of the Krol Group is characterized by argillaceous marly sequence of the Lower Krol Formation (= Krol A). The Formation grades upward into purple green slates and yellow weathered dolomites with pockets of gypsum of the Hanumangarhi Formation (= Krol B). The Formation constitutes a marker horizon in the Krol belt. The Upper Krol Formation (Krol C, D, and E) is characterized by an assemblage of dolomitic limestone at the base followed by carbonaceous shales, fenestral dolomite showing cross bedding, brecciation and oolites and cryptalgal laminites. The upper most part is made up of massive stromatolitic dolomites locally cherty and phosphatic at places. The youngest Tal Formation comprises purple green slates interbedded with cross-bedded fine-grained sandstone and siltstone. The lower most southern part of the watershed comprises Siwalik Formation with massive sandstones.

Faults

Thrusts

Dolerite Dyke

Siwalik GroupKrol Group

BlainiGroup

Index

Lower Krol Formation (A)

Kailakhan Formation (Infrakrol)

Middle Krol Formation (B)

Upper Krol Formation (C,D,E)

Pangot Formation

Lariakanta Formation

Bhumiadhar Formation

Geology and Structurl SettingDabka Watershed

After Pant, C.C. (2002)

Third order Sub Basins

Km0 1 2

1:25000

0.5

Dabka Watershed

Existing Land Use

Index

Oak

Pine

Mixed

Scrub Land

Drainage/River bed

Agricultural Land

Barren Land


LineamentsDabka Watershed


Index

Lineaments


Lineaments

Index

Third order Sub Basins (TOSBs)8

9

10

1112

21

18

1920

17

1516

14

13

23

3

21

22

4

5

6

7

Third Order Sub-BasinsDabka Watershed

Third order Sub Basins (TOSBs)

Fourth to Sixth order Basins

Index


Figure 4: Third order Sub Basins, Lineaments, Geology and Land use (Clockwise from upper Left)



77

Land Use: The forest emerged as the major land use/land cover also in the year 2010. A geographical area of 36.77 km2, which accounts for nearly 53 % of total area of the watershed, has been classified as forests. Due to complexities of terrain and other geomorphic features the forests of the watershed are diversified in nature. Out of the total forest 22.20 % (15.33 km2) is under mixed forest, 19.56 % (13.51 km2) is under Oak forest, and 11.48 % (7.93 km2) is under Pine forests. The hilly and mountainous parts of the watershed are covered with Oak and Pine species, whereas, in the lower elevations in the south mixed type of vegetation is very common. Agriculture and settlement are now confined to 20.40 km2 or 29.54 % of the total area. Scrub land, barren land and Riverbeds and water bodies respectively extend over 6.22 km2 (9.01 %), 3.39 km2 (4.91 %), 2.28 km2 (3.30 %) of the total geographical land surface of the study area (Fig. 4).

Morphometry-informatics Morphometric module consists of morphometric analysis for several drainage basin parameters include drainage pattern, stream order, stream number, stream length, mean stream length, drainage pattern, drainage density, stream frequency, stream length ratio, relief ratio, elongation ratio, bifurcation ratio, form factor, circularity ratio and sinuosity index as describing following sections: Drainage basin, drainage divide, and drainage pattern: The entire area of a river basin whose runoff drains into the river in the basin is considered as a hydrologic unit and is called a drainage basin, watershed or catchment area. The boundary line along a topographic ridge separating two adjacent drainage basins is called drainage divide. The DRB possesses a triangular shaped catchment area, which develop greater flood intensity at the outlet (at Bagjala). The greater flood intensity is because of the analogous length of tributaries and the run off reaches almost at once to the outlet. Stream Order: The first order streams are those that do not have any tributary. The smallest recognizable channels (stream) are called first order and these channels normally flow during wet weather (Chow et al., 1988). A second order stream forms when two first order stream join and a third order when two second order streams are joined and so on (Strahler, 1964). Where a channel of lower order joins a channel of higher order, the channel downstream preserves the higher of the two orders and the order of the river basin is the order of the stream draining its outlet, the highest stream order in the basin (Chow et al, 1988). It may be noted that Dabka river is a sixth order stream and the third to Sixth order streams are perennial and all others are ephemeral in nature (Fig. 3 and Table 1). The first order streams (495 numbers) can be identified only during monsoon period and stream ordering designates discharge from a drainage network. Stream Number: The order wise total number of stream segment is known as the stream number. As mention above that Dabak river is a Sixth order stream includes as many as 495 first, 105 second, 22 third, 5 fourth and 2 fifth order streams (Fig. 3 and Table 1). The data reveals that the number of stream segments decreases with increase in stream order. The decrease in the number of stream segments is experienced because when a channel of lower order joins a channel of higher order, the channel downstream retains the higher of the two orders (Chow et al, 1988). Table 1 holds excellent the law of stream numbers which states that the number of stream segment of each order form an inverse geometric sequence with states the order number (Horton



78

1945). There is a total of 1945 stream in DRB. Some TOSBs with high proportion of first order stream and it may be due to structural weakness present in DRB. For the detailed study of other morphometric parameters the TOSBs were taken the number of polygons, perimeter and area of 23 TOSBs and the remaining portion (23rd polygon) were determined and the location of TOSBs were observe (Fig. 4) and the drainage parameters of the TOSBs were compiled (Table 2).

Table 2: Results of morphometric analysis of 23 third order basins

Morphometric parameters

Sub

-bas

ins

(TO

SB

s)

Sub

-bas

in

Are

a (K

m2 )

Leng

th o

f Rb

(Km

.)

Per

imet

er (

Km

.)

Str

eam

ord

er

No.

of s

trea

m

(Seg

men

ts)

Leng

th o

f S

trea

m (

Km

.)

Mea

n S

trea

ms

Leng

th (

Km

.)

Str

eam

s Le

ngth

R

atio

(or

der)

Bifu

rcat

ion

Rat

io

Ste

ams

Invo

lved

in

Rb.

Dra

inag

e D

ensi

ty

(Km

/Sq

Km

.)

Dra

inag

e fr

eque

ncy

(Str

eam

s/sq

km

.)

1 13 3.5 0.2692308 6.5

2 2 1.5 0.75 2.7857 2 14 4 11.33 1

1.5 6.4 17.25 3 1 1 1 1.3333 3

1 8 2 0.25 4

2 2 1 0.5 2 2 10 2.8 8.67 2

1.25 5.3 15.1 3 1 0.5 0.5 1 3

1 6 1.5 0.25 3

2 2 0.5 0.25 1 2 8 3.52 13.75 3

0.85 4.5 11.3 3 1 1 1 4 3

1 14 8 0.5714286 4.6667

2 3 3.5 1.1666667 2.0417 3 17 5.68 9.55 4

2.2 12 25 3 1 1 1 0.8571 4

1 13 5 0.3846154 4.3333

2 3 1 0.3333333 0.8667 3 16 5.6 16 5

1.25 6 14 3 1 1 1 3 4

1 28 8 0.2857143 4.6667

2 6 1.5 0.25 0.875 6 34 5.33 18.23 6

2.25 10.1 20 3 1 2.5 2.5 10 7

1 8 2.3 0.2875 4

2 2 0.5 0.25 0.8696 2 10 5.33 17.34 7

0.75 7 17 3 1 1.2 1.2 4.8 3

1 32 10 0.3125 8

2 4 2.5 0.625 2 4 36 5.47 15.48 8

2.65 11 22.4 3 1 2 2 3.2 5

1 11 2 0.1818182 5.5

2 2 1.5 0.75 4.125 2 13 5 14.55 9

1.1 10 15 3 1 2 2 2.6667 3

1 8 2 0.25 4

2 2 0.5 0.25 1 2 10 1.47 11.35 10

1.15 9 16 3 1 1.2 1.2 4.8 3



79

1 6 1.5 0.25 3

2 2 0.5 0.25 1 2 8 3.05 12.95 11

0.85 6.08 11.1 3 1 0.6 0.6 2.4 3

1 11 3.2 0.2909091 3.6667

2 3 1.2 0.4 1.375 3 14 4.52 16.37 12

1.15 6.46 13 3 1 0.8 0.8 2 4

1 13 3.6 0.2769231 3.25

2 4 1.6 0.4 1.4444 4 17 3.16 10.23 13

2.15 12 19.5 3 1 1.6 1.6 4 5

1 28 10 0.3571429 4.6667

2 6 5 0.8333333 2.3333 6 34 4.22 9.11 14

4.5 19 33 3 1 4 4 4.8 7

1 9 2.5 0.2777778 4.5

2 2 1 0.5 1.8 2 11 4.2 14 15

1 5.35 13 3 1 0.7 0.7 1.4 3

1 8 3 0.375 4

2 2 2.5 1.25 3.3333 2 10 7.37 16.25 16

0.8 5.05 11 3 1 0.4 0.4 0.32 3

1 14 4.5 0.3214286 4.6667

2 3 1.5 0.5 1.5556 3 17 7.42 12.35 17

1.75 7 17.1 3 1 7 7 14 4

1 24 6.5 0.2708333 3.4286

2 7 2.2 0.3142857 1.1604 7 31 4.8 16.59 18

2.35 12.06 22.2 3 1 2.6 2.6 8.2727 8

1 6 2.8 0.4666667 3

2 2 0.4 0.2 0.4286 2 8 4.7 12.94 19

0.85 5.12 11.45 3 1 0.8 0.8 4 3

1 21 8 0.3809524 4.2

2 5 2.5 0.5 1.3125 5 26 6.76 18.82 20

1.7 6.48 18.35 3 1 1 1 2 6

1 13 5 0.3846154 4.3333

2 3 1.3 0.4333333 1.1267 3 16 4.88 11.11 21

1.8 11.38 19.3 3 1 2.5 2.5 5.7692 4

1 9 3.8 0.4222222 4.5

2 2 2.5 1.25 2.9605 2 11 7.88 16.47 22

0.85 5.16 13 3 1 0.4 0.4 0.32 3

1 11 3.5 0.3181818 5.5

2 2 1.5 0.75 2.3571 2 13 5.8 16.84 23

0.95 7.15 14.48 3 1 0.8 0.8 1.0667 3

Total 35.65 189.59 389.53 135 393 171.5 55.722182 122.97 163.88 464 108.96 308.95

Ave 1.55 8.243 16.936 5.8696 17.087 7.4565 2.4227036 5.3466 7.1252 20.174 4.7374 13.433

Stream Length (Lu): Horton’s law of stream lengths states that the mean lengths of streams segment of each the order. Generally Lu increases as the order of segment increases. Except the TOSBs 7, 8, 9, 10, and 20 Lu decreases as the order of stream segments increases and it constitutes about 24% of the TOSBs. The 48 stream orders of TOSBs have Lu less than 1 Km



80

(57 %), 24 stream orders have a value between 1 and 2 Km for first order streams and for others Lu given in Table 2.

Drainage density (Dd): Drainage density (Dd) is the total length of the stream in a given drainage basin divided by the area of drainage basin (Horton, 1932).

Dd= ∑L/A, Where ∑L- total length of the stream, A- Area of drainage basin. Table 2 depicts the distribution of third order basins under different drainage density groups. The Dd value of DRB is 1.82 Km/km2. The study of TOSBs revealed that average Dd is 2.67 km/km2 and it varies in between 1.47 km/km2 to 7.88 km/km2 (Table-2). The highest Dd is for the TOSB 15, 16, 20, and 22. With a value of 7.37, 7.42, 6.76, 7.88 respectively is situated nearest to the highest rainfall occurring region such as Ghughukhan ,Maniya and Binayak. The TOSB 10 with lowest Dd value with 1.44, nearer to the lowest rainfall occurring region such as Fathehpur. The Table 3 reveals the relationship between rainfall and Dd. At present Ghughukhan rain gauge station records highest rainfall.

The Dd generally increases with rainfall (R) Thus Dd ∞ R Dd= KXR Dd/R = K, where K is a constant and its value is always less than one The Dd and R studies reveal that Dd controls runoff following a particular period of precipitation and the increasing Dd shows increasing size of mean annual flood. Table 3: Relation of rainfall and drainage density (Dd)

Code of TOSB

Dd Nearest Rain Gauge

Annual mean Rainfall (mm)

Data Recorded

22 7.88 Ghughukhan 2749.80 5Year (2005-

2010)

13 3.16 Maniya 2357.10 5Year, (2005-

2010)

10 1.47 Aniya 854.06 5Year, (2005-

2010)

Stream frequency (Df): It is the number of stream segments per unit area (Horton, 1932, 1945). The stream frequency, Df = ∑N/A, where N is the number of stream segments and A denotes the drainage area. The average Df for DRB is 2.45. The lowest Df value is for the TOSB 8 with a value of 8.67 and the highest for the TOSB 20 with a value of 18.82. The frequency value wise numbers of TOSBs are tabulated (Table 2).. Relation between Drainage Density and Frequency: The relation of Dd and Df revealed that the Df is directly proportional to Dd (Table 4) Thus drainage frequency is double the value of drainage density and its variation occurs due to rainfall, relief, infiltration rate, and initial resistivity of terrain to erosion, total drainage area of the basin and above all the Dd of the basin



81

itself. The low values of Df indicate poor stream networks and high indicate denser networks in the catchment area. Stream length ratio (RL): Stream length ratio is the ratio of mean length of streams of one order to that of the next lower order that tends to be constant thorough the successive order of a watershed (Horton, 1945). The stream length ratio RL = Lu/Lu-1, Where Lu is the mean stream length of order u and Lu-1 is the mean stream length of next lower order. The average length ratio of TOSBs is 5.35 with highest value of 14.00 for TOSB of 17 (indicates lower order sources for the next higher order streams) and the lowest 0.32 for the TOSBs of 16 & 22 (indicates limited length of lower order streams) Table 2. Relief ratio (Rh): The difference in elevation between the highest and lowest points in a basin is called basin relief. It indicates the overall steepness of drainage basin and is an ndication of intensity of degradation processes operating on slopes of the basin and is ratio between the total relief of the basin and its longest dimension parallel to the principal drainage line. Rh = H/Lb

Table 4: Relation between Dd & Df

Sub-basins Drainage Density Drainage Frequency Relation (TOSBs) (Km/Sq Km.) (Streams/sq km.) (R=Df/Dd)

1 4 11.33 2.83 2 2.8 8.67 3.10 3 3.52 13.75 3.91 4 5.68 9.55 1.68 5 5.6 16 2.86 6 5.33 18.23 3.42 7 5.33 17.34 3.25 8 5.47 15.48 2.83 9 5 14.55 2.91 10 1.47 11.35 7.72 11 3.05 12.95 4.25 12 4.52 16.37 3.62 13 3.16 10.23 3.24 14 4.22 9.11 2.16 15 4.2 14 3.33 16 7.37 16.25 2.20 17 7.42 12.35 1.66 18 4.8 16.59 3.46 19 4.7 12.94 2.75 20 6.76 18.82 2.78 21 4.88 11.11 2.28 22 7.88 16.47 2.09 23 5.8 16.84 2.90



82

Where H is the total relief and Lb is the basin length. The Rh value of DRB is 0.173. The low Rh values are resulted by resistant bedrock and low slope and Rh values usually increases with decreasing with decreasing drainage area (Table 2).

Elongation ratio (Re): It is the ratio of diameter of a circle having the same area as of the basin and maximum basin length (Schumm, 1956). The Re is given by Re = d/Lb, Where d is diameter of a circle having the same area as of the basin, and Lb maximum basin length parallel to the principal drainage line. It is a measure of the shape of the river basin and the value ranges between 0.6 and 1. Value ranges from 0.6 to 0.8 are regions of high relief and the value close to 1.0 are regions of very low relief with circular in shape and are efficient in the discharge of runoff than and elongated basin because concentration time is less in circular basins. Thus Re values help in flood forecasting. The elongation ratio and shape of basin are given in Table 5.

Table 5: Elongation ratio and shape of river

Bifurcation ratio (Rb): It is defined and the number of streams in and order to the number of streams in the next high order (Horton, 1945) and is given by the Rb = Nu/Nu+1, Where Rb is bifurcation ratio, Nu is number of segments in an order Nu+1 is the number of segments in the next higher order. If within a unit, bifurcation ratios are equal, it is called Hortron’s net and the values usually range between 3 and 5.0 for networks formed in homogeneous rock (suffered less structural disturbances) and with more than 10 where structural controls play dominant role with elongate narrow basins (Chow, et al., 1988). Horton’s net condition is not present in any of the FOSBs, the average Rb is 3.8 and lowest with a value of 2.00 for TOSBs 1,2,3,7,9,10,11,14,15,16,19,22, , and highest with a value 8.00 for TOSB 23 (Table 2). The variation is due to lithological and geological development of TOSBs. Form Factor (Rf): It is the dimensionless ratio of basin area to the square of basin length (Horton, 1932) and is calculated by Rf = A/Lb2 Where A is the drainage area and Lb is the length of the river basin. The Rf value of 0 indicates a highly elongated shape and the value of 1.0, a circular shape with high peak flows for short duration but for elongated basin with low Rf with a flatter peak flows for longer duration. The flood flows of elongated basins can be easily managed than that of circular. The Rf value of DRB is shows 0.12 showing its elongated shape and its flood flows can be managed efficiently.

Elongation ratio Shape of basin <0.7 Elongated 0.8-0.7 Less elongated 0.9-0.8 Oval >0.9 Circular

Elongation ratio Shape of basin <0.7 Elongated



83

Circularity ratio (R e): It is the ratio of area of river basin to the area of circle having the same perimeter as the basin (Miller, 1935). Like form factor, it is also a dimensionless ratio to express outline of drainage basin (Strahler, 1964) and Re is uniform between 0.6 and 0.7 for homogenous rock types and 0.40 and 0.5 for quartzitic terrain and is influenced by length and Df of streams, geological structures, vegetation, climate, relief and slope of the basin. Sinuosity index (S): It is the ratio of channel length and river valley length (Muller, 1968). Sinuosity index reveals the topographic and hydraulic conditions of streamlines and it varies from 1.1 to 4.0 or more and those having S less than 1.5 called sinuous and with 1.5 or more than 1.5 are called meandering. Stream channels usually originate in sinuous form, which depends on underlying rock structure, climate, vegetation and time. The average Sinuosity index of DRB is 1.11. Data Integration and Natural Hazard Vulnerability A ssessment The morphometric results have been integrated and superimposed with other GIS base maps to natural hazard vulnerability assessment. Mainly three types of natural hazard identified within the third order sub basins i.e. erosion, landslide and flash flood hazard. A brief description on each type of hazard vulnerability within all the twenty three third order sub-basins given as below: Erosion Hazard Vulnerability: Increasing first order streams, increasing drainage density and frequency are the main morphometric parameter for erosion hazard vulnerability but the frizzled geo-ecological parameters (i.e. stressed and crushed geology, active faults and thrust, degraded land use pattern and lineaments etc.) are accelerating factor for the hazard vulnerability. Therefore out of total 23 sub basins 15 sub basins have high erosion vulnerability whereas 4 sub basins found for moderate and 4 sub basins found for low erosion hazard vulnerability (Fig. 5 and Table 6). Landslide Hazard Vulnerability: Although the study area is highly vulnerable for seismic landslide activity due to active lineaments such as thrusts (MBT) and number of faults but it experienced that the area is equally vulnerable for non-seismic landslide during rainy season because of degraded land use pattern whereas the morphometric factors for landslide vulnerability are same as for erosion hazard vulnerability. Fig. 5 and Table 6 depicting the spatial distribution of the landslide vulnerability within the third order sub basins. Out of total 23 sub basins maximum 19 sub basins have high landslide vulnerability whereas only 2 sub basins found for moderate and 2 sub basins found for low landslide hazard vulnerability (Fig. 5 and Table 6).



84

8

9

10

1112

21

18

1920

17

1516

14

13

23

3

2

122

4

5

6

7

Erosion Hazard VulnerabilityDabka Watershed

High

Moderate

Index

Low

Km0 1 2

1:25000

0.5Km

2

High

Moderate

Index

Low

8

9

10

1112

21

18

1920

17

1516

14

13

23

3

2

122

4

5

6

7

Landslide Hazard VulnerabilityDabka Watershed

High

Moderate

Index

Low

8

9

10

1112

21

18

1920

17

1516

14

13

23

3

2

122

4

5

6

7

Multiple Hazard VulnerabilityDabka Watershed

High

Moderate

Index

Low8

9

10

1112

21

18

1920

17

1516

14

13

23

3

2

122

4

5

6

7

FloodHazard VulnerabilityDabka Watershed

Figure 8: Natural Hazards Vulnerability (Flood, Erosion, and Landslide=Multiple:

Clockwise from Upper Left to Lowe Left)

Flood Hazard Vulnerability: Mainly two types of floods are common throughout the Himalaya i.e. flash flood and river-line flood which are among the more devastating types of hazard as they occur rapidly with little lead time for warning, and transport tremendous amounts of water and debris at high velocity. Intense rainfall (IRF) is very frequent cause for flash flood and river-line flood in the study area which play a key role for flash flood and river-line flood. The main meteorological phenomenon causing intense rainfalls in the region are cloudbursts, stationarity of monsoon trough and monsoon depressions. Flash flood in the region cause great loss to life and property and poses serious threat to the process of development with have far-reaching economic and social consequences. In order to that it is quit important to assess the flood hazard vulnerability due to morphometric parameters and geo-environmental factors. The major morphometric parameter of flood high hazard vulnerability is decreasing Elongation ratio (Re). The spatial distribution of flood hazard vulnerability with in the third order sub basins suggesting that out of total 23 sub basins 6 sub basins have high flood vulnerability whereas only 5 sub basins found for moderate and 12 sub basins found for low flood hazard vulnerability (Fig. 5 and Table 6).



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Table 6: Several Types of Hazards Vulnerability Assessment in 23 third order Sub Basins Different Natural Hazard Vulnerability Thir

d orde

r Sub Basins

Erosion Hazard Vulnerability

(I)

Landslide Hazard

Vulnerability

(II)

Flash Flood Hazard

Vulnerability (III)

Multiple Hazard

Vulnerability

(I+II+III)

1 High High low High 2 High High low High 3 High High low High 4 High High Moderate High 5 High High Moderate High 6 High High Moderate High 7 High High Moderate High

8 Moderate Moderate Low Modera

te 9 High High Moderate High

10 Moderate Moderate High Modera

te 11 Low Low Low Low 12 Low Low Low Low 13 High High High High 14 High High High High 15 High High low High 16 High High low High 17 High High High High

18 Moderate Moderate Low Modera

te 19 High High low High 20 High High low High

21 Moderate Moderate High Modera

te 22 High High low High 23 High High High High

Multiple Hazard Vulnerability: Above study reviles that each third order sub basin not equally vulnerable for all three types of natural hazards (Fig. 5 and Table 6). In view of that a combined multiple hazard vulnerability map has been carried out through integration and overlaying GIS layers of all these three natural hazards vulnerability (i.e. erosion+ landslide+ flood) for each third order sub-basin (Fig. 5 and Table 6). This map suggesting that out of total 23 sub basins maximum 17 sub basins have high natural hazards vulnerability whereas only 4 sub basins found for moderate and 2 sub basins found for low natural hazards vulnerability.

Conclusion Throughout the study area third order sub basins found highly vulnerable for several types of natural hazards and also responsible to accelerate the vulnerability for down order river basins. Therefore the study concentrated on third order sub basins (TOSBs) morphometric analysis and integrated the results with geo-environmental background of the sub basins through GIS database



86

management system (DMS). The study concluded that each third order sub basin not equally vulnerable for all three types of natural hazards. In view of that a combined multiple hazard vulnerability map has been carried out through integration and overlaying GIS layers of all these three natural hazards vulnerability (i.e. erosion+ landslide+ flood) for each third order sub-basin and suggesting that out of total 23 sub basins maximum 17 sub basins have high natural hazards vulnerability whereas only 4 sub basins found for moderate and 2 sub basins found for low natural hazards vulnerability.

Acknowledgement This study constitutes part of multidisciplinary Collaborated project, Department of Science and Technology (D.S.T.) Gov. of India, No.ES/11/599/01 Dated 27/05/2005, “Geo-environmental Appriasal of the Dabka Watershed, Kumaun Lesser Himalaya, District Nainital: A Model Study for Sustainable Development” funded to Prof. C.C. pant under collaboration of Department of Geography and Geology Kumaun University Nainital. Dr. Pradeep Goswami, Senior Scientist, Center for climate change, Kumaun University Nainital helped in GIS analysis for which authors indebted to him. Thanks to Shri M.S. Bargali, project assistant helped during the intensive field work. References Auden, J.B. 1934. The Geology of the Krol belt. Geol. Soc. India, 67: 357-454. Bisht, M.K.S, 1991. Geohydrological and geomorphological investigations of the Dabka catchment district Nainital, with special reference to problem of erosion. Unpublished PhD. thesis, 37-115. Bora, C.S. and L.S. Lodhiyal, 2010. Ecological trends of under canopy species of Eucalyptus plantations in Bharbhar and Tarai region of India Central Himalaya. E-International Scientific Research Journal, 2 (2): 118-127. Biswas, S., S. Sudhakar, and V.R. Desai 1999. Prioritisation of subwatersheds based on morphometric analysis of drainage basin: A Remote Sensing and GIS approach, Journal of Indian Society of Remote Sensing, 27(3): 155166. Chopra, R., R. Dhiman, and P.K. Sharma 2005. Morphometric analysis of subwatersheds in Gurdaspur District, Punjab using Remote Sensing and GIS techniques, Journal of Indian Society of Remote Sensing, 33(4): 531539. Cruz, R.A.D. 1992. The determination of suitable upland agricultural areas using GIS technology. Asian pacific Remote Sensing Journal, 5:123-132. Melton, M.A. 1958. Correlation structure of morphometric properties of drainage system and their controlling agents. J. Geol., 66: 442-460.



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Morisawa, M.E. 1962. Quantitative geomorphology of some watershed in the Appalachian plateau, Geol. Soc. Bull., 73: 1025-1046. Horton, R.E. 1932. Drainage Basin Characteristics. Transfixions of Am. Geophys. Union, 13: 350.

Miller, V. C., 1953. A quantitative geomorphic study of drainage basin characteristics in the clinch mountain area, Technical report3, Department of Geology, Columbia University. Nag, S. K., 1998, Morphometric analysis using remote sensing techniques in the Chaka subbasin, Purulia district, West Bengal, Journal of Indian Society of Remote Sensing, 26(12): 69-76. Nookaratnam, K., Y.K. Srivastava, V. Venkateswarao, E. Amminedu, K.S.R. Murthy, 2005. Check dam positioning by prioritization of microwatersheds Using SYI model and morphometric analysis – Remote sensing and GIS perspective, Journal of the Indian Society of Remote Sensing, 33(1): 25-28. Rawat, Pradeep K., C.C. Pant 2007. Geohydrology of Dabka watershed, using remote sensing and GIS”, in Rawat M.M.S and Pratap D. (Ed.), Management Strategy for the Indian Himalayan Development and Conservation proceedings of National conference held in Srinagar Uttarakhend, India 2007, Trans Media Publication, Srinagar Uttarakhend, India: 161-183. Rawat, Pradeep K., P.C. Tiwari, C.C. Pant, A.K Sharama, P.D. Pant, 2011. Climate change and its geo-hydrological impacts on mountainous terrain: A case study through Remote Sensing and GIS modelling. E-International Scientific Research Journal, 3 (1): 51-69. Schumm, S. A., 1956. Evolution of drainage systems and slopes in Badlands at Perth Amboy, New Jersey. Geological Society of America, Bulletin, 67: 597-646. Shrimali, S.S., S.P. Aggarwal,and J.S. Samra 2001. Prioritizing erosionprone areas in hills using remote sensing and GIS – a case study of the Sukhna Lake catchment, Northern India, JAG, 3(1): 54-60. Srinivasa, V. S., S. Govindaonah, and H. G Home 2004. Morphometric analysis of subwatersheds in the Pawagada area of Tumkur district South India using remote sensing and GIS techniques. Journal of Indian Society of Remote Sensing, 32(4): 351-362. Strahler, A. N. 1964. Quantitative geomorphology of drainage basins and channel networks, section 4II, In: Handbook of Applied Hydrology, edited by V.T. Chow, McGraw Hill: 439. Smith, K.G. 1953. Standard for grading texture of erosional topography, Am. J. Soc., 5(298):655-668. Valdiya, K.S. 1980. Geology of Kumaun Lesser Himalaya. Wadia Institute of Himalaya Geology, Dehradun UP.:291.



88

Seaweed Bath Soap Product Formulation and Development

Rogelio M. Estacio Associate Professor

Don Mariano Marcos Memorial State University, Bacnotan, La Union, Philippines

Abstract The process of making bath soap comprises the following steps: preparation of alkaline solution; preparation of seaweed gel, papaya (Carica papaya Linn), atsuete (Bixa orellana Linn) and coconut oil as primary ingredients of the product, and mixing these ingredients to produce a thick solution, pouring the solution into the molder, cooling and solidifying the solution at room temperature, aging , and packaging the end- product. The “Seaweed Bath Soap” was an offshoot product of the project entitled “ Seaweed Gel Extract Product Formulation and Development.” The soap product containing a mixture of seaweed gel, papaya and atsuete extract were brought to the Cagayan Valley Herbal Processing Plant.- Philippine Institute for Traditional and Alternative Health Care, Carig, Tuguegarao City for bioassay analysis and testing. After which, it was subjected to sensory evaluation by trained panelists of Don Mariano Marcos Memorial State University- North La Union Campus. Result of the study revealed that the soap product was found to be very much acceptable in its overall quality attributes.

Keywords: Seaweeds bath soap, seaweed gel extract, seaweed product formulation, herbal soap, DMMMSU soap

Introduction

In the Ilocos Region, seaweeds such as sargassum, gracilaria and eucheuma are abundant. Eucheuma is cultured in some parts of the province. Favorable growth of this seaweed is noted but still limited to meet the export demand. Sargassum, however, is found washed up on beaches in large quantity or floating near shore. Sargassum is found throughout the world’s ocean and seas and none is known to be poisonous. It is usually ignored by coastal dwellers and treated as waste in the coastal area. The objective is to utilize seaweeds such as eucheuma, gracilaria and sargassum seaweeds, and turned them into something that would result to a better income opportunity for the fish farmers in the region. This work can also create employment by putting small and medium enterprises of the product in coastal communities as livelihood projects of housewives, out of school youths and jobless adults.



89

Seaweed bath soap is a natural product for cleansing. It is actually a salt that foams. This crystalline nature soap is made of seaweed gel extract that is mixed with caustic soda and natural oil to produce an opaque, premium bath soap that is mild for sensitive skin. The seaweeds eucheuma, gracilaria and sargassum are red-to-brown grass of the sea that provide food for man. Aside from being consumed as food these are utilized as raw material in the manufacture of industrial products such as alginate, agar and carrageenan. They contain protein which help to fight premature aging of the skin by restructuring collagen and generating elasticity, skin suppleness which in turn reduces and softens wrinkles. They also contain betacarotene to help slow skin aging, treat acne and irritated skin, as well as eczema problems. It is also used as detoxifier when it is eaten or applied to the skin.

The papaya extract is known as an effective skin whitening. Papaya contains vitamins A, that benefits the skin through increasing the rate of new cell formation. It also balances and regulates skin firmness, tones and improve smoothness.

The atsuete contains volatile fatty oil with palmitin and traces of stearin alkaloids saponin and tannin for homogeneous color of the skin aside from fascinating the product.

The soap product provides the benefits of exfoliation, cleansing, smoother, healthier looking skin that’s is more receptive to moisturizing lotion. Exfoliation is part of natural skin care and the secret to smooth, soft healthy looking skin. The seaweed bath soap gently scrubs away dead skin and other skin impurities caused by environmental pollutions, sun exposures and stresses of everyday life. By using this soap, a younger skin is exposed. It also stimulates blood circulation.

Analysis showed that the quality attributes of the product such as physical evaluation (texture, color, odor, hardness and size) and after-effect evaluation (exfoliation, irritation, freshness, irritation, freshness, lather and allergynisity) were very much acceptable .

Moreover, the soap is cheaper compared to other commercial soap in the market. Review of Related Literature Seaweeds are rich in vitamins A, B1, B2, B6, Folic acid and Niacin. It supplies 60 trace elements and is a primary source of B12 and significant amount of vitamins E and K. It is also an excellent source of over 60 minerals, especially potassium, calcium, iodine, magnesium, phosporous iron, zinc, manganese (Trono, Gavino, 1989,). Seaweed bath soap products hydrate and feed the body through the most vital organ – the skin. Our entire skin care bath soap is 100 percent natural, free of dyes, animals-by-products and contains no artificial fragrances, making it safe for all skin types. Regular use of seaweed bath soap increases the levels of moisture in the skin and promote a healthy glowing complexion. Seaweeds contain much larger concentration of what is present in seawater, and in a form, which can easily be assimilated, the potassium-sodium content of sea vegetable is usually quite close to



90

that occurring naturally in human body. Many marine algae are a source of B12, which are rarely found in land vegetables. Many species of seaweeds contain protein which helps fight premature aging of the skin by restructuring collagen and generating elasticity. This increase the skin suppleness which in turn reduces and softens wrinkles. Due to its iodine and sulphur amino acid content, seaweeds are stimulating, revitalizing and nourishing to the skin. It also offers antibacterial and skin healing benefits. Objectives General: To develop new non-food products from seaweed (eucheuma, greacilaria, and sargassum) extract. Specific:

1. To formulate new products, e.g. bath soap with seaweeds extract; 2. To determine the acceptability of the formulated product; and 3. To determine the cost and return benefits of the product.

Expected Output: A novel seaweed bath soap that would promote healthy skin.

Methodology/ Procedure:

a. Preparation of supplies and materials The basic soap ingredients (fats, oil and alkali), seaweed and equipments molder, mixing bowl, weighing scale and stirring rod to be used were prepared and set at the processing laboratory of DMMMSU-NLUC. Gracilaria spp, Sargassum and Eucheuma seaweeds were gathered at the shoreline of Balaoan, La Union. Other raw materials were bought locally. The seaweeds were prepared by washing, drying, bleaching, and cooking for phycocolloids extraction.

b. Seaweed bath soap product formulation

b.1. Bath soap formulation



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Table 1. Percentage composition of the ingredients for each seaweed used. Treatment Seaweeds Papaya Atsuete

T1

45% 35% 20%

T2

55% 25% 20%

T3

65% 15% 20%

T4 conrol

Leading brand Leading brand Leading brand

b.2. Procedure 1. Dissolve caustic soda flakes in distilled water by continuously stirring until

completely dissolved and cooled; 2. Add this to the coconut/oil and mix in a single direction for five minutes; 3. Add seaweeds extract, papaya and atsuete extract and continue stirring the solution

for 30-40 minutes; 4. Pour the solution into the molders; and are left to cool and harden. This is now the

cooling and solidifying stage; 5. Afterwhich, remove the soap from the molder and age the soap for 3 to 4 weeks.

The process will remove the irritation effects of caustic soda; 6. Finally, the soap is packed for analysis and evaluation.

c. Representative samples were brought to DOST- Cagayan Valley Herbal Processing

Plant, Carig, Tuguegarao City for bioassay analysis and testing.



92

Figure 1. Flow Chart – the step- by- step process of making seaweed bath soap

7.

Mixing the prepared ingredients through a plastic container

Aging (saponification) the solution,

Removing the solution to the said molder

Cooling and solidifying the solution at room

temperature

Pouring the solution into the molder

Stirring the mixed ingredients continuously to

produce a thick solution

Preparing alkaline solution, seaweed, papaya and atsuete

extract as ingredients

Packaging the finish product



93

d. Test and Evaluation

1. Sensory evaluation – The formulated bath soap was initially evaluated by 6 panelists who tried the product. Below is the rating scale used by the panelists.

Table 2. Scale Used: 0 – 5 POINT VALUE RANGE DESCRIPTIVE

RATING 5 4.20 – 5.00 Very High 4 3.40 – 4.19 High 3 2.60 – 3.39 Moderate High 2 1.8 – 2.59 Low 1 0 – 1.79 Very Low

2. Sampling tools and technique. The panelists, composed of faculty, staff and students

of the University, used the modified hedonic rating scale to evaluate the products. The quality attributes or criteria for evaluation was adopted based on the recommendation of the Philippine Institute of Traditional and Alternative Health Care for bath soap product.

3. Data Analysis – Data on the sensory evaluation results were analyzed using the analysis of variance (ANOVA)

Results and Discussion

A. Acceptability of the seaweed bath soap. Sensory evaluation results by the panelists is reflected in Tables 3, 4 and 5. Five (5) quality attributes on physical evaluation and five (5) quality attributes on the effect of the seaweed bath soap were presented and served as basis for the acceptability test of the product.

Table 3.a. Physical Evaluation.

Mean response on the different quality attributes of the gracilaria formulated soap. Treatment Texture Color Odor Hardness Size Mean Descriptive

Equivalent T1

4.5 4.5 4.16 4.5 4.16 4.36 Very High

T2

4.16 4.83 4.16 4.16 4.5 4.36 Very High

T3 3.5 4.0 4.66 4.33 4.16 4.13 High

T4 4.35 4.25 4.26 4.26 4.15 4.26 Very High



94

Table3.b. Effect Evaluation.

Mean response on the different quality attributes of the gracilaria formulated soap. Treatment Exfoliation Irritation Freshness Lather Allergynisity Mean Descriptive

Equipvalent T1 4.66 4.5 4.33 4.5 4.66 4.53 Very High T2 4.66 4.33 4.0 4.66 4.33 4.39 Very High T3 4.0 3.83 3.66 4.0 4.66 4.03 High T4 4.1 4.15 4.0 4.15 4.25 4.13 High

Table 4.a. Physical Evaluation.

Mean response on the different quality attributes of the eucheuma formulated soap Treatment Texture Color Odor Hadness Size Mean D. E. T1 4.83 4.83 4.0 4.66 4.66 4.59 VH T2 4.0 4.33 4.5 4.5 4.5 4.36 VH T3 4.33 4.66 4.0 4.5 4.5 4.39 VH T4 4.25 4.25 4.25 4.25 4.25 4.25 VH

Table 4.b. Effect evaluation.

Mean response on the different quality attributes of the eucheuma formulated soap.

Table 5.a. Physical evaluation. Mean response on the different quality attributes of the sargassum formulated soap.

Treatment Texture Color Odor Hardness Size Mean D. E. T1 4.83 4.83 4.0 4.66 4.66 4.59 VH T2 4.0 4.33 4.5 4.5 4.5 4.36 VH T3 4.33 4.66 4.0 4.5 4.5 4.39 VH T4 4.25 4.25 4.25 4.25 4.25 4.25 VH

Treatment Exfoliaation Irritation Freshness Lather Allergynisity Mean D.E. T1 4.33 4.83 4.5 4.66 4.83 4.63 VH T2 4.0 3.66 3.66 3.83 4.66 3.96 High T3 4.66 4.16 4.16 4.66 4.33 4.39 VH T4 4.16 4.25 4.33 4.25 4.25 4.25 VH



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Table 5.b. Effect evaluation.

Mean response on the different quality attributes of the sargassum formulated soap. Treatment Exfoliation Irritation Freshness Lather Allergynisity Mean D.E. T1 4.33 4.83 4.5 4.66 4.83 4.63 VH T2 4.0 3.66 3.66 3.83 4.66 3.96 High T3 4.66 4.16 4.16 4.66 4.33 4.39 VH T4 4.0 4.15 4.25 4.25 4.25 4.18 H Result indicated that the mean value of all formulated product is 4.41 which suggests that the product is highly acceptable. The five (5) physical quality attributes ( texture, color, odor, hardness and size) are indicative to a good bath soap quality attributes considering the “non-traditional” materials used in developing bath soap. On the other hand, the five (5) effect quality attributes of the seaweed bath soap is very high with regards to the acceptability of the soap. The exfoliation, irritation, freshness, lather and allergynisity are characteristics of bath soap. The irritation and allergynisity quality attribute is very high with a mean value of 4.23 and 4.58, respectively that indicate that the formulated seaweed bath soap is considered safe for all skin types. The exfoliation and lather quality attributes of the formulated soap is generally acceptable by the panelists with a mean of 4.49 and 4.36, respectively in which such attributes reflect good quality of the product. Meanwhile, the quality attribute of the soap that may need to be improved is the “freshness.” Though the product is generally accepted by the panelists, such attribute must be improved for acceptance by the general public.

Table 5.a. Physical Evaluation. Mean acceptability of the seaweed bath soap by the panelists on the quality attributes of the products.

Formulated soap

Texture Color Odor Hardness Size Mean Descriptive equivalent

Gracilaria soap

4.05 4.43 4.32 4.33 4.27 4.28 Very high

Eucheuma soap

4.44 4.60 4.10 4.55 4.66 4.47 Very high

Sargassum soap

4.38 4.60 4.16 4.55 4.55 4.44 Very high

Commercial soap

4.29 4.25 4.26 4.26 4.22 4.26 Very high



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Table 5.b. Effect Evaluation. Mean acceptability of the seaweed bath soap by the panelists on the quality attributes of the products.

Formulated soap

Exfoliation Irritation Freshness Lather Allergynisity Mean D.E.

Gracilaria soap

4.44 4.22 3.99 4.38 4.55 4.31 VH

Eucheuma soap

4.55 4.27 4.05 4.38 4.60 4.37 VH

Sargassum soap

4.33 4.21 4.10 4.38 4.60 4.32 VH

Commercial soap

4.09 4.19 4.20 4.22 4.25 4.19 Hgh

Table 5 presents the mean score of respondents with respect to the three (3) bath soap products. Results indicated that all formulated bath soap preparations are highly accepted by the respondents with the highest mean of 4.47 and the lowest is 4.26 which has very high descriptive equivalent. Irritation and allergynisity are given very high score by the respondents, maybe because of the no erythema occurrence to the skin. Cost and return analysis

FIXED CAPITAL Cost/Unit(P) TOTAL AMOUNT(P)

1 food processor 2,500.00 2,500.00

1 plastic container 10Lcap 300.00 300.00

1 stainless casserole 1,000.00 1,000.00

1 laddle (stainless long handle)

100.00 100.00

50 pcs plastic molder 50.00 2,500.00

II. WORKING CAPITAL (one cycle operation)

Quantity particular Amount 12 kgs Caustic soda 3,000.00 50 ltr. Minola oil 5,000.00 3kg Dried gracilaria 200.00 1kg Dried atsuete seed 100.00 1 contract labor 5 days @ 190.00 950.00 600 pcs. Paper boards 2,040.00



97

III. TOTAL PROJECT COST P17,690.00 IV. Depreciation Cost per cycle

Particular Cost Number of Services

Salvage value Depreciation cost

Food processor 2,500.00 100 50.00 24.50 Plastic container 300.00 100 3.00 2.97 Stainless casserole

1,000.00

200

10.00

4.95

laddle 100.00 200 2.00 molder 2,500.00 200 50.00 12.25

Total depreciation Cost P46.67 Projected Income per cycle Total number of production per cycle 1,200 pcs/cycle Selling Price (farm gate) P25.00 per piece Cost of sale per cycle P 17,690.00 Total Sales 30,000.00 Net Income 12,310.00 Break even price 14.74 ROI 41 %

Economic analysis The utilization of low grade/rejected/washed-out/highly abundant seaweeds will pave a new area of developing alternative livelihood and resource utilization for the benefits of coastal communities. The basic soap ingredients such as oil and alkaline as well as the equipments can be bought locally. The seaweeds can be gathered easily the coastal areas. Thus, the total cost for a single operations of the soap making is very minimal and affordable by the producers. The return of investment is 41% if price is P25.00/pc. and break even at P14.74/pc. Table 6.a. Erythema test Skin reaction of test animals after application of test soap samples with code numbers T1 gracilaria T2 gracilaria T3 sargassum.

Skin Reactions

S C O R E

F1-1 F1-2 F3-2

A) erythema 30min 24h 3day 7days 30min 24h 3day 7days 30min 24h 3day 7days No erythema

0 - 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10

Very Slight eryrhema

1 1/10 - - - - - - - - - -



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Well defined erythema

2 - - - - - - - - - - -

Moderate to severe erythema

3 - - - - - - - - - - -

Erythema with eschar

4 - - - - - - - - - - -

Table 6.b Edema test. Skin reaction of test animals after application of test soap samples with code numbers T1 gracilaria T2 gracilaria T3 sargassum.

Skin Reactions

S C O R E

F1-1 F1-2 F3-2

A) edema 30min 24h 3day 7days 30min 24h 3day 7days 30min 24h 3day 7days No edema 0 - 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 Very Slight edema

1 - - - - - - - - - - - -

Well defined edema

2 - - - - - - - - - - - -

Moderate to severe edema

3 - - - - - - - - - - - -

Edema with eschar

4 - - - - - - - - - - - -

Tables 6.a & 6.b. represent the skin reactions of mature guinea pig (about 4 months old male). Results indicated that the formulated bath soap causes one of the ten (1/10) animals reacting with very slight erythema which detected within the first 30 minutes with reversible reaction during the succeeding periods of evaluation. The skin reaction was evaluated in 30 minutes, 24hours, 3days and 7 days for reactions of erythema and or edema. Table 7. Mean weight (g)/hr of dissolved soap material observed with the seaweed soap

samples. T1-1 (g/hr dissolve)

T1-2 (g/hr dissolve)

T3-2 (g/hr dissolve)

14.35 15 16.5 21.12 18 19.5 8.59 10 9.5 Total 44.06 43 45.5 Mean 14.68 14.33 15.16

The mean weight (g) lost/hr (14-15g) of dissolution time for soap samples are not comparably different, which means that the quality of the formulated soap is almost equal.



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Analysis of variance (ANOVA) on the mean score of the panelists on the physical quality attributes of the products shows that there were no significant differences; Fc is lesser than Ft. But on the effect quality attribute, analysis of variance shows that there is significant difference in eucheuma formulated soap (Fc is greater than Ft).

Summary, Conclusion and Recommendations

Summary The seaweed bath soap product was formulated out of seaweeds, papaya and atsuete extracts combined with coconut oil. The use of the local materials as soap ingredient will optimize the utilization and increase the current effort of seaweeds farmers. A formulation protocol was developed in coming up with the seaweed bath soap product. The product was brought to DOST- Cagayan Valley Herbal Processing Plant, Carig, Tuguegarao City for bioassay analysis and testing. The product was sensory-evaluated by 10 trained panelists of the DMMMSU-NLUC, Bacnotan, La Union. The result indicated that all seaweed bath soap preparations including the control are highly acceptable by the respondents.

Conclusions

Results indicated the following: The seaweed soap product was generally accepted as to its quality attributes, though other factor attributes such as freshness shall be improved. There are no significant differences with regards to the quality attributes and among soap preparations presented for evaluation. The application of the soap formulations (gracilaria, sargassum, eucheuma) did not cause skin edema in guinea pig during the rest of the evaluation. The patch application with test soap samples T1 gracilaria caused one of the ten(1/10) animals reacting with very slight erythema detected within the first 30 minutes with reversible reaction detected during the succeeding periods of evaluation. Recommendations

The seaweed bath soap must be tested by a wider consumer group to serve as basis for further improvement of the quality attributes of the product; and

Stability and packaging studies must be undertaken for quality development and standardization prospect of the product.



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References 1. http://www.perfects.net/Aoqili-Seaweed-Soap-defat-Soap-All-Natural-5,3-02-bar-

html 2. http://www.aromatic.com/seaweed.html 3. http://www.ihatecellulite.com/cellulite-seaweed-soap.html 4. http://www.bluespenoriginals/seaweed.soap.html 5. http://bathgifts.us/ 6. http://ezinearticle.com/?slimming-seaweed-soap---Abetter-Alternativeandid=827378 7. http://soapoperabathshop.blogspot.com/ 8. http://www.redonbit.com/news/health/819342/seaweed_substance_helps_againsts_ski

n_cancer/ 9. http://www.ehow.com/facts_5700892_benefits_seaweed-bath_html 10. Trono, Gavino, 1989, Field Guide and Atlas of the Seaweed Resource of the

Philippines.

Seaweed Bath Soap `



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In Vivo Fluorescence Imaging Of Fruit Fly With Soluble Quantum Dots

Tapas K. Mandal, Nragish Parvin and Mitali Saha

Department of Chemistry, National Institute of Technology Agartala, Agartala- 799055, India

Corresponding address:- *Tapas K. Mandal

Department of Chemistry, National Institute of Technology,Agartala-799055,India;

Cell: +91 8974729766 E-mail : [email protected]

Abstract The ability of multi colour fluorescence imaging with water soluble carbon quantum dots (WSCQDs) in organisms and biological tissues has been explored using Drosophila melanogaster (fruit flies). Here we present strategies to visualize different developmental stages and their various internal organs in vivo and in vitro condition with multiple, distinct colors. Their viability and growth were not reduced by oral quantum dots ingestion. We demonstrate a new methodology in the field of bioimaging by using synthesized water soluble carbon quantum dots (WSCQDs) that will bring a revolution in the history of biomedical science.

Keywords: Noninvasive, bioimaging, carbon, quantum dots, water soluble, fruit fly

Introduction: Exploration of quantum dots in biological systems got attention ever since its discovery. The role of WSCQDs, in biological systems and its implications are currently under evaluation primarily on the work interfacing chemistry, physics and biology. The emergence of fluorescence carbon nanoparticles/ dots shows high potential in biological labeling, bioimaging, and other different optoelectronic device applications (Batalov et al., 2009; Selvi et al., 2008; Mochalin and Gogotsi 2009). These carbon nanoparticles are biocompatible and chemically inert, (Lim et al., 2009 ; Kong et al., 2005) which has advantages over conventional cadmium-based quantum dots (Medintz, et al., 2005). However, these application of fluorescent carbon nanoparticles are poorly studied compared with other carbon or cadmium based materials. In addition, the understanding of the uses of fluorescence character in carbon nanoparticle is far from sufficient. (Zhou et al., 2007; Zhao et al., 2008). For example, the understand dynamic processes in live cells, such as intercellular and intracellular trafficking, microstructure remains unclear. To prove these difficult imaging tasks, a robust water soluble QDs are needed. Several synthesis strategies have been used, such as surface functionalization with water-soluble ligands (Sun et al., 2006),



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silanization (Cao et al., 2007) and encapsulation within block-copolymer micelles (Liu et al., 2007). Here, we investigated the photo physical properties of water-soluble CQDs prepared by a synthesis method based on Common routes in making fluorescent water soluble carbon quantum dots followed by oxidation of carbon soot ( collected from waste plant materials burning) with nitric acid (Ray et al., 2009). We observed that these small carbon particles enter into cells without any further functionalization and the fluorescence property of the particles can be used for fluorescence-based cell-imaging applications. The ability of multi color fluorescence imaging with WSCQDs in organisms and biological tissues has been explored by using Drosophila melanogaster (fruit flies). In vivo emission imaging has made detailed study of a biological species by fluorescence microscopy. Therefore we show all the in vivo images of various internal organs through out the developmental phases of Drosophila melanogaster using a new fluorescent material like WSCQDs and tally with a control experiment. We successfully acquire in vivo images of the developing three larval stages till the adult hood by using the minimally invasive imaging modality of ordinary fluorescence microscopy. The whole-body imaging of a probe in real time means that the efficacy of therapeutic treatments can be seen directly without the need for any invasive procedure. Experimental Procedures / Materials and Methods: Synthesis of water soluble Carbon Particles: Carbon soot 50 mg (collected from burning waste plant materials) was mixed with 30 ml of 5 M nitric acid in a 50ml three-necked flask. It was then refluxed at 140 °C for 10 h with magnetic stirring. After that, the black solution was cooled and centrifuged at 8000 rpm for 7 min to separate out unreacted carbon soot. The light brownish-yellow supernatant was collected, which shows green fluorescence under UV exposure. The aqueous supernatant was mixed with acetone (water/acetone volume ratio was 1:3) and centrifuged at 16000 rpm for 10 min. The black precipitate was collected and dissolved in 30 ml of water. The colorless and nonfluorescent supernatant was discarded. This step of purification separates excess nitric acid from the carbon nanoparticles. This concentrated aqueous solution, having almost neutral pH, was taken for further use. The same synthesis technique was also performed for 18 h of reflux. The supernatant obtained from the 18 h reflux, was dark yellow. We weighed the unreacted carbon soot, which was removed as precipitate, in order to find out the yield of soluble carbon nanoparticles.

The weight was ∼50 mg for the 18 h reflux times (yield ∼22%). This solution has particles having sizes ranging from 20 to 220nm and is called as-synthesized carbon quantum dots (CQDs) (see Scheme 1). Figure-2 shows AFM and TEM images of WSCQDs. Fly cultured with water soluble CQDs: Flies were of the Canton S strain (obtained from the laboratory of Dr Pradip Singha , Department of biological science ,IIT Kanpur) that had been reared in the laboratory for many generations. Stocks were maintained in an room temperature at 20- 25°C under a L14:D10 photoperiod in 250-ml bottles on WSCQDs mixed 60g of a cornmeal–agar medium seeded with yeast. Cornmeal agar medium was made according to a recipe modified from (Lewis, 1960).



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Agar (8.00g) and 0.5mg WSCQDs mixed water (1000 ml) were added to a saucepan and heated until boiling. 50 grams of organic cornmeal, 40g dextrose, 25g dried yeast were mixed together and added when the agar was boiling. The mixture was simmered for 5 min and then removed from the heat and allowed to cool to at room temperature . 2.5 gms of Nipagin and 9 ml propionic acids in 15 ml of 95% ethanol were then added and stirred into the food. Flies that were to be used in experiments were reared as follows. Twenty virgins adult were assigned randomly to media containing vial. Vials were plugged with cotton wool bungs and placed in a room temperature / incubator at 20-25°C under a L14:D10 photoperiod (the lights came on at 08:00 GMT). Drosophila were treated for two- three days before to lay eggs. These eggs were allowed to grow under WSCQDs treated food to complete their life cycle. Another set of experiment (control) has done without any WSCQDs, others conditions were same like treated and their life cycle was monitored. The organism and their all life cycle stages were washed thrice with the sterilized PBS (pH 7.4) for fluorescence microscopy (LEICA DC200). Fluorescence microscopy: Images of life cycle stages of drosophila were captured by using a Leica inverted microscope (Leica DC200, Leica microscopy system ltd, CH-9435, Heerbrugg) with an attached RS Photometrics Sensys camera, KAF1401E G1. The intensity of fluorescence was quantified by using the 488, 561 and 633nm band pass (BP) emission filter functions of the Leica microsystem imaging solution software (Leica Q fluoro version V1.0a, Leica microsystem imaging solution ltd, Germany).

Result and Discussion: The fruit fly Drosophila melanogaster is one of the most valuable organisms in genetic and developmental biology studies. Transgenic methods are in use to image with bleachable organic fluorphore or fluorescent protein, full image of all the stages of the life cycle of the living wild organism is lacking. WSCQDs have a high emission range fluorescence property. The fluorescence property of the particles were used to track their position in cells using a conventional fluorescence microscope. We acquire in vivo images of the eggs through all the larval stages till adult hood under oral ingestion (figure-3). Figure-4. showed multicolored fluorescence images providing clearer internal structure of Drosophila. In contrast, the fluorescence signals of the cells without addition of the CQDs were invisible in control experiments. Furthermore, these images reveal WSCQDs bind to the cells, but it is nonspecific. The possible mechanisms are that the WSCQDs with surface carboxylic acids bind on the surface of cells (Jessica et al., 2007; Liu and Vu, 2007). It indicated that the water-soluble CQDs bind on the surface of cells. Since the surface of WSCQDs were the functional carboxylic Group was free, which can be easily coupled with amine groups the surface cell of an organism, such as proteins, peptides and amino acids. In fact, one cell membrane carries numerous proteins and one protein typically bind on numerous water soluble CQDs.



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We observed the viability rate of the both control and treated organisms were same. While both of them completed their life cycle within 12-14 days. Their behavioral pattern is same with the normal fly. So WSCQDs does not show any toxic effect during the life cycles of Drosophila).

Conclusion: Before our these experiments a non-invasive method to create an image of a body structure from a laboratory animal using relatively simple equipment is not known. Bio–imaging began since the discovery of X-rays by Roentgen in 1895. The magnetic resonance imaging (MRI) technique has been introduced to overcome the relatively high permeability of X-rays and its deleterious effects on biological tissue. The imaging can noninvasively monitor cellular or genetic activity and subsequently use the results to track gene expression, the spread of disease, or the effect of a new drug in vivo. Our imaging process could give in vivo multicolor fluorescence images. Water soluble carbon quantum dots will become key probes for multicolor fluorescence microscopy. It is suitable for long term imaging because it is not photo bleaching. Also it has not cytotoxic effect. The whole-body imaging of a probe in real time means that the efficacy of therapeutic treatments can be seen directly without the need for any invasive procedure. Our approach can be used for milligram-scale to bio imaging . These fluorescence imaging process can useful for medical applications. These process can obtain in vivo images of cells without any invasive surgery. Also these process have the potential in biomedical applications where cadmium-based quantum dots show toxic effects. However, synthetic methods of these particles need to be much more advanced so that large quantities of these particles with different emission colors were easily prepared.

Acknowledgements: T. K. M., N.P. and M.S. are grateful to Prof. R. Gurunath, Prof. S. Sarkar and Prof. B. Prakash, IIT Kanpur for providing necessary laboratory facilities. N.P. T.K.M thanks NIT Agartala for providing a fellowship. Thanks to Prasenjit Samanta and Santanu Mondal of D.A.V college, Kanpur, for helping us.

References: Batalov, A., Jacques, V., Kaiser, F., Siyushev, P., Neumann, P., Rogers, L. J., McMurtrie, R. L., Manson, N. B., Jelezko, F. and Wrachtrup, (2009). Low temperature studies of the excited-state structure of negatively charged nitrogen-vacancy color centers in diamond. J. Phys. ReV. Lett. 15;102(19):195506. Cao, L., Wang, X., Meziani, M. J., Lu, F., Wang, H., Luo, P. G., Lin, Y., Harruff, B. A., Veca, L. M., Murray, D., Xie, S. Y. and Sun, Y. P.(2007). Carbon dots for multiphoton bioimaging. J. Am. Chem. Soc. 129, 11318. Jessica, P., Ryman-Rasmussen, Nancy, A., Riviere, J.E. and Monteiro-Riviere. (2007). Variables Influencing Interactions of Untargeted Quantum Dot Nanoparticles with Skin Cells and Identification of Biochemical Modulators. Nano Lett. 7:1344–1348.



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Kong, X. L., Huang, L. C. L., Hsu, C. M., Chen, W. H., Han, C. and Chang, H. C. (2005). High-affinity capture of proteins by diamond nanoparticles for mass spectrometric analysis,” Anal. Chem. 77, 259-265. Lewis, E.B. (1960). A new standard food medium. D. I. S. 34: 117--118. Lim, T.S., Fu, C.-C., Lee, K.-C., Lee, H.-Y., Chen, K., Cheng, W.-F., Pai, W. W., Chang, H.-C. and Fann, W. (2009). Fluorescence enhancement and lifetime modification of single nanodiamonds near a nanocrystalline silver surface. Phys. Chem. Chem. Phys. 14;11(10):1508-14. Liu, H., Ye, T. and Mao, C. (2007). Fluorescent Carbon Nanoparticles Derived from Candle Soot. Angew. Chem. 46 (34), 6473-6475 Liu, H.Y. ,and Vu, T.Q.( 2007). Quantum Dot Hybrid Gel Blotting: A Technique for Identifying Quantum Dot-Protein/Protein-Protein Interactions .Nano Lett. 7:1044–1049. Mochalin, V. and Gogotsi, Y.( 2009). Wet Chemistry Route to Hydrophobic Blue Fluorescent Nanodiamond. J. Am. Chem. Soc.131, 4594-95. Medintz, I. L., Uyeda, H. T., Goldman, E. R. and Mattoussi, H. (2005). Quantum dot bioconjugates for imaging, labelling and sensing. Nat. Mater. 4(6):435-46. Ray, S. C., Saha, A., Jana, N. R. and Sarkar R.(2009). Fluorescent Carbon Nanoparticles: Synthesis, Characterization, and Bioimaging Application, J. Phys. Chem. C.113, 18546–18551 Selvi, B. R., Jagadeesan, D., Suma, B. S., Nagashankar, G., Arif, M., Balasubramanyam, K., swaramoorthy, M. and Kundu, T. K. (2008). Intrinsically Fluorescent Carbon Nanospheres as a Nuclear Targeting Vector: Delivery of Membrane-Impermeable Molecule to Modulate Gene Expression In Vivo. Nano lett., 8(10):3182-85. Sun, Y. P., Zhou, B., Lin, Y., Wang, W., Fernando, K. A. S., Pathak, P., Meziani, M. J., Harruff, B. A., Wang, X., Wang, H., Luo, P. G., Yang, H., Kose, M. E., Chen, B., Veca, L. M. ans Xie, S. Y. (2006). Quantum-sized carbon dots for bright and colorful photoluminescence. J. Am. Chem. Soc. 128(24):7756. Zhou, J., Booker, C., Li, R., Zhou, X., Sham, T. K., Sun, X. and Ding, Z. (2007). An electrochemical avenue to blue luminescent nanocrystals from multiwalled carbon nanotubes (MWCNTs). J. Am. Chem. Soc. 129(4):744-5. Zhao, Q.-L., Zhang, Z.-L., Huang, B.-H., Peng, J., Zhang, M. and Pang, D.-W. (2008). Facile preparation of low cytotoxicity fluorescent carbon nanocrystals by electrooxidation of graphite. Chem. Commun. 41, 5116-5118.



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Figure Legends

Figure 1. Schematic diagram of WSCQDs treated drosophila (fluorescing) and untreated drosophila (not fluorescing). Figure 2. AFM topography images of water soluble C-Dots(left) and HRTEM image C.Dots (right). Figure 3. Fluorescence images of various developmental stages of drosophila treated with WSCQDs. From left to right egg, larva, pupa, female imago and male imago respectively.

Figure 4. Various internal organs of D. melanogaster larva treated with water soluble quantum dots. In vivo image, merge of three lights (488, 561 and 633nm). Where at-atrium, bn-brain, as-anterior spiracle, tc-trachea, pxpharynx, sd- salivary duct, sg-salivary gland, Ep-esophagus, pvc-proventiculus, gc-gastric ceaca, mg-midgut, mi-mid intestine, gd-gonad, utr-ureter, mt-malpighian tubule, hg-hind gut, as- anus. Scale bar 0.5mm.

Figure-1



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Figure-2

Figure-3

Figure-4



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Biofertilizers in Action: Contributions of BNF in Sustainable Agricultural Ecosystems

A.M., Ellafi, 1 Gadalla, A2 and Galal2, Y.G.M. 1Biotechnology Research Center, Tripoli, Libya

2Atomic Energy Authority, Nuclear Research Center, Soil and Water Research Department, Abou-Zaabl, 13759, Egypt.

Abstract Biofertilizers are considered to be cost effective, ecofriendly and renewable sources of plant nutrients supplementing chemical fertilizers in sustainable agricultural systems. This refers to microorganisms, which increase crop growth through different mechanisms, i.e. biological nitrogen fixation, growth-promoting or hormonal substances increased availability of soil nutrients. Their importance lies in their ability to supplement/ mobilize soil nutrients with minimal use of non-renewable resources and as components of integrated plant nutrient systems. The most important group of biofertilizers that have played vital role of maintaining soil fertility in agriculture via BNF process. Contributions of BNF through the application of different nitrogen fixing microorganisms (biofertilizers groups) were estimated under different environmental conditions given using isotopic (15N isotope dilution) and non-isotopic (N difference) methods. Symbiotic plant-microbe interactions such as Rice-Azolla, Legume-Rhizobium either prennial crops or fixing trees were examined on field and greenhouse experiments. Similarly, free-living or associative N2 fixing microorganisms were evaluated for potential N2 fixation with non-legumes, i.e. rice, maize, barely and wheat. Also, growth-promoting effect was considered for plants, particularly cereal crops inoculated with diazotrophs and/or arbuscular mycorrhiza fungi (VAM). Such microflora have the ability to provide considerable amounts of sparing nutrients especially P in rhizoplane of inoculated plants. Application of 15N tracer techniques gave us a chance to confirm some of the mechanisms responsible for enhancement of plant growth and nutrient acquisition. From our viewpoint, it is important to encourage the use of biofertilizers especially under circumstances of lacks in soil and water resources like we have in our region and in the same time, to spread out the concept of low input agriculture to the poor farmers. Therefor, there is a need to develop reliable biofertilizers with scientifically defined modes of action and incorporating BNF to maximize their efficacy.

Keywords: Agro-ecosystems, Biofertilizers, BNF, Isotopic techniques

Introduction Beneficial plant-microbe interactions in the rhizosphere are primary determinants of plant health and soil fertility. Various soil microorganisms that are capable of exerting beneficial effects on plants have a potential for use in agriculture and can lead to increased yields of a wide variety of crops. Soil-plant-microbe interactions are complex and there are many ways in which the outcomes can influence plant health and productivity (Kennedy 1998). Microbial groups that



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affect plants by supplying combined nitrogen include the symbiotic N2-fixing rhizobia in legumes, actinomycetes in non-leguminous trees, and blue-green algae in symbiosis with water ferns. Additionally, free-living fixing bacteria of the genus Azospirillum affect the development and function of grass and legume roots, thereby improving minerals and water uptake (Okon et al., 1998). Other microorganisms that are known to be beneficial to plants are the phosphate solubilizers (Pesudomonas spp. and Bacillus megatherium), plant-growth-promoting pesudomonads and mycorrhizal fungi.

These different types of microorganisms are of economic importance in improving crop productivity and can replace costly chemical fertilizers, improving water utilization, lowering production costs, and reducing environmental pollution, while ensuring high yields. Some groups of beneficial rhizosphere microorganisms are engage in well-developed symbiotic interactions in which particular organs are formed, such as mycorrhizas and root nodules, whilst others develop from fairly loose associations with the root. The interaction between rhizobial bacteria and the roots of leguminous crops has been well researched (Brockwell et al., 1995), but for the mycorrhizal relationship it has only recently become a significant topic of research (Smith and Read 1997). Other plant root-microbe interactions arise from specific interactions between groups of bacteria or fungi that are adapted to live in the rhizosphere. Such rhizobacteria or rhizofungi are adapted to exploit this niche and often act synergistically in combination with mycorrhizal. Both the growth-promoting rhizobacteria (PGPR) and plant-growth-promoting fungi (PGPF) affects the plant health through interactions with potential phytopathogens (Azcon-Aguilar and Barea 1996). Others produce compounds that directly stimulate plant growth, such as vitamins or plant hormones (Barea 1997, 2000). Others, such as the fungi Trichoderma, may stimulate plant growth in more than one mechanism (Ousley et al., 1994).

Advanced methodologies, such as 15N techniques applied in such topics of biofertilization offers reliable techniques for verifying the mechanisms involved in plant-growth promotion occurred and consequently gave an exact estimation of biologically fixed nitrogen.

In this context, we will overview the situation of different biofertilizers systems applied under semi-arid conditions of our area using the conventional and isotopic methods. Thus, the biofertilizers effectiveness on plant health and soil fertility, as a most cheap source of nutrients, has been discussed.

Rhizobium-Legume symbiosis Sustainable agriculture relies greatly on renewable resources and on-farm nitrogen

contributions are achieved largely through biological nitrogen fixation (BNF .(Biological nitrogen fixation helps in maintaining and/or improving soil fertility by using N2, which is in abundance in the atmosphere. Annually, BNF is estimated to be around 175 million tones N of which close to 79 % is accounted for by terrestrial fixation. In this respect, Fig (1) illustrates the distribution of N2-fixed in various terrestrial systems and recognize the importance of BNF in the context of the global N cycle. The BNF offers an economically attractive and ecologically sound means of reducing external N inputs and improving the quality and quantity of internal resources (Wani et al., 1995). Experimental estimates of the proportion of plant N derived from N2-fixation (Pfix.) and the amounts of N2-fixed by important tropical and cool season crop legumes



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are presented in Table (1). Available data of N2-fixed in forage legumes, cover crops and N2-fixing trees indicates similar values to

Fig 1. Distribution of 139 million tonnes of N2 fixed in terrestrial systems. Source: Burns and Hardy (1975)

Table 1. Range of experimental estimates of the proportion (Pfix) and amount of N2 fixed by important pulses and legume oilseeds. Source: Peoples et al. (1995)

Species Pfix Amount N2 fixed

(%) (kg N ha-1) Cool-season legumes

Chickpea (Cicer arietinum ) 8 - 82 3 - 141

Lentil (Lins culinaris) 39 - 87 10 –192

Pea (Pisum sativum) 23 - 73 17 –244

Faba bean (Vicia faba) 64 - 92 53 – 330

Lupin (Lupinus angustifolius) 29 - 97 32 – 288

Warm–season legumes

Soybean (Glycine max) 0 - 95 0 – 450

Groundnut (Arachis hypogaea) 22 - 92 37 – 206

Common bean (Phaseolus vulgaris) 0 - 73 0 –125

Pigeon pea (Cajanus cajan) 10 - 81 7 – 235

Permanent Grasslands Forsts & WoodlandsLegumes Unused LandNon-Legumes



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Green gram (Vigna radiata) 15 - 63 9 – 112

Black gram (V. mango) 37 - 98 21 – 140

Cowpea (V. unguiculata) 32 - 89 9 - 201 that of crop legumes (Tables 2, 3). Legumes have been an important component of agriculture since ancient times because of its role in improving soil fertility via their N2-fixing ability (Wani et al., 1995). Review made by those investigators showed different proportions of N2-fixed raged from low to moderate and high levels. For example, pigeon pea cultivars fixed 4 - 53 kg N ha-1 season-1 while depleting 20 - 49 kg N ha-1 from the soil pool. In the case of chickpea, different cultivars fixed 23 - 40 kg N ha-1 season-1 and removed 63 - 77 kg N ha-1 season-1 from soil. Groundnut fixed 190 kg N ha-1 season-1 when pod yields were around 3.5 t ha-1 (Nambiar et al., 1986), however, it relies for its 20 - 40 % (47 - 127 kg N ha-1 season-1) of the N requirement on soil or from fertilizer (Giller et al., 1987). Our results, in this regard, showed that N2-fixation in groundnut was vigorous with Bradyrhizobium inoculation either solely of in combination with mycorrhizal fungi (El-Ghandour et al., 1997), and the values of N derived from air, as estimated using 15N isotope dilution, were on line with those reported by Giller et al., (1987). Residual effect of 15N-labelled urea or ammonium sulfate on growth and N2-fixation by modulating soybean was examined (Galal and El-Ghandour 1997), and the data of N derived from air was ranged from 42 to 65 % as affected by Bradyrhizobium inoculation either solely or in combination with Azotobacter chroococcum strain. Similar, dual inoculation with B. japonicum and Azospirillum brasilense enhanced growth and N2-fixation of nodulating soybean cultivated in sterilized and/or non-sterilized soils. It seems that A. brasilense act as helper bacteria for developing B. japonicum performance in the rhizosphere of nodulating soybean (Galal 1997).

Table 2. Range of experimental estimates of the proportion (Pfix) and amount of N2 fixed by important forage legumes. Modified after: Peoples et al. (1995)

Species Pfix

(%)

Amount N2 fixed

(kg N ha-1)

Period of measurement

Temperate forages Lucerne/ alfalfa (Medicago sativa) 46 - 92 90 – 386 Annual White clover (Trifoliumrepens) 62 - 93 54 - 291 Annual Subterranean clover (T. Subterranean) 50 - 93 2 - 206 Annual Vech (Vicia sativa) 75 106 Not available

In a pot experiment, El-Ghandour and Galal (1997) reported that more than 80 % of the nitrogen

requirement of faba bean plants (different genotypes) was gained from air (% Ndfa). Thus, the addition of 15N rice straw enhanced the N2-fixation potential as compared to 15N-ammonium nitrate fertilizer. Combined inocula of rhizobia and mycorrhizae fungi had enhanced growth and N2-fixation of inoculated faba bean comparable to single inocula.

Faba bean grown in farmer’s fields well responded to inoculation with Rhizobium applied in two ways (liquid culture or peat-based) under gradual increase of nitrogen fertilizer up to 40 kg N ha-

1. In this field experiment, nitrogen fixation estimated by N difference method was negatively affected by the high level of fertilizer applied (40 kg N ha-1).In this respect, the inoculant types were slightly differentiated (El-Ghandour et al. 2001). Similar field trial with mungbean (Vigna radiata L. Wilczek) was conducted under drip irrigation system to investigate the effect of



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rhizobial inoculants either applied in peat based inoculum or through the irrigation water (inocugation). Nodulation was excellently performed with both types of inoculum. Application of isotope dilution approach showed the superiority of inocugation method over the peat-based inoculum since the percentages of N derived from air and utilized by seeds were 73% and 50%, respectively (Thabet and Galal 2001).

Table 3. Range of experimental estimates of the proportion (Pfix) and amount of N2 fixed by important N2-fixing trees, green manures and cover crops. Modified after: Peoples et al. (1995) Species Pfix

(%)

Amount N2 fixed

(kg N ha-1)

Period of measurement

Trees Acacia holosericea 30 3 - 6 6.5 months Casuarina equisetifolia 39 -65 9 - 440 6 – 12 months Gliricidia (Gliricidia sepium) 52 - 64 86 - 309 Annual - hedgerow for forage 69 - 75 99 - 185 3 – 6 months - alley crop hedgerow 43 170 Annual Leucaena (Leucaena leucocephala) 34 - 78 98 - 230 3 – 6 months Green manures and cover crops Azolla spp. 52 - 99 22 - 40 30 days Sesbania cannabina 70 - 93 126 - 141 Seasonal average Sesbania rostrata 68 - 94 70 - 324 45 – 65 days Sesbania sesban 13 - 18 7 - 18 2 months Asymbiotic diazotrophs Several groups of asymbiotic N2-fixing bacteria have been identified in soils and flooded systems and those genera which include N2-fixing species were reviewed by Roper and Ladha (1995). The heterotrophic diazotrophs depend on carbon, e.g. from crop residues, for energy. The most common isolates from soils are Azotobacter, Azomonas, Beijerinckia and Derxia, Clostridium and Bacillus, Klebsiella and Enterobacter, and Azospirillum, Desulfovibrio and Desulfotomaculum (Roper and Halsall 1986).

Nitrogen fixation by asymbiotic bacteria has been observed in greenhouse and field experiments under dry land cropping systems. Biological N2 fixation associated with crop residues (legumes or cereals) was investigated in pot experiments with wheat (Galal 2002) and chickpea cultivars (El-Ghandour and Galal 2002). In these experiments, both residues of wheat and rice were labelled with 15N and used as organic N sources in comparison with either 15N-labelled ammonium sulfate or ammonium nitrate as chemical nitrogen fertilizers. Dual inoculation with Rhizobium and mycorrhizae fungi significantly affected nodulation and colo0nization percentages of chickpea cultivars (El-Ghandour and Galal 2002). Rhizobium inoculation extended to be used with wheat gave the best results of growth parameters and N2 fixation when combined with Azospirillum brasilense as heterotrophic diazotrophs (Galal 2002). The economical return of Azospirillum brasilense (as liquid media or commercial product) was estimated with maize crop grown under field conditions. The obtained data showed that inoculation combined with the half dose of recommended N fertilizer rates was the most effective and low cost agricultural inputs (Abdel Monem et al. 2001). The nitrogen uptake by wheat plants was significantly increased by application of soybean residues and inoculation with



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Azospirillum brasilense (Galal and Thabet 2002). This field trial concluded that soybean residue as enriched N material, and Azospirillum brasilense inoculation enhanced growth, grain and N yields of wheat cultivars grown in poor fertile sandy soil.

All studies on lowland rice reported a positive N balances but no one determine what proportion of this N may be derived from free-living N2-fixing cyanobacteria in the flood water, heterotrophic N2-fixers in the soil or those associated with the plant (Boddey et al. (1995). Against the acetylene reduction (AR) assay, the 15N isotope dilution technique has the potential to estimate contribution of BNF to the plants over the whole growth season and unlike the N balance and acetylene reduction techniques, it estimates fixed N actually incorporated into the plant tissue (Chalk 1985). The main problem with this technique lies in labelling the soil with 15N, if the enrichment varies with area, depth or time, different plants (the control and different rice varieties) may have different N uptake patterns and do not obtain the same 15N enrichment in the soil derived N, an assumption essential to the application of the technique (Boddey 1987). Many diazotrophs has been isolated from the rhizospherte and roots of rice such as Azotobacter, Azospirillum, Pseudomonas, Klebsiella and Enterobacter, but the presence of these microorganisms in association with rice roots does not necessarily mean that the plants obtain significant contribution from biological fixation. In this respect, Boddey et al. (1986) counted numbers of Azospirillum brasilense above 106 cells g fresh root-1 of wheat plants grown in 15N-labelled soil and in the same time, plant N uptake was significantly increased by inoculation, but 15N enrichment data showed that the response was not due to BNF inputs.

Galal and El-Ghandour (2000) examined the effect of inoculation of Azospirillum brasilense on grain yield, biological nitrogen fixation and NPK uptake by two rice cultivars (Giza 172 and IR 28), grown under greenhouse conditions (pot experiment). 15N data confirmed the enhancement of N derived from fertilizer and 15N recovery due to inoculation with Azospirillum as compared to the uninoculated treatment. The proportion of N derived from air not exceeds 28% indicating that the effective mechanism is the promotion of plant growth and nutrients uptake rather than BNF. Similar findings were observed when comparative study was held between Azospirillum brasilense, Azolla pinnata and arbuscular mycorrhizae fungi, as individual inoculum, using 15N tracer technique in pot experiment with japonica rice variety, Giza 171 (Galal; 2000).

Azolla

The aquatic fern Azolla is probably used as a green manure on < 2% of the world’s rice crop, but this still represents around 2 to 3 million ha (Giller and Wilson 1991), Under optimal conditionsa Azolla doubles in biomass every 3 to 5 days and one crop can be expected to accumulate between 70 and 110 kg N ha-1 (Ventura and Watanabe 1993). With experimental values of Pfix commonly >70% (Kumarasinghe and Eskew 1993; Roger and Ladha 1992).Azolla represents a potentially important source of N for flooded rice. However, there is little information available concerning inputs of N by Azolla in farmer’s fields. Since growth and N2-fixing capacity of Azolla can be affected by many environmental variables, mineral nutrition (particularly phosphorus), insect predators and pathogens, it is uncertain whether experimental potentials are ever realized in farmer’s paddies (Giller and Wilson 1991).



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Estimation of N2 fixed by Azolla and utilized by rice plants using 15N techniques indicated that N derived from Azolla pinnata was identical to those derived from 15N-labelled urea and more than 80% of the N utilized by rice was gained from fixation. This result was true under both sterilized and nonsterilized soils (Galal 1997). Azolla have a potential to fix atmospheric air in adequate quantities (more than 60% of total N uptake) which compensated a considerable amount of N requirements for rice production (Galal, 2000; Galal and El-Ghandour, 2000).

References Abdel Monem, MAS, Khalifa, HE, Beider, M, El-Ghandour, IA and Galal, YGM (2001) Using biofertilizers for maize production: Response and economic return under different irrigation treatments. J. sustainable Agric. 19: 41-48. Azcon-Aguilar, C. and Barea, J.M. (1996). Arbuscular mycorrhizas and biological control of soil-borne plant pathogens: an overview of the mechanisms involved. Mycorrhiza 6: 457-464. Barea, J.M. (1997). Mycorrhiza / bacteria interactions on plant growth promotion. In: Ogoshi A., Kobayashi L., Homma Y., Koclama E., Kondon N., Akino S. (eds) Plant growth-promoting rhizobacteria, present status and future prospects. OECD, Paris, pp. 150-158. Barea, J.M. (2000). Rhizosphere and mycorrhiza of field crops. In: Toutant J.P., Barazs E., Galante E., Lynch J.M., Schepers J.S., Werner D., Werry P.A. (eds) Biological resource management: connecting science and policy, (OECD) INRA Editions and Speringer, Berlin Heidelberg New York, pp. 110-125. Boddey, RM (1987) Methods for quantification of nitrogen fixation associated with gramineae. CRC Crit. Rev. Plant Sci. 6: 209-266. Boddey, RM, de Oliveira, OC, Urquiaga, S, Reis, VM, de Olivares, FL, Baldani, VLD and Döbereiner, J (1995) Biological nitrogen fixation associated with sugar cane and rice: Contribution and prospects for improvement. Plant and Soil 174: 195-209. Boddey, RM, Baldani, VLD, Baldani, JI and Döbereiner, J (1986) Effect of inoculation of Azospirillum spp. on the nitrogen assimilation of field grown wheat. Plant and Soil 95: 109-121. Brockwell, J., Bottomley, P.J. Thies, J.E. (1995). Manipulation of rhizobia microflora for improving legume productivity and soil fertility: a critical assessment. Plant and Soil 174: 143-180. Burns, RC and Hardy, RWF (1975) Nitrogen fixation in bacteria and higher plants. Springer Verlag, Berlin, 189 p. Chalk, PM (1985) Estimation of N2 fixation by isotope dilution: An appraisal of techniques involving 15N enrichment and their application. Soil Biol. Biochem. 17: 389-410



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El-Ghandour IA and Galal YGM (2002) nitrogen fixation and seed yield of chickpea cultivars as affected by microbial inoculation, crop residue and inorganic N fertilizer. Egypt. J. Microbiol. (Accepted) El-Ghandour IA, Galal YGM, Aly SS, Gadalla AM and Soliman S (2001) Rhizobium inoculants and mineral nitrogen for growth, N2-fixation and yield of faba bean. Egypt. J. Microbiol. 36: 243-254. El-Ghandour, I.A., Galal, Y.G.M.(1997). Evaluation of biological nitrogen fixation by faba bean (Vicia faba L.) plants using N-15 dilution techniques. Egypt J. Microbiol. 32: 295-307. El-Ghandour, I.A., Galal, Y.G.M., Soliman, S.M. (1997). Yields and N2-fixation of groundnut (Arachis hypogaea L.) in response to inoculation with selected Bradyrhizobium strains and mycorrhizal fungi. Egypt J. Microbiol. 32: 467-480. Galal, YGM (2002) Assessment of nitrogen availability to wheat (Triticum aestivum L.) from inorganic and organic N sources as affected by Azospirillum brasilense and Rhizobium leguminosarum inoculation. Egypt. J. Microbiol. (Accepted) Galal, YGM (2000) Rice biofertilization: A comparative study using 15N tracer technique. In: El-Nawawy et al. (Eds.) Proceedings of the Tenth Microbiology Conference, pp. 87-99. Galal, Y,G.M. (1997). Dual inoculation with strains of Bradyrhizobium japonicium and Azospirillum brasilense to improve growth and biological nitrogen fixation of soybean (Glycine max L.). Biol. Fertil. Soils 24: 317-322. Galal, YGM (1997) Estimation of nitrogen fixation in an Azolla-rice association using the nitrogen-15 isotope dilution technique. Biol. Fertil. Soils 24: 76-80. Galal YGM and Thabet EMA (2002) Effect of soybean residues, Azospirillum and fertilizer N on nitrogen accumulation and biological fixation in two wheat cultivars. Egypt. J. Microbiol. (Accepted) Galal YGM and El-Ghandour, IA (2000) Biological nitrogen fixation, mycorrhizal infection and Azolla symbiosis in two rice cultivars in Egypt. Egypt. J. Microbiol. 35: 445-461. Galal, Y.G.M., El-Ghandour, I.A. (1997). Biological N2-fixation and growth of soybeans as affected by inoculation and residual 15N. Egypt J. Microbiol. 32: 453-466. Giller, KE and Wilson, KJ (1991) Nitrogen fixation in tropical cropping systems. CAB International, Wallingford, UK, 313 p. Giller, K.E., Nambiar P.T.C. Sritivasa Rao, B., Dart, P.J., Day, J.M. (1987). A comparison of nitrogen fixation in genotypes of groundnut (Arachis hypogae L.) using 15N-isotope dilution. Biol. Fertil. Soils 5: 23-25.



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Kennedy, A.C. (1998). The rhizosphere and spermosphere. In: Sylvia DM, Fuhrmann JJ, Hartel PG, Zuberer DA (eds) Principles and applications of soil microbiology, Prentice Hall, New Jersey, pp. 389-407. Kumarasinghe, KS and Eskew, DL (1993) Isotopic studies of Azolla and nitrogen fertilization of rice. Kluwer Academic Publishers, Dordrecht, 145 p. Nambiar, P.I.C., Rego, T.G., Sritivasa Rao, B. (1986). Comparison of the requirement and utilization of nitrogen by genotypes of sorghum (Sorghum bicolor) and nodulating and non-nodulating groundnut (Arachis hypogae L.). Field Crops Res. 15: 165-179. Okon, Y., Bloemberg, G.V. and Lugtenberg, B.J.J. (1998). Biotechnology of biofertilization and phytostimulation. In: A. Altman (ed.) Agricultural Biotechnology. Marcel Dekker, Inc. New York, pp. 327-349. Ousley, M.A., Lynch, J.M., Whipps, J.M. (1994). Potential of Trichoderma spp. as consistent plant-growth stimulators. Biol. Fertil. Soils. 17: 85-90. Peoples, MB, Herridge, DF and Ladha, JK (1995) Biological nitrogen fixation: An efficient source of nitrogen for sustainable agricultural production? Plant and Soil 174: 3-28. Roger, PA and Ladha, JK (1992) biological N2 fixation in wetland rice fields: estimation and contribution to nitrogen balance. Plant and Soil 141: 41-55. Roper MM and Ladha JK (1995) Biological N2 fixation by heterotrophic and phototrophic bacteria in association with straw. Plant and Soil 174: 211-224. Roper MM and Halsall DM (1986) Use of products of straw decomposition by N2-fixing (C2H2 reducing) populations of bacteria in three soils from wheat-growing areas. Aust. J. Agric. Res.37: 1-9. Smith, S.E., Read, D.J. (1997). Mycorrhizal symbiosis. Academic Press, London. Thabet EMA and Galal YGM (2001) Field trial to evaluate mungbean (Vigna radiata L.Wilczek) response to rhizobial inoculation using N-15 tracer technique. Isotope & Rad. Res. 33: 339-348. Ventura, W and Watanabe, I (1993) Green manure production of Azolla microphulla and Sesbania rostrata and their long-term effects on the rice yields and soil fertility. Biol. Fertil. Soils 15: 241-248. Wani, S.P., Rupela, O.P., Lee, K.K. (1995). Sustainable agriculture in the semi-arid tropics through biological nitrogen fixation in grain legumes. Plant and Soil 174: 29-49.



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Successional Changes in Herb Vegetation Community in an Age Series of Restored Mined Land- A Case Study of

Uttarakhand India

Shikha Uniyal Gairola* Dr. (Mrs.) Prafulla Soni**

*Research scholar Forest Ecology and Environment Division

Forest Research Institute Dehradun, Uttarakhand

India e-mail- [email protected]

**Scientist G & Head

Forest Ecology and Environment Division Forest research Institute Dehradun, Uttarakhand

e-mail- [email protected]

Corresponding author e-mail [email protected]

Abstract Present study was done with an objective to study the successional changes in herbaceous vegetation in an age series of restored mined land and also analyzes them by subjecting the vegetation data to cluster analysis. Succession is a slow process naturally and in the absence of human interventions and aid, disturbed areas such as abandoned surface-mined sites proceed through a process of primary succession, which carries important implications for long term site stability, soil fertility, and compositional changes in vegetation and plant productivity. In the field of ecology, community composition changes over time. The study of succession addresses this change, which is influenced by the environment, biotic interactions and dispersal. The present study was carried out in an age series of 23, 22, 21 and 20 years old mine restored sites at Dehradun district in Uttarakhand and an adjoining natural forest was also studied for comparison of composition of herbs in all sites. The results of the study reveals that with widespread distribution and dominance of some of the prominent naturals invaders as component of both - the mined sites as well as the undisturbed natural site, the final composition of the community at the restored sites are compiled solely from the existing population of the species and the succession on restored area results in the similar community as that found on undisturbed forest in the same vicinity.

Key words: Age series; Community composition; Natural invaders; Restored mined land Site; stability; Succession; Undisturbed forest;



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Introduction Humans have disturbed, preempted or damaged much of the earth's terrestrial ecosystems. Some of this damage is permanent and it is clear that degradation thresholds have been crossed in many habitats and that natural succession alone cannot restore viable and desirable ecosystems without intervention (Van Andel and Aronson 2005). Natural succession of mine spoil is a slow process. Initially, the mine spoils are colonized only by a few herbaceous species especially the hardy grasses and nitrogen-fixing legumes. The growth of grasses and legumes ameliorates the spoil fertility by the addition of organic matter and nutrients to it, subsequently paving way for other herbaceous species to colonize (Arvind Singh,2004). The present study has been undertaken in restored area of rock phosphate mine at Maldeota in Doon Valley that has an elevation ranging from 650m to about 1050m above mean sea level (MSL). It is situated in the north east of Dehradun, Uttarakhand (India) at a distance of about 18km on the west bank of perennial river Bandal. The area affected by open cast mining was about 15 hectares till 1982 when ecorestoration was initiated. Ecological restoration of this mine site has been done by using integrated technical and biological measures. (Soni and Vasistha, 1985). Present study was done in the year 2005 and 2006 and data was collected during post monsoon seasons during both the years. A comparative study of herbaceous vegetation has been done between a 23 years old restored site (site1), 22 years old restored site (site 2), 21 years old restored site (site3) and 20 years old restored site (site 4). For comparison an adjoining natural forest (site 5) has also been studied.

Materials and Methods For the present investigation, the restored areas of different ages were selected, besides the adjoining natural forest (undisturbed by mining) as control site for comparing the impact of restoration and successional changes in shrubs in all age series of restoration. Five quadrat of 1x1 meter was laid in the selected sites according to quadrat method (Misra, 1968). Importance Value Index (IVI) was calculated separately for each species of the community. Importance Value Index (IVI) was calculated by the summation of relative values of frequency, density and dominance (Curtis and McIntosh, 1950; Curtis and Cottam, 1956; Phillips, 1959).

The formulae used for the various calculations were: -

Density = studied quadrats ofnumber Total

species a of individual ofnumber Total

Frequency% = studied quadrats ofnumber Total

species a of occurrence of quadrats ofNumber × 100

Abundance = occurrence of quadrats ofNumber

species a of sindividual of no. Total



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Results In site 1, during first year among herbaceous vegetation highest IVI was found for Murraya koenigii (97.18) while lowest IVI was calculated for Achyranthes aspera (9.52). During the second year of study the minimum IVI was observed for Cynadon dactylon i.e. 5.02 and maximum IVI was observed for Ageratum conyzoides (Table 5.1). In site 2, among herbs during the first year highest IVI was found for Adhatoda zeylanica (63.78) while lowest IVI was calculated for Cymbopogon martini (6.39). Similarly, during the second year of study the minimum IVI was observed for Eupatorium glandulosum i.e. 4.74 and maximum IVI was observed for Lantana camara (60.99) (Table 5.7). In site 3, among herbaceous vegetation in first year highest IVI was found for Murraya koenigii (74.24) while lowest IVI was calculated for Corchorous aestuans (6.76) (Table 5.13). During second year of the study minimum IVI was observed for Aerva scandens i.e. 9.03 and maximum IVI was observed for Achyranthes aspera (68.03) (Table 5.16). In site 4, among herbs in first year highest IVI was found for Bidens pilosa (81.61) while lowest IVI was calculated for Frageria (4.42). During second year of the study the minimum IVI was observed for Murraya paniculata i.e. 4.67 and maximum IVI was observed for Murraya koenigii (49.31) (Table 5.22). In site 5, among herbaceous vegetation during post-monsoon season in first year highest IVI was found for Bidens pilosa (118.49) while lowest IVI was calculated for Ageratum conyzoides (3.78). During second year of the study in minimum IVI was observed for Rumex hastatus i.e. 5.32 and maximum IVI was observed for Achyranthes aspera (77.06) (Table 5.28).

Cluster Analysis. Cluster analysis divides data into cluster that are meaningful and useful and helps in understanding relationships between and within the community. Classification of sites was done through cluster analysis. In the present study cluster analysis was used to distinguish the sites on the basis of herb layer (RS in the figure denotes the restored sites). 5.2.1 Cluster analysis for herbs Among herbs, (figure 5.1) during the period of study, first division of the cluster was at 59.17% similarity segregating 22 years old restored site (site 2) from other four sites i.e. 23 years old restored site (site 1), 21 years old restored site (site 3), 20 years old restored site (site 4) and natural forest (site 5). This segregation may be due to the presence of Agave sisalana and Deutizia staminia in site 2 and absence of Bidens pilosa. The second division of cluster was at 53.62% which segregated site 3 from other study sites. This may be due to the presence of Melia composita seedling, Corchorous aestuans, Oxalis corniculata, Urtica aphyla and absence of Oplismenus burmanii. Third division of cluster was at 47.41% which segregated site 4 from other sites. This may be due to the presence of Frageria sp., and Randia dumetorum in this site. The fourth division was observed at 47.25%. This division segregated site 1 from site 5. Presence of species like Sida cordifolia, Adhatoda zeylanica and Oplismenus compositus may be the reason for this segregation.



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Figure 1 Cluster analysis of herbs during postmonsoon season in October 2005 During the study period, among herbs, the first division of cluster was at 36.87% similarity segregating site 3 from other four sites i.e. 23 years old restored site (site 1), 22 years old restored site (site 2) 20 years old restored site (site 4) and and natural forest. This segregation may be due to the presence of Cyperus rotundus, Boerhavia diffusa in site 3 and absence of Adhatoda zeylanica. The second division of cluster was at 48.16% which segregated site 4 from other study sites. This may be due to the presence of Cissampelos pareira, Syzygium cumini seedling, Toona ciliata seedling. Third division of cluster was at 52.44% which segregated site 5 from other sites. This may be due to the presence of Rumax hastatus, Ipomoea fistulosa and absence of Lantana camara seedling. The fourth division was observed at 59.25%. This division segregated site 2 from site 1. Presence of species like Justicia simplex, Setaria glauca may be the reason for this segregation while Aerva scandens



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Figure 2 cluster analysis of herbs during pre monsoon in the study period (October 2006) was absent from site 2. However, site 2 and site 1 were the most similar sites observed during the study.

Discussion Among herbs (Site 1) Murraya koenigii, Lantana camara, Ageratum conyzoides and Bidens pilosa were the dominant herbs found in this site (Table 5.1 and 5.4). The invasion of large number of native species including trees, shrubs and herbs and grasses may attribute that the system is still progressing towards successional phase. Invasion in the successional phase is relatively easy than invasion in to climax phase of the system (Ramakrishnan, 1991). Among herbaceous vegetation (site 2) a total of 22 species were found and none of the planted species were found in the restored area. This may be due to the process of natural succession. Murraya koenigii, Adhatoda zeylanica, Oplismenus compositus Barleria cristata showed the highest density. (Table 5.7 and 5.10). In herbaceous vegetation Bidens pilosa, Achyranthes aspera and Commelina benghalensis were found dominant in site 3. Among herbaceous vegetation, during the study period in site 4 Bidens pilosa, Cymbopogon martini, Murraya koenigii, Oplismenus compositus, Eupatorium glandulosum and Lantana camara were the densest species found during the study period (Table 5.19 and 5.22). Due to restoration activity the diversity of the plant community generally increases. It was due to invasion of native plant species from surrounding areas as the site got ameliorated after restoration providing favorable condition for their establishment. Bhatt et al. (1991) and Banerjee et al. (1996) have supported these findings. In site 5 i.e. the natural forest Bidens pilosa had the maximum density. The maximum number of species in the natural site and the restored sites were similar which supports the fact that plant species from adjoining areas must have invaded the restored sites. Bhatt 1990 has reported the presence of Eriophorum comosum, Pennisetum purpureum and Saccharum spontaneum after 8 years of restoration in the same area but after 23 years of



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succession these species has been replaced by higher successional species. The critical examination of the data shows that although some of the planted species like Agave sisalana, Dodonea viscosa and Rumex hastatus are still present but their density has declined considerably through the entire period of successional development. The widespread dominance of natural invaders like Eupatorium glandulosum, Desmodium gangeticum Artemisia vulgaris, Boehmeria platyphylla, Woodfordia fruticosa, Lantana camara indicates that the restored site is proceeding towards similar characteristics of the adjacent natural forest. It is interesting to note that while natural invaders recorded an increase in the percentage contribution to overall density, the species introduced initially showed an increasing mortality. These findings support the earlier studies which show that planted species do not persist because local species required less maintenance and provide compatibility with surrounding sites (Luken, 1990).

References Banerjee, S.K. A.J. Williams; S.C. Biswar; R.B. Manjhi and T.K. Mishra, 1996. Dyanmics of

Natural ecorestoration in coal mine overburden of dry deciduous zone in M.P. India. Ecol. Env. & Cons. 2 (97-104).

Bhatt, V., 1990. Biocoenological succession in reclaimed rock phosphate mine of Doon Valley. Ph.D. thesis, H.N. Bahuguna Garhwal University, Srinagar (U.K.)

Bhatt, V., Soni, P., Vasistha H.B. and Kumar, O., 1991. Preliminary investigation of the status of soil inhabitants in reclaimed mine spoils. J. Nat. Con., 3(1): 10

Curtis, J.T. and Cottom, G., 1956. Plant Ecology workbook laboratory field reference manual. Minnesota, Burgers Publishing Co. pp 193.

Curtis, J.T. and McIntosh, R.P., 1950. The interactions of certain analytic and synthetic phytosociological characters. Ecology 31: 434-455.

Luken, O.J., 1990. Directing ecological succession. Champman and Hall, University press Cambridge. 127-251.

Misra, R., 1968. Ecology Work Book, Oxford and IBH Publishing Co. New Delhi.(pH) Philip, E.A., 1951 Methods of vegetation study. Henry Holf and Co. ing. Ramakrishnan, P.S., 1991. Biological invasion in the Tropics (Ed.) An overview. In: Ecology of

Biological Invasion in the Tropics (Ed.) Ramakrisnan, P.S. International Scientific Publication. New Delhi

Singh, A., 2004. Herbaceous biomass yield on an age series of naturally revegetated mine spoils in a dry tropical environment, Journal of Indian Institute of science, 84, 53-56 pp.

Soni, P. and Vasistha, H.B., 1985. Reclamation of rock phosphate mine at Maldeota. In: (Eds) Sharma, M.R. & Gupta, B.K. Proc. Recent advances in plant science. Bishen Singh and Mahendra Pal Singh, Dehradun.

Van Andel J and Aronson J (Eds) 2005:Restoration Ecology: The New Frontier. Oxford: Blackwell Publishing.



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Table 1. Floristic structure of herbs in site 1(23 years old restored site).

Herbs Frequency Density ha-1 Abundance IVI

Year I st II nd I st II nd I st II nd I st II nd

Achyranthes aspera. L. 20.00 20.00 2000 2000 1.00 1.00 9.52 8.53

Adhatoda zeylanica Nees. 20.00 20.00 2000 2000 1.00 1.00 9.69 9.67

Aerva scandens Wall. - 20.00 - 2000 - 1.00 - 8.05

Ageratum conyzoides Linn. 20.00 60.00 2000 12000 1.00 2.00 21.80 42.57

Artemisia vulgaris Linn. - 20.00 - 2000 - 1.00 - 8.98

Bidens pilosa L. 20.00 80.00 22000 20000 11.00 2.50 64.79 39.83

Commelina benghalensis L. - 20.00 - 2000 - 1.00 - 5.61

Cymbopogon martini Stapf. 20.00 - 4000 - 2.00 - 13.15 -

Cynadon dactylon (L.) Pers. - 20.00 - 2000 - 1.00 - 5.02

Eupatorium glandulosum Michx.

- 20.00 - 2000 - 1.00 - 6.00

Lantana camara L. - 100.00 - 14000 - 1.40 - 50.77

Malvestrum coromandelianum .Gareke.

- 20.00 - 2000 - 1.00 - 6.44

Mallotus philippensis (Lam.) Muell.-Arg.

20.00 - 2000 - 1.00 - 12.08 -

Murraya koenigii Spreng. 40.00 60.00 60000 10000 15.00 1.67 97.18 32.31

Murraya paniculata (L) Jacq. 20.00 20.00 2000 2000 1.00 1.00 11.70 11.04

Oplismenus compositus (L.) P. Beauv

20.00 40.00 2000 6000 1.00 1.50 18.20 16.17

Oxalis corniculata (L.) L - 20.00 - 4000 - 2.00 9.17

Sida acuta Burm. 60.00 20.00 8000 2000 1.33 1.00 30.65 5.48

Sida humilis Willd. 20.00 40.00 4000 4000 2.00 1.00 11.23 11.14

Urena lobata L. - 60.00 - 6000 - 1.00 - 17.24

Woodfordia fruticosa Kurz. - 20.00 - 2000 - 1.00 - 5.96



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Table 2. Floristic structure of herbs in site 2 (22 years old restored site).


Year Ist II

nd I

st II

nd I

st II

nd I

st II

nd

Achyranthes aspera L. 40.00 80.00 8000 10000 2.00 1.25 26.77 22.44

Adhatoda zeylanica Nees. 60.00 100.00 18000 22000 3.00 2.20 63.78 38.59

Aerva scandens Wall. 60.00 - 6000 - 1.00 - 19.37 -

Agave sisalana Perrine 20.00 - 2000 - 1.00 - 6.40 -

Ageratum conyzoides Linn. 20.00 - 4000 - 2.00 - 8.23 -

Barleria cristata Linn. 40.00 - 10000 - 2.50 - 21.48 -

Bidens pilosa L. - 100.00 - 20000 - 2.00 - 56.10

Boehmeria platyphylla D.Don 20.00 40.00 6000 6000 3.00 1.50 10.60 11.25

Commelina benghalense L. - 20.00 - 2000 - 1.00 - 4.78

Cymbopogon martini Stapf. 20.00 - 2000 - 1.00 - 6.39 -

Deutzia staminea R. Br. ex. Wall.

20.00 - 2000 - 1.00 - 7.59 -


20.00 20.00 2000 2000 1.00 1.00 8.09 4.74

Justicia simplex D.Don 20.00 60.00 4000 6000 2.00 1.00 9.74 13.64

Lantana camara L. 60.00 100.00 8000 36000 1.33 3.60 27.23 60.99


20.00 - 2000 - 1.00 - 10.66 -



40.00 60.00 12000 8000 3.00 1.33 20.39 27.70

Oxalis corniculata (L.) L - 20.00 - 2000 - 1.00 - 5.72

Desmodium gangeticum DC. - 20.00 - 4000 - 2.00 - 11.51


Toona ciliata M.Reem. - 20.00 - 2000 - 1.00 - 5.00

Urena lobata L. - 40.00 - 6000 - 1.50 - 12.25



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Table 3. Floristic structure of herbs in site 3 (21 years old restored site) Herbs Frequency Density ha-1 Abundance IVI

years Ist II

nd I

st II

nd I

st II

nd I

st II

nd


Adiantum sp. 20.00 - 20000 - 10.00 - 14.03 --


Aerva scandens Wall. - 20.00 - 4000 - 2.00 - 9.03

Bidens pilosa L. 60.00 - 18000 - 3.00 - 22.41 -

Boerhavia diffusa L. - 60.00 - 10000 - 1.67 56.28

Corchorus olitorius Linn. 20.00 - 6000 - 3.00 - 6.76 -

Commelina benghalensis L.

- 100.00 - 12000 - 1.20 - 32.81

Cyperus rotandrus L. - 40.00 - 12000 - 3.00 - 20.62


- 20.00 - 4000 - 2.00 - 31.09

Lantana camara L. 20.00 100.00 6000 12000 3.00 1.20 15.89 36.82


20.00 - 4000 - 2.00 - 13.63 -

Melia composita Willd. leaf.

20.00 -- 2000 - 1.00 - 7.53 -

Murraya koenigii Spreng. 100.00 - 40000 - 4.00 - 74.24 -

Oplismenus burmannii (Retz.) P. Beauv.

100.00 - 46000 - 4.60 - 41.40 -

Randia dumetorum Lamk. 40.00 - 10000 - 2.50 - 34.33 -

Sida cordifolia Linn. 60.00 - 16000 - 2.67 - 19.22 -

Urtica aphyla L. 20.00 - 10000 - 5.00 - 23.12 -



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Table 4. Floristic structure of herbs in site 4 (20 years old restored site).


Year Ist

IInd

Ist

IInd

Ist

IInd

Ist

IInd


Adiantum sp. 20.00 - 2000 - 1.00 - 4.74 -

Adhatoda zeylanica Nees. - 20.00 - 2000 - 1.00 - 9.05

Aerva scandens Wall. 20.00 60.00 4000 12000 2.00 2.00 6.46 26.82


Bidens pilosa L. 80.00 - 74000 - 9.25 - 81.61 -


Boehmeria platyphylla D.Don

- 20.00 - 2000 - 1.00 - 6.98

Cissampelos pareira L. var. hirsute (DC.) Forman

- 40.00 - 6000 - 1.50 - 11.62

Cymbopogon martini Stapf.

20.00 - 46000 - 23.00 - 40.91 -

Dicliptera peristrophe Nees

20.00 - 6000 - 3.00 - 7.84 -


- 40.00 - 28000 - 7.00 - 36.28

Euphorbia hirta L. - 40.00 - 6000 - 1.50 - 11.54

Frageria vesca Linn. 20.00 - 2000 - 1.00 - 4.42 -

Justicia simplex D.Don 20.00 - 4000 - 2.00 - 5.93 -

Lantana camara L. 20.00 40.00 2000 12000 1.00 3.00 7.06 16.83


20.00 - 2000 - 1.00 - 4.63 -


Murraya paniculata (L) Jacq.

- 20.00 - 2000 - 1.00 - 4.67


80.00 40.00 44000 24000 5.50 6.00 44.34 33.82

Oxalis minuta Thunb. 20.00 - 6000 - 3.00 - 8.90 -


Syzygium cumini (L.) Skeels

- 20.00 - 2000 - 1.00 - 4.95

Toona ciliata M.Reem. - 20.00 - 2000 - 1.00 - 5.49

Urena lobata L. - 20.00 8000 - 4.00 - 11.01



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Table 5. Floristic structure of herbs in site 5 (Natural forest) Herbs Frequency Density ha-1 Abundance IVI

Year Ist II

nd I

st II

nd I

st II

nd I

st II

nd


Adhatoda zeylanica Nees. - 100.00 16000 1.60 49.89

Aerva scandens Wall. 40.00 60.00 16000 12000 4.00 2.00 11.22 15.99

Ageratum conyzoides Linn. 20.00 - 2000 - 1.00 - 3.78 -

Apluda mutica L.. 40.00 - 10000 - 2.50 - 9.84 -


Bidens pilosa L. 100.00 100.00 198000 24000 19.80 2.40 118.49 35.95

Boehmeria platyphylla D.Don

40.00 40.00 4000 4000 1.00 1.00 7.81 8.38

Commelina benghalensis L.

- 40.00 - 4000 - 1.00 - 8.11

Cyperus rotandrus L. - 40.00 - 4000 - 1.00 - 7.98

Dicliptera roxburghiana Nees

80.00 - 18000 - 2.25 31.45


40.00 40.00 12000 6000 3.00 1.50 22.37 17.48

Ipomoea fistulosa Mart. ex Choisy

- 20.00 - 4000 - 2.00 - 5.34


20.00 - 2000 - 1.00 - 10.15 -

Murraya koenigii Spreng. - 60.00 - 12000 - 2.00 - 38.84


80.00 60.00 28000 6000 3.50 1.00 21.20 11.92

Oxalis corniculata (L.) L - 40.00 - 4000 - 1.00 - 8.34

Randia dumetorum Lamk. 20.00 - 2000 - 1.00 10.83 -

Rumex hastatus D. Don. - 20.00 - 4000 - 2.00 - 5.32

Sida humillis Willd. 20.00 40.00 4000 6000 2.00 1.50 4.42 9.42

```



128

Short-Term Dynamics of the Active and Passive Soil Organic Carbon Pools in a Volcanic Soil Treated With Fresh

Organic Matter

Wilfredo A. Dumale, Jr.1, 2, *, Tsuyoshi Miyazaki 2, Taku Nishimura 2 and Katsutoshi Seki3

1 Department of Plant Science, Nueva Vizcaya State University, Bayombong 3700, Nueva Vizcaya, Philippines

2 Department of Biological and Environmental Engineering, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 Japan

3 Faculty of Business Administration, Toyo University, 5-28-20 Hakusan, Bunkyo-ku, Tokyo 112-8606, Japan

* Corresponding author,

e-mail: [email protected]; [email protected]

Abstract In a 110-day constant temperature experiment (20° C), we determined the effect of fresh organic matters (FOM): 0 (control); 1.81 g leaf litter (LL) carbon kg-1; and 2.12 g chicken manure (CM) carbon kg-1 in the stable soil organic carbon [mineral-associated organic carbon (MAOC)], labile soil organic carbon [soil microbial biomass carbon (SMBC)], and carbon dioxide (CO2) evolution of a volcanic ash soil from Tsumagoi, Gunma Prefecture, Japan (138°30’ E, 36°30’ N). Overall, CO2 evolution and SMBC increased after the treatment of soil with FOM, whereas MAOC decreased below its original level three days after FOM application. These data support the view that fresh OM promotes increases in SMBC and CO2 in the rapidly cycling active carbon pool and further suggest that the MAOC fraction, though stable as conventionally believed, can be a source of CO2. Our findings challenge the convention that only labile SOC is the source of short-term CO2 evolution from soils.

Keywords: mineral-associated organic carbon, soil microbial biomass carbon, soil organic carbon, CO2 evolution

Introduction

Soil organic carbon (SOC) is the largest pool within the terrestrial carbon cycle (Gerzabek et al., 2001), consisting of a heterogeneous mixture of organic matter originating from plant, microbial and animal residues (Baldock and Skjemstad, 2000). A variety of terrestrial ecosystem models have been developed recently to study the impacts of management and/or climate change on SOC turnover under different climates, topographies and management (Sherrod et al., 2005). For example, the CENTURY model is a terrestrial SOC model which partitions SOC into three



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conceptual pools: active, slow, and passive, which differ in turnover times (Parton et al., 1988). The relationships of the measurable fractions of these conceptual pools and their measurable fractions with the particle size fractions were summarized by Dumale et al. (2009) (Table 1). The mineral-associated organic carbon (MAOC) is the measurable fraction of the passive SOC pool (Sherrod et al., 2005). The MAOC fraction can be measured by physically separating the <53 mm particle size fraction, which is the silt-and clay-sized fraction (Haile-Mariam et al., 2008). The associated SOC of the combined silt and clay is the MAOC (Cambardella and Elliot, 1992).

Table 1. Matrix table indicating relationships of conceptual SOM pools, their measurable fractions, and particle size fractions (Dumale et al., 2009)

Conceptual SOM pools** Description Active Slow Passive

1.Turn-over time hours to months; 2- to 4-years

Decadal; 20- to 50-years

centuries to millennia; 800–2000 years

2.Representative SOM Fraction***

SMBC (soil microbial biomass carbon)

POMC (particulate organic matter carbon)

MAOC (mineral-associated organic carbon)

3.Description of the fraction

active soil organic matter (SOM) consisting of live microbes and microbial products

protected fraction that is more resistant to decomposition

physically-protected or chemically resistant and has long turnover time

4.Chemical composition

chloroform-labile, microwave-irradiation-labile SOM, amino compounds, phospholipids

amino compounds; glycoproteins; aggregate protected POM; acid/base hydrolyzable; mobile humic acids

aliphatic macromolecules; charcoal; sporopolleins; lignins; high molecular, condensed SOM, humin, nonhydrolyzable SOM, fine silt, coarse-clay associated SOM

5.SOM fraction association with soil particle sizes

Fumigated and extracted SMBC

2mm–53µm; Sand-sized or larger

<53 µm, silt and clay-sized**** referred to as MAOC in this paper

** The term “pool” is used to refer to the theoretically separated, kinetically delineated components of SOM *** The term “fraction” is used to describe measurable organic matter components associated with the pool **** Silt and clay-sized particles were <53 µm diameter based on the USDA Soil Texture Classification System

The particle size fractions play different roles in stabilization of soil organic matter (SOM). The major part of the SOM is usually associated with the clay- and silt-sized fractions (Ohm et al., 2007). Fine-textured soils have higher organic C and N contents than coarse-textured soils when supplied with similar input of organic material (Hassink, 1997). It was assumed that the difference was due to the ability of fine-textured soils to provide greater protection to soil organic matter (Hassink, 1997; van Veen and Kuikman, 1990), and physical protection of SOM is due to its ability to associate with clay and silt particles (Li et al., 2007; Zhao et al., 2006). The SOM associated with silt- and clay-sized fractions is often older than in the sand fractions, which



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is attributed to the stabilization mechanisms through surface interactions (Baldock and Skjemstad, 2000; Lützow et al., 2006; Rumpel et al., 2002). The silt- and clay-associated C was older in the light fraction (LF) and particulate organic matter (POM) (Haile-Mariam et al., 2008). Further, the clay-associated residues have the highest mean residence times (MRT). Most of the input of carbon to soil from different sources is subject to microbial attack, explaining the extra CO2 mineralization soon after addition to soil. A part, however, are retained and stabilized into the soil over long period of time. Previously, it was suggested that this extra CO2 originates from the labile SOC fraction. However, from more recent studies, it seems unlikely that only the labile pool is affected, since it cannot fully account for the extra CO2 released (Hamer and Marschner, 2005). The extra CO2 evolution can originate from the various pools of SOM (Kuzyakov, 2006). Some studies have found that organic matter (OM) application does not increase SOC (Foereid et al., 2004; Fontaine et al., 2004; Fontaine et al., 2003; Bell et al., 2003; Campbell et al., 1991). Others have reported gains in SOC after years of OM addition to soil (Gerzabek et al., 2001; Gerzabek et al., 1997; Dalenberg and Jager, 1989). We separated the soil microbial biomass carbon (SMBC) as a measure of the labile soil organic carbon using a modification of the fumigation extraction technique (Vance et al., 1987) and the mineral-associated organic carbon (MAOC) fraction as a measure of the stable soil organic carbon using combined chemical dispersion and physical fractionation (Sherrod et al., 2005; Haile-Mariam et al., 2008; Cambardella and Elliot, 1992). Our objectives are to (1) determine the short–term influence of fresh organic matter (FOM) application on the dynamics of MAOC, and (2) study the dynamics of SMBC and CO2 evolution in soils applied with fresh organic matters. We hypothesized that although the MAOC is stable soil organic carbon due to physical protection in the silt and clay fractions, it does contribute to C turnover in the short-term, although conventionally believed to turn over in centuries to millennial time scales.

Materials and Methods

Soil sampling and FOM preparation Soil samples collected from the 0–5- and 6–20-cm layers of an upland field located in Tsumagoi, Gunma Prefecture, Japan (138°30’ E, 36°30’ N) were air-dried in the shade, sieved through a 2-mm mesh screen, and stored at 4°C until experimentation. Some of the physico-chemical properties of the soil are presented in Table 2. Most of the plant residue was removed by flotation, followed by drying of the soil. Leaf litter (362.7 g kg-1 C; 18.0 g kg-1 N; 20.1 C/N ratio) and chicken manure (424.9 g kg-1 C; 52.5 g kg-1 N; 8.1 C/N ratio) were used as FOM. The FOM was air-dried for one week in the shade and then finely ground and passed through a 0.5-mm mesh screen. FOM was stored at 4°C until use.



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Table 2. Some physico-chemical properties of the Tsumagoi soil, Gunma Prefecture, Japan.

Depth (cm)

Soil texture

Particle density (gcm-3)

Bulk density (gcm-3)

Total C (gkg-

1)

Total N (gkg-1)

C/N ratio

Land use/ Common vegetation

0–5 2.48 0.44 70.57 4.76 14.83

5–20

sandy loam 2.48 0.5 88.9 5.7 15.6

Agricultural experimental field; cabbage

Incubation experiment

Transparent 500-mL glass bottles with plastic lid were used for incubation. (Figure 1). Three holes, one 12.5-mm diameter and two 10-mm diameter, were bored on the lid in triangular fashion. A “cock-rubber stopper” assembly, inserted into the 10-mm holes, served dually as air outlet of “old air” inside the bottles and air inlet of “new moist air” after every sampling day. This “cock-rubber stopper” assembly was made by inserting a three-way plastic cock (Top Corp., Japan) into a 14 x 15.5 x 10.5 mm rubber stopper.

Figure 1. The experimental unit. Acrylic tubing fitted with a septum mounted on cable grand served as the gas sampling port (A); Viewed from the bottom of the lid are the gas sampling port,



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inlet and outlet “cock-rubber stopper” assembly (B); the triangular boring in the bottle lid (C); the “cock-rubber stopper” assembly (D); and the assembled experimental unit (E). Also, a self-designed 35-mm length acrylic tubing sealed with a rubber septum was fitted in the 12.5-mm diameter hole in the bottle lid through a cable grand. This tubing served as the gas sampling port for CO2 evolution measurement. All assembled incubation bottles were tested leak-free by immersing in a pail of water. Each experimental unit consisted of 20-g soil samples adjusted to 50% of the soil’s water-holding capacity. Incubation was conducted for 110 days at 20°C constant temperature. Prior to sealing each incubation bottle, FOM was evenly incorporated to the soil according to treatment rates. Experimental units allowed for three replicates per treatment on each sampling day. Parameters were measured by destructive sampling at 3, 13, 21, 44, 70, 85, and 110 days after FOM application. For MAOC, measurement was also conducted at day zero.

Separation and measurement of the MAOC fraction Combined chemical dispersion and particle size separation methods based on the work of several authors (Sherrod et al., 2005; Haile-Mariam, et al., 2008; Cambardella and Elliot, 1992; Bell et al., 2003) were used to separate the combined silt- and clay-sized fractions which contain the MAOC. On each sampling day, 5-g subsample was placed in 100-mL plastic bottle and dispersed with 50 mL of sodium hexametaphosphate (5 g/L). The suspension was shaken in a reciprocating shaker (Yamato shaker model SA-31, Yamato Scientific Co., Ltd., Japan) overnight at 240 rpm. The soil suspensions were sieved in a 53-µm screen (Tokyo Screen Co. Ltd., Japan). During sieving, the particles retained in the screen were repeatedly rinsed with distilled water to ensure thorough separation of the <53 µm particle size fraction. The resulting suspension was dried overnight at 70° C. The dried samples were finely ground manually using mortar and pestle and passed through an 80-µm sieve (Tokyo Screen Co. Ltd., Japan). MAOC was measured by dry combustion using a Sumigraph NC-90A NC analyzer (Sumika Inc., Japan). Gas sampling and CO2 evolution measurement Gas samples for CO2 evolution measurement were drawn from incubation bottles using a 10-mL plastic syringe (Nipro, Japan) fitted with 0.70 x 38.00 mm needle (Nipro, Japan). Transparent 7-mL capacity glass vials were used as sample containers. Prior to sampling, the vials were vacuumed by subjecting to 2 millibars suction for about 10 min and sealed with a rubber septum. Prior to drawing gas samples, the air inside each incubation bottle was homogenized by alternate pumping and sucking using the sampling syringe 4–5 times. Seven mL of gas sample were drawn and injected into the sample vials. From there, 1 mL of gas sample was drawn and injected into a 16A Gas Chromatograph (Shimadzu Inc.). Each sampling day, after drawing gas samples, the air inside the bottles were flashed out and substituted with moist air through the



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inlet and outlet cocks mounted in the bottle lid. The inlet cock was connected to an air source passing through a tank of distilled water to moisten the air and maintain moisture inside the incubation bottles. The outlet cock was simultaneously opened while moist air flowed through the inlet cock at 2.5 kgf cm-2 for 3 min to ensure flushing out of “old air” from the bottles.

SMBC fumigation, extraction, and measurement The soil microbial biomass carbon (SMBC) fumigation and extraction technique used was slightly modified from the fumigation-extraction method described by Vance et al. (1987). Each sampling day, 5-g subsamples were placed in small Petri dishes and placed inside a glass desiccator containing 40 mL of ethanol-free chloroform (CHCl3) in a small beaker. To enhance vapor production, the beaker of CHCl3 was immersed in a cup of hot water. The desiccator was sealed and placed in the dark at 25°C for 24 h. After 24 h the beaker of CHCl3 was removed, and the residual CHCl3 vapor in the soil was removed by repeated evacuation using a vacuum pump connected to the desiccator. For extraction, the samples were transferred to 100-mL plastic bottles, diluted with 50 mL of potassium sulfate (0.5 M K2SO4), and shaken in an oscillating shaker at 240 rpm. After 30 min, the suspension was filtered using Whatman No. 42 filter paper followed by membrane filtration using 0.2-µm Millex syringe-driven filter units. A separate set of unfumigated samples was also prepared for use as control. The filtered samples were analyzed using a Total Organic Carbon Analyzer (Shimadzu TOC-VCSN, Shimadzu, Inc.). SMBC was calculated using the formula, SMBC = 2.64Ec, where Ec is the difference between the organic carbon extracted from the fumigated and non-fumigated samples (Vance et al., 1987).

Statistical treatment of data Data were subjected to statistical analysis following the split-split plot design to compare and determine any significant differences between and among treatment means. The analysis of variance (ANOVA) was done using the SAS software (SAS Institute). Comparisons of means were done using the least significant difference (LSD) or the Duncan’s multiple range test (DMRT) where appropriate.


CO2 evolution rate and cumulative CO2 evolution The addition of leaf litter and chicken manure increased the CO2 flux in soil (Figure 2). Soils that received chicken manure had exceptionally higher CO2 production than did soils that received leaf litter. The peak of CO2 production occurred during the first three days of incubation, except for soils from the 0–5-cm layer that received chicken manure, which peaked at day 13 of incubation. The 6–20-cm layer that received chicken manure exhibited the highest CO2 production, reaching as high as 122 mg kg-1 day-1 during the first three days after OM application. In the 0–5-cm layer, CO2 production peaked at 74.6 mg kg-1 day-1 at 3 to 13 days after FOM application. There was little difference in CO2 production between soils from the 0–5-



134

cm (0.49) and 6–20-cm (0.52 mg CO2 kg-1 day-1) layers that received leaf litter during the 120-day incubation period. The carbon equivalent of the total CO2 produced in the 0–5- and 6–20-cm layers of soil and between the control and leaf litter treatments ranged from 109 to 178 mg kg-1 for the 120-day incubation period. For soils that received chicken manure, the carbon equivalent reached as high as 527 and 828 mg carbon kg-1 for the 0–5- and 6–20-cm layers, respectively.

0

20

40

60

80

100

120

140

160

180

0 10 20 30 40 50 60 70 80 90 100 110

Time (days)

CO

2 ev

olut

ion

rat

e (m

g kg

-1 da

y-1) 0-5 cm No OM (control) 5-20 cm No OM (control)

0-5 cm Chicken manure 5-20 cm Chicken manure0-5 cm Leaf litter 5-20 cm Leaf litter

Figure 2. Carbon dioxide evolution rate (mg kg-1 day-1) of Tsumagoi soil, Gunma Prefecture, Japan over 110 days of incubation following application of leaf litter and chicken manure.

Soils that received chicken manure produced 199.7% and 531.9% more CO2 than did controls from the 0–5- and 6–20-cm soil layers, respectively. Approximately, 32–64% of the total evolved CO2 was released during the first 21 days after FOM addition. In soils that received leaf litter, both the 0–5- and 6–20-cm layers had total evolved CO2 levels similar to those of the controls. Thus, in contrast to chicken manure, leaf litter has negligible effects on CO2 production when applied to soil. In the 0–5-cm layer, CO2 evolution rate in the control was highest 0–3 days after FOM addition (Table 3). Beyond this period, CO2 evolution rates were statistically lower until the end of incubation. CO2 evolution rates were comparable during the 4–110-day periods. This is exactly the trend in the leaf litter-applied 0–5-cm layer. In the chicken manure-applied soils, CO2 evolution rates significantly varied, and highest during the early stage of incubation (0–13 days after FOM addition), when rate was in the range 68.24 to 74.55 mg kg-1 day-1. Starting from two weeks after FOM application, CO2 evolution rate significantly dropped by more than half of the 4–13-day period level, decreasing with time until the end of incubation. From 14–110 days after FOM addition, evolution rates did not significantly differ. In the 0–5-cm layer without manure (control), CO2 evolution rate was in the range 5.43 to 19.26 mg kg-1day-1 during the 0–44-day period, but from 4–110 days after FOM application, rates were



135

also statistically the same. In the leaf litter-applied soils, CO2 evolution rate during the 0–3-day period (23.03 mg kg-1day-1) was significantly highest than anytime during the incubation period. Rates during the period 4–110 days after FOM application, ranging from 3.87 to 8.75 mg kg-

1day-1, were all statistically the same. In the chicken manure-treated soils, rates were significant from each other in the 0–3-, 4–13, and 14–21-day periods. Starting from 22 days after incubation, CO2 evolution rates did not vary significantly. These results were in agreement with earlier reports. Addition of manure increased CO2 flux of the soils and that the largest difference between manured and control soils occurred at week 1, when the manured soils had from 42 to more than 400 % higher CO2 fluxes (Calderon et al., 2004). Similarly, after a short lag phase (3 days) after cellulose addition, the cellulose decomposition followed an exponential dynamic until the rate of CO2 production had markedly decreased (at day 17) likely due to cellulose exhaustion (Fontaine et al., 2004). Conversely, cumulative values of evolved CO2-C increased rapidly from day 0 to 14, thereafter the increase was less for the rest of the incubation (Rudrappa et al., 2006). Maximum CO2 production rate in the urine+dairy farm effluent-applied soils incubated at 28° C was attained starting immediately after application until day 5 (Clough and Kelliher, 2005).

Table 3. Effects of time and fresh organic matter application on the CO2 evolution rate in the 0–5- and 5–20-cm layers of Tsumagoi soil, Gunma Prefecture, Japan.

CO2 evolution rate (mg kg-1day-1)

0–5-cm 5–20-cm

Days after FOM addition

No OM (control)

Leaf litter Chicken manure

No OM (control)

Leaf litter Chicken manure

0–3 22.77 a 25.89 a 68.24 a 19.26 a 23.03 a 121.67 a

4–13 5.34 b 7.8 b 74.55 a 6.29 ab 8.2 b 107.25 b

14–21 5.24 b 7.55 b 36.3 b 7.52 ab 8.75 b 49.31 c

22–44 2.33 b 4.45 b 8.27 c 5.43 ab 5.33 b 17.94 d

45–70 2.28 b 3.27 b 7.45 c 3.27 b 4.56 b 10.79 d

71–85 3.35 b 3.76 b 7.89 c 2.09 b 5.6 b 12.28 d

86–110 1.54 b 2.38 b 4.49 c 3.56 b 3.87 b 8.4 d

In a column, means followed by different letters are significant at 5% level using DMRT

Rates of organic matter decomposition depend upon several factors, ranging from the type of organic amendments to the soil type and properties, the climatic conditions and land management practices (Pedra et al., 2007). In addition, the quantity and nature of the soil clay affects the amount of C stabilized in soil, since fine textures soils often contain higher amounts of OM than sandy soils (Mtambanengwe et al., 2004)



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Sources of CO2 efflux from soils According to Kuzyakov (2006), there are four main contributors to CO2 efflux classified as microbial: (1) microbial decomposition of soil organic matter in root free soil without undecomposed plant remains, frequently referred to as “basal respiration”; (2) microbial decomposition of soil organic matter in root affected or plant residue affected soil, called “rhizosphere priming effect” or “priming effect”; (3) microbial decomposition of dead plant remains; and (4) microbial decomposition of rhizodeposits from living roots, called “rhizomicrobial respiration”. Root respiration is and the dissolution of calcium carbonate (CaCO3) also contributes to CO2 efflux from soils. However, this CaCO3 contribution during pedogenesis is only marginal since soil-CO2 flux measurements are usually done in sub-annual, annual, and decadal time scales. Soil microbial biomass carbon The soil microbial biomass as an active soil organic matter (SOM) fraction and agent of CO2 production in soil is divided into two main groups: heterotrophic and autotrophic organisms. The most important heterotrophs in the soil can be subdivided into two broad groups: (1) soil microorganisms (bacteria, fungi, actinomycetes and protozoans) and (2) soil macrofauna, the contribution of which to total CO2 efflux from soils is usually a few percent (Ke et al., 2005; Konate et al., 2003; Andren and Schnurner, 1985). Most of the CO2 evolved by heterotrophic soil organisms is respired by microorganisms such as bacteria, non-mycorrhizal and mycorrhizal fungi, and actinomycetes. This component of soil CO2 flux is collectively called microbial respiration (Kuzyakov, 2006). The SMBC increased dramatically in the early stages of incubation (Figure 3). The application of chicken manure caused a greater increase in the SMBC than did the application of leaf litter. Peak microbial growth occurred 13 days after the application of FOM. SMBC concentration in the 5–20-cm layer that received chicken manure peaked at 1509.2 mg kg-1. The 0–5-cm layer that received chicken manure peaked at a SMBC concentration of 1059.5 mg kg-1. In soils that were treated with leaf litter, the peak SMBC concentration was 631.2 and 886.9 mg kg-1 for the 0–5- and 5–20-cm layers, respectively. Control soils had SMBC peaks of 123.62 (0–5-) and 875.16 mg kg-1 (5–20-) also at 13 days after FOM application.



137

0

200

400

600

800

1000

1200

1400

1600

1800

0 10 20 30 40 50 60 70 80 90 100 110

Time (days)

So

il m

icro

bial

bio

mas

s ca

rbo

n

(mg

kg-1

)

0-5 cm No OM (control) 5-20 cm No OM (control)

0-5 cm Chicken manure 5-20 cm Chicken manure

0-5 cm Leaf litter 5-20 cm Leaf litter

Figure 3. Changes in the soil microbial biomass (SMBC) (mg kg-1) of Tsumagoi soil, Gunma Prefecture, Japan over 110 days of incubation following application of leaf litter and chicken manure.

The peak in SMBC at 13 days after the addition of FOM was followed by a drop at day 21. From day 21, different patterns in SMBC were observed. SMBC in the 0–5-cm layer of control and leaf litter-applied soils generally increased again at 44 days after incubation and peaked 85 days after FOM application while in the chicken manure-applied soil, SMBC continued to drop until day 70 and peaked at day 85. In 5–20-cm layer control, SMBC peaked at day 70, while for the leaf litter- and chicken manure-applied soils, SMBC continued to increase until the end of incubation. In all FOM treatments, the increase in SMBC during the 4–13-day period was highest (Table 4). Following this peak was a decline at the start of the 4th week (22 days after FOM addition). Following this decline was a significant increase again. For the control and leaf litter-applied soils, this was observed starting from 45 days after incubation onwards. In the case of chicken manure-applied soils, the marked increase of SMBC for the second time was observed starting from 71 days until the end of incubation.



138

Table 4. Effect of time and fresh organic matter application on the soil microbial biomass carbon (SMBC) of Tsumagoi soil, Gunma Prefecture, Japan.

SMBC (mg kg-1) Days after FOM

addition No OM (control) Leaf litter

(1.81 g kg-1) Chicken manure

(2.12 g kg-1)

0–3 451.84 b 500.98 b 416.46 c

4–13 799.39 a 759.09 a 1284.36 a

14–21 249.96 d 223.12 d 409.2 c

22–44 357.9 cd 397.58 c 474.45 c

45–70 448.23 bc 436.22 bc 331.41 c

71–85 583.57 b 565.11 b 761.82 b

86–110 483.47 b 544.28 b 817.52 b

In a column, means followed by different letters are significant at 5% level using DMRT According to Fontaine et al., (2004) the supply of cellulose highly stimulated the microbial activity. In their experiment, the production of unlabelled extra CO2 induced by glucose was completed after 3 days and amounted to about 15-19 % of the microbial biomass-C. Further, the addition of cellulose as small as <5 % of the native soil C induced a two-fold increase of total biomass, which was not sustainable, since it decreased starting from day 21 until the end of incubation. The soils amended with chicken manure showed exceptionally high CO2 production, indicating the presence of readily available C for microbial consumption and cell division. This is revealed by the lower C/N ratio of the chicken manure compared to that of the leaf litter. The occurrence of two distinct peaks in SMBC was seen in all treatments. The first SMBC peaks occurred 13 days after FOM application. These peaks coincided with the period of high CO2 evolutions. Several authors stated the direct relationship between CO2 production and SMBC (Kuzyakov et al., 2000). Similar results have been reported in previous studies (e.g. Calderon et al., 2004). However, the timing of occurrence of the second SMBC peaks was different. The second SMBC peaks in the 0–5-cm depth occurred at day 70 in all treatments, while in the 5–20-cm layer, SMBC in chicken manure-applied soils continued to increase from day 70 until the end of incubation; the control had second peak at day 70 and the leaf litter-applied soil had sustained SMBC increase from day 22 until day 110. The first SMBC peak 13 days after incubation was most likely due to the availability of substrates originating from the FOMs and from the labile SOM in the case of the control soils. The drop in SMBC at day 21 was probably due to the exhaustion of these readily-available substrates.



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The occurrence of a second peak suggests that soil microorganisms started to use an alternative source of energy, since the readily-available components of the applied manure could have been used up, leading to a drop in SMBC at 21 days after FOM application. In this scenario, although the actual microbial structure that caused the first SMBC peak was not identified, we propose that the dominant microbial structure that caused the first SMBC peak was different from that in the second peak. Several authors observed similar trend in terms of the surge in SMBC early in the incubation period. Annual application of manure caused a rapid increase in SMBC following application and potentially mineralizable C reached maximum fluxes within a month after manure application (Lee et al., 2007). The microbial population is easily activated even by trace amounts of readily-available source of energy. Trace amounts of simple and easily degradable substances such as glucose or amino acids, and more complex soil and root extracts, could shift the soil microorganisms from dormancy to activity, causing more to be evolved as CO2 than was contained in the substrate (De Nobili et al., 2001). This response of the microbial biomass is presumably in anticipation of the coming of a bigger source of energy available for further reproduction and respiration. This could partly explain the response of SMBC almost immediately after FOM application. In conditions without any external application of readily-available substrates, favorable conditions of soil moisture or aeration would trigger this initial microbial response. Kinetics and dynamics of the mineral-associated organic carbon

Original MAOC level was lower in the 0–5-cm layer (33.93 g kg-1) than in the 5–20-cm layer (38.19 g kg-1) of the Tsumagoi soil (Table 5).

Table 5. Initial total organic carbon (TOC) and mineral-associated organic carbon (MAOC) of

the 0–5- and 5–20-cm layers of Tsumagoi soil, Gunma Prefecture, Japan.

Depth (cm)

TOC (g kg-1)

MAOC (g kg-1)

Labile SOC* (g kg-1)

0–5 70.57 33.93 36.64

5–20 88.9 38.19 50.71

* TOC less MAOC

The short-term kinetics of MAOC of Tsumagoi soil are shown in Figure 4. The behavior of the MAOC three days after the application of FOM is significant (Table 6) and an interesting point of discussion. MAOC is conventional understood as a stable entity and have long turnover times due to protection by silt and clay. Statistical comparison of treatments means challenge our conventional knowledge of the stability of MAOC. There was strong evidence that significant portion of MAOC is turned over in short time scale. The decline in MAOC three days after FOM addition suggests that a portion of MAOC is prone to turnover in a matter of days, though it is believed that MAOC has turnover times of centuries to millennial timescales (Table 1).



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30

35

40

45

0 10 20 30 40 50 60 70 80 90 100 110

Time (days)

Min

eral

-ass

oci

ated

org

anic

car

bon

(g k

g-1

)

0-5 cm No OM (control) 5-20 cm No OM (control)

0-5 cm Chicken manure 5-20 cm Chicken manure

0-5 cm Leaf litter 5-20 cm Leaf litter

Figure 4. Mineral-associated organic carbon (MAOC) (g kg-1) of Tsumagoi soil, Gunma Prefecture, Japan over 110 days following application of leaf litter and chicken manure.

Results showed that the mineral-associated organic carbon changes from time to time within a short time scale, indicating that there are sites within the mineral phase that are accessible to the microorganisms. It may be safe to say that the mineral phase is also continually interacting with the soil organic matter, as indicated by the significant differences in MAOC at particular time periods. Between measurement dates during the incubation period, “add and subtract” changes in the MAOC particularly in the early stage of incubation were observed. These changes could have been due to the labile SOM that moves and associates with the particle size fractions. SOM is a continuum of materials from very young to very old with ongoing transfers between pools (Haile-Mariam et al., 2008). This means that SOM moves between particle size fractions. Owing to artificial, biological, and other pedoturbations, the transfer of SOC between the particle size fractions is a continuous process in the soil continuum. However, it is assumed that the transfer of SOC from the silt- and clay sized fractions should be less than the transfer from the sand fractions to the finer-sized fractions, due to the physical protection of SOM by the silt and clay fractions (Hassink, 1997; can Veen and Kuikman, 1990). The organo-silt and organo-clay fractions in FOM are slow to mineralize due to physical protection (Mando et al., 2005). This could result to the heterogeneity of SOC in the fine soil fractions because SOC from the sand-size fraction, from where SOM moves to the silt- and clay-sized fractions, is dominated by particulate plant material that has a lower extent of decomposition (Guggenberger et al., 1995) and has younger radiocarbon ages (Lützow et al., 2006).



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Most notable was the significant decline of the MAOC three days after FOM addition compared with the day zero level (Table 6). Two possible fates of the lost MAOC can be interpreted – (1) some microbial structures utilized this MAOC for microbial cell division, and (2) microbial degradation to CO2. This cannot be verified using the experimental design used in this experiment because to prove this may require isotopic fractionation of evolved CO2 and comparing the isotopic signature of the MAOC. This finding, however, proved that the stable MAOC may be a source of microbial energy in the short-term, although this stable fraction is conventionally believed to have long mean residence and turnover times.

Table 6. Effect of time on the mineral-associated organic carbon (MAOC) of Tsumagoi soil, Gunma Prefecture, Japan.

Days after FOM addition

MAOC (g kg-1)

0 36.06 cd

3 34.05 e

13 39.03 a

21 37.58 b

44 36.99 bc

70 36.91 bc

85 35.64 d

110 35.64 d

Means followed by different letter(s) are significant at 5% level by DMRT

Conclusions

The occurrence of second SMBC peaks in this experiment involving one-time only addition of fresh organic matters is very meaningful, and suggests a shift in the microbial community structure as the readily-available substrates from FOM became exhausted a few days after application. This suggests that the new soil microbial biomass growth found energy from a new source, which could be the MAOC, a stable SOM fraction. Regarding this process, it is suggested that most energetic compounds of FOM are used by r-strategist microorganisms that only decompose FOM. K-strategists arise only in the later stage of the FOM decomposition process when energy-rich compounds have been exhausted and only polymerized compounds remain (Fontaine et al., 2003). Our finding of a significant MAOC decline three days after FOM application puts into question the convention that only the labile SOC contributes to CO2 evolution in soils applied with FOM. Further, this suggests that physical protection of SOC in the silt and clay fractions is not a guarantee of its resistance to turnover in the short-term time scale, although previously believed



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as such. This could have big impact on the overall terrestrial carbon dynamics if the most stable SOC with long turnover times are lost in exchange of the less stable SOC that moves into the fine soil fractions during carbon input to soil.

References

Andren, O., and J. Schnurer, 1985. Barley Straw Decomposition with Varied Levels of Microbial Grazing by Folsomia fimetaria(L.) (Collembola isotomidae). Oecologia, 68: 57–62.

Baldock J. A., and J. O. Skjemstad, 2000. Role of the Soil Matrix and Minerals in Protecting Natural Organic Materials Against Biological Attack. Organic Geochemistry, 31:697–710.

Bell, J. M., J. L. Smith, V. L. Bailey, and H. Bolton, 2003. Priming Effect and C Storage in Semi-arid No-till Spring Crop Rotations. Biology and Fertility of Soils, 37:237–244.

Calderon F. J., G. W. McCarty, J. A. S. van Kessel, J. B. Reeves, 2004. Carbon and Nitrogen Dynamics during Incubation of Manured Soil. Soil Science Society of America Journal, 68:1592–1599.

Cambardella, C. A., and E. T. Elliot, 1992. Particulate Soil Organic Matter Changes Across a Grassland Cultivation Sequence. Soil Science Society of America Journal, 58:1167–1173.

Campbell, C. A., G. P. Lafond, R. P. Zentner, and V. O. Biederbeck, 1991. Influence of Fertilizer and Straw Baling on Soil Organic Matter in a Thick Black Chernozem in Western Canada. Soil Biology and Biochemistry, 23:443–446.

Clough T. J., and F. M. Kelliher, 2005. Dairy Farm Effluent Effects on Urine Patch Nitrous Oxide and Carbon Dioxide Emissions. Journal of Environmental Quality, 34:979–986.

Dalenberg, J. W., and G. Jager, 1989. Priming Effect of some Organic Additions to 14C-labelled Soil. Soil Biology and Biochemistry, 21:443–448.

De Nobili, M., M. Contin, C. Mondini, and P. C. Brookes, 2001. Soil Microbial Biomass is Triggered into Activity by Trace Amounts of Substrate. Soil Biology and Biochemistry, 33:1163–1170.

Dumale, W. A. Jr., T. Miyazaki, T. Nishimura and K. Seki, 2009. CO2 Evolution and Short-term Carbon Turnover in Stable Soil Organic Carbon from Soils Applied with Fresh Organic Matter. Geophysical Research Letters. 36:L01301.

Foereid, B., A. de Neergaard, and H. H. Jensen, 2004. Turnover of Organic Matter in a Miscanthus field: Effect of Time in Miscanthus Cultivation and Inorganic Supply. Soil Biology and Biochemistry, 36:1075–1085.

Fontaine S., G. Badoux, L. Abbadie, and A. Mariotti, 2004. Carbon Input to Soil may Decrease Soil Carbon Content. Ecological Letters, 7:314–320.

Fontaine S., A. Mariotti, and L. Abbadie, 2003. The Priming Effect of Organic Matter: A Question of Microbial Competition? Soil Biology and Biochemistry, 35:837–843.

Gerzabek, M. H., G. Haberhauer, and H. Kirchman, 2001. Soil Organic Matter Pools and Carbon-13 Natural Abundances in Particle Size Fractions of a Long-term Agricultural Field Experiment Receiving Organic Amendments. Soil Science Society of America Journal, 65:352–358.

Gerzabek, M. H., F. Pichlmayer, H. Kirchmann, and G. Haberhauer, 1997. The Response of Soil Organic Matter to Manure Amendments in a Long-term Experiment at Ultuna, Sweden. European Journal of Soil Science, 48:273–282.



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Guggenberger, G., W. Zech, L. Haumaier, B. T. Christensen, 1995. Land-use Effects on the Composition of Organic Matter in Particle-size Separates of Soils: II. CPMAS and Solution 13C NMR Analysis. European Journal of Soil Science, 46:147–158.

Haile–Mariam, S., H. P. Collins, S. Wright, and E. A. Paul, 2008. Fractionation and Long–term Laboratory Incubation to Measure Soil Organic Matter Dynamics. Soil Science Society of America Journal, 72:370–378.

Hamer, U., and B. Marschner, 2005. Priming Effects in Soils after Combined and Repeated Substrate Additions. Geoderma, 128:38–51.

Hassink, J., 1997. The Capacity of Soils to Preserve Organic C and N by their Association with Clay and Silt Particles. Plant and Soil, 191:77–87.

Ke, X., K. Winter, and J. Filser, 2005. Effects of Soil Mesofauna and Farming Management on Decomposition of Clover Litter: A Microcosm Experiment. Soil Biology and Biochemistry, 37:731–738.

Konate, S., X. Le Roux, B. Verdier, and M. Lepage, 2003. Effect of underground Fungus-growing Termites on Carbon Dioxide Emission at the Point- and Landscape-scales in an African Savanna. Functional Ecology, 17: 305–314.

Kuzyakov, Y., 2006. Sources of CO2 Efflux from Soil and Review of Partitioning Methods. Soil Biology and Biochemistry, 38:425–448.

Kuzyakov, Y., J. K. Friedel, and K. Stahr, 2000. Review of Mechanisms and Quantification of Priming Effects. Soil Biology and Biochemistry, 32:1485–1498.

Lee, D. K., J. J. Doolittle, and V. N. Owens, 2007. Soil Carbon Dioxide Fluxes in Established Switchgrass Land Managed for Biomass Production. Soil Biology and Biochemistry, 39:178–186.

Li, L. X. Zhang, P. Zhang, J. Zheng and G. Pan, 2007. Variation of Organic Carbon and Nitrogen in Aggregate Size Fractions of a Paddy Soil under Fertilization Practices from Tai Lake Region, China. Journal of Food Science and Agriculture, 87:1052–58.

Lützow, M. V., I. Kıgel–Knabner, K. Ekschmitt, E. Matzner, G. Guggenberger, B. Marschner and H. Flessa, 2006. Stabilization of Organic Matter in Temperate Soils: Mechanisms and their Relevance under Different Soil Conditions – A Review. European Journal of Soil Science, 57:426–45.

Mando, A., B. Quattara, A. E. Somado, M. C. S. Woperis, L. Stroosnijder, H. Breman, 2005. Long-term Effects of Fallow, Tillage and Manure Application on Soil Organic Matter and Nitrogen Fractions on Sorghum Yield under Sudano-Sahelian Conditions. Soil Use and Management, 21:25–31.

Mtambanengwe, F., P. Mapfuno, and H. Kirchmann, 2004. Decomposition of organic matter in soil as influenced by texture and pore size distribution. In Managing nutrient cycles to sustain soil fertility in sub-Saharan Africa Ed., Bationo, A. Centro Internacional de Agriculture Tropical. pp: 261–276.

Ohm, H., U. Hamer, and B. Marschner, 2007. Priming Effects in Soil Size Fractions of a Podzol Bs Horizon after Addition of Fructose and Alanine. Journal of Plant Nutrition and Soil Science, 170:551–59.

Parton W. J., J. W. B. Stewart, and C. V. Cole, 1988. Dynamics of C, N, P, and S in Grassland Soils: A Model. Biogeochemistry, 5:109–131.

Pedra, F., A. Polo, A. Ribiero, and H. Domingues, 2007. Effects of Municipal Solid Waste Compost and Sewage Sludge on Mineralization of Soil Organic Matter. Soil Biology and Biochemistry, 39: 1375–1382.



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Rudrappa L., T. J. Purakayastha, D. Singh, and S. Bhadraray, 2006. Long-term Manuring and Fertilization Effects on Soil Organic Carbon Pools in a Typic Haplustert of Aemi-arid Sub-tropical India. Soil Tillage and Research, 88:180–192.

Rumpel, C., H. Knicker, I. Kıgel-Knabner, J. O. Skjemstad and R. F. Huttl, 1998. Types and Chemical Composition of Organic Matter in Reforested Lignite-rich Mine Soils. Geoderma, 86:123–142.

Sherrod L. A., G. A. Peterson, D. G. Westfall, and L. R. Ahuja, 2005. Soil Organic Carbon after 12 Years in No-till Dryland Agroecosystems. Soil Science Society of America Journal, 69:1600–1608.

van Veen, J. A., and P. J. Kuikman, 1990. Soil Structural Aspects of Decomposition of Organic Matter by Microorganisms. Biogeochemistry, 11:213–233.

Vance, E. D., P. C. Brookes, and D. S. Jenkinson, 1987. An Extraction Method for Measuring Soil Microbial Biomass C. Soil Biology and Biochemistry, 19:703–707.

Zhao, L., Y. Sun, X. Zhang, X. Yang, and C. F. Drury, 2006. Soil Organic Carbon in Clay and Silt Sized Particles in Chinese Mollisols: Relationship to the Predicted Capacity. Geoderma, 132:315–323.



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Rainwater Harvesting, Quality Assessment And Utilization In Region I

*Adriano T. Esguerra, **Antonio E. Madrid and *** R odolfo G. Nillo, Mme

*Professor VI, **Associate Professor V , ***Instructor; Don Mariano Marcos Memorial State University

Bacnotan, La Union

Corresponding author Email: [email protected]

Abstract The project harnessed the potential of house rooftops as rainwater harvesters for household use, principally as drinking water. It likewise assessed the system’s technical soundness, environmental dimensions, economic feasibility as well as its social and political acceptability. Technically, the rainwater harvesting system consisting of rooftops, gutters, down spouts, filter and storage tank is capable of collecting/impounding rainwater to supply and support the drinking water needs of 8-12 members of the family throughout the six-month dry period (January-June) of the year. In terms of rainwater microbiological quality, total coliforms and Escherichia coli were of low concentrations (i,e., less than 1.1 MPN/100 ml) meeting the allowable limits set by the Philippine National Standards for Drinking Water (PNSDW). Other quality and aesthetic characteristics of collected/stored rainwater such as the presence of inorganic and organic substances through total dissolved solids as well as its total hardness adequately met the PNSDW values indicating potability of the harvested rainwater. The harvester is economically feasible especially so if construction materials would be limited to locally available ones. Economic analysis showed that the cost of the rainwater harvesting system could be recovered in two years at most. Cost of the system could be significantly lower if more than three families would share in the construction and that the harvested rainwater would be utilized for purposes other than for drinking. Demonstrating the importance of the system to the community, neighboring families were convinced that it provided water for drinking purposes microbiologically safer than the existing water they have been drinking for years. Result of the survey confirmed the desire of the community to put up similar system as they stressed that their health is of paramount importance and subscribed that the construction cost is not an issue at all. Local government units were likewise of the perception that the system would work in the locality and that they are willing to support the initiative of making the system an important and innovative part of their development plan. Keywords: water, water scarcity, rainwater harvesting,



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Introduction

Water is essential to all life – human, animal and vegetation. It is therefore important that adequate supplies of water be developed to sustain such life. The development of water sources must be within the capacity of the nature to replenish and to sustain. The application of innovative technologies and the improvement of indigenous ones should therefore include management of the water sources to ensure sustainability and to safeguard the sources against pollution. There is now increasing interest in the low cost alternative – generally referred to as “rainwater harvesting”. (http://members.rediff.com/asitsahu/)

Rainwater harvesting, in its broadest sense, is a technology used for collecting, conveying and storing rainwater for human use from rooftops, land surfaces or rock catchments using simple techniques such as jars and pots as well as engineered techniques. Rainwater harvesting has been practiced for more than 4,000 years, owing to the temporal and spatial variability of rainfall. It is an important water source in many areas with significant rainfall but lacking any kind of conventional, centralized supply system. It is also a good option in areas where good quality fresh surface water or groundwater is lacking. The application of appropriate rainwater harvesting technology is important for the utilization of rainwater as a water resource.

Rainwater harvesting is simple to install and operate. Local people can be easily trained to implement such technologies, and construction materials are also readily available. Rainwater harvesting is convenient in the sense that it provides water at the point of consumption of family members have full control of their own systems, which greatly reduces operation and maintenance problems. Running costs, also, are almost negligible. Water collected from roof catchments usually is of acceptable quality for domestic purposes. As it is collected using existing structures not specially constructed for the purpose, rainwater harvesting has few negative environmental impacts compared to other water supply project technologies. Although regional or other local factors can modify the local climatic conditions, rainwater can be a continuous source of water supply for both the rural and poor. Depending upon the household capacity and needs., both the water collection and storage capacity may be increased as needed within the available catchmentarea.(http://www.gdrc.org/uem/water/rainwater/introduction.html)

The project was carried out at Barangay Sapilang, Bacnotan, La Union from October to December 2009.

Objectives of the Project

The project aimed at piloting/showcasing rooftop rainwater harvesting as an adaptation strategy against impact of climate change in an upland ecosystem. It showcased how a house rooftop could be effectively and efficiently harnessed to harvest rainwater for domestic or household use.

Specifically, it sought to determine the project’s technical soundness, environmental safety, economic feasibility, social as well as political acceptability in terms of the assessed quality (microbiological and physic-chemical) and level of utilization of the harvested rainwater.



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Materials and Methods

Site Identification and Characterization

The study site is located at Barangay Sapilang, Bacnotan, La Union, about 200 meters west of the Don Mariano Marcos Memorial State University- North La Union Campus (Figure 1). It is 16o43’N longitude and 120o21’E latitude. The area falls under Type 2 Climate, with two pronounced seasons, that is, dry from November to April and wet the rest of the year. The area is an upland rainfed usually grown with rice during wet season and vegetable crops during the dry months of the year. Fruit crops such as banana and citrus also abound. Raising of cattle and small ruminants on top of the native chicken and pigs is a common scene in the area. The source of water is a spring which has been observed inadequate to supply the water requirements of the barangay during the entire dry season. As of 2007, the total population of the barangay under study is 858, equivalent to 140 households.

Project Preparation and Construction

Upon identification of the project site, survey of the house to serve as the rainwater harvesting unit was done. Two adjacent houses, about three meters separating them, were selected for the purpose. Assessment of the capability of the households was done through a personal face-to-face interview. On top of the series of questions asked was the willingness of the households to undertake simple data gathering during the project implementation as well as their share of responsibility in maintaining the project even after its completion.

Figure 1. Map of La Union showing location of the Municipality of Bacnotan



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Figure 2. Map of the Municipality of Bacnotan showing the location of barangay Sapilang

Figure 3. Location plan of the Rainwater Harvesting Project



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Considering the subject households’ willingness to work with the project management vis-à-vis data gathering and responsibility sharing, the necessary preparation of the project commenced. The roofings as well as their accessories such as gutters and downspouts of the two subject houses were then assessed for proper technical planning and budget considerations. Added to these were the needed calculations on the water requirements of the households as well as their animals and crops. This requirement was made basis in the design and development of the rainwater collecting tank.

The necessary installation of the needed parts of the subject houses such as roofing, gutters, and downspouts alongside the construction of the rainwater collecting tank and its accessories began after the completion of the project technical plan. The DMMMSU-NLUC Planning Office was requested to do the technical plan for the project.

Project Implementation

The project implementation consisted of two parts, the quality assessment and the utilization of the harvested rainwater.

Upon completion of the construction and installation of the necessary accessories of the project, project implementation and data gathering followed. Simple manual for the project implementation to include operation and maintenance activities as well as data gathering instructions was provided to the households. The first few days of the project implementation was confined to do’s and don’ts of the operation and maintenance of the rainwater harvesting system as well as the what, where and how to gather data. To ensure accurate gathering of data, qualified research assistants were assigned to assist during the implementation and data gathering period.

Quality Assessment. Determination of the microbiological and physic-chemical data such as total coli form count, Escherichia coli (E. coli ) count, total hardness, total dissolved solids and acidity of both rainwater before and after rooftop harvesting formed part of the quality assessment phase. Rainwater samples (at 500 ml each) were collected and taken to the Department of Science and Technology Laboratory, Region I at San Fernando City, La Union for analysis. The results of the analyses served as guide in determining the utilization of the rainwater after its collection by way of rooftop as harvester.

Utilization. Utilization of the harvested rainwater was for drinking water and for other household use such as for cooking, dishwashing, house cleaning, bathing, etc. Other uses were in the form of vegetable crop irrigation and provision for backyard animal water requirement.

Other Information and Observation

Kinds of materials used and their costs as well as other economic parameters were also gathered for the purpose of assessing the economic feasibility of the rooftop as rainwater harvester for household use (i.e., cooking, drinking, bathing, washing, and others to include irrigating vegetable crops and supplying water needs of backyard animals).



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In terms of social and political acceptability of the rooftop rainwater harvesting project, a simple survey instrument was prepared and was later distributed to the barangay constituents. About 30 percent of the total households (or 40 households) who served as respondents of the study were randomly selected to determine the perceived level of acceptability of the harvester. Barangay as well as municipal officials were also referred to as to the acceptability of the same rainwater harvesting project in their jurisdictions. The level of acceptability ranges from 1-5, with 5 as the highest level described as highly acceptable and 1 as the lowest interpreted as not acceptable at all. For the purpose of getting fresh and accurate responses from these two groups of respondents, they were all invited to the project site to see how the rainwater harvester worked.

Data Treatment and Analysis

Descriptive analysis was done for all the data obtained in the project, which includes frequency, percentage and means determination.


Rainwater Harvesting

Figure 1 shows the completed rainwater harvesting system utilizing house rooftops to collect rainwater for multiple household uses. It consists of the following major parts, namely: (a) rooftop as catchment, (b) gutter and downspout as rainwater conduit to the tank, (c) filter, and (d) collecting tank. Each of these composite parts is described below.

a. Catchment

The rainwater catchments are the rooftops of the two houses made of painted galvanized iron (G.I.) corrugated sheets which directly receive the rainfall providing water to the system. The two houses are of gable-type roofs and were designed to withstand the dead load as well as the forces of wind and rain. The rooftops of the two houses (owned by Mr. Nillo family and Mr. Antipolo family) have total surface areas of 56 sq m (7m x 8m) and 30 sq m (3m x 10m), respectively.

b. Gutters and Downspouts

Gutters are channels placed at the edge or end of the gable sloping roof to collect and transport rainwater to the concrete collecting tank. These are called “Spanish gutter” and in semi – circular shape. Downspouts, on the other hand, are pipelines or drains linked to the gutters that carry rainwater from the catchment or rooftop area of the two houses to the harvesting system. They are made up of polyvinyl chloride (PVC).



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Figure 1. Overview of the Rainwater Harvesting System

c. Filter

The harvester as a system requires first flush device to ensure that runoff from the first spell of rain is flushed out and does not enter the system. This was done since the first spell of rain carries a relatively larger amount of pollutants from the air and catchment surface. The filtering device measures 2 meters long, 0.5 meter wide and 0.5 meter deep. It is divided into two compartments by a concrete wall having 0.3 meter high allowing water to overflow upon reaching this level moving to the second compartment prior to entering the concrete collecting tank. The first compartment was filled with three different filtering materials. These are dried empty shells placed at the bottom , coarse white sand at the middle and gravel with medium size at the top and a thickness of 2 cm, 3 cm, and 4 cm, respectively. It is covered with a pre – fabricated reinforced concrete.

d. Rainwater Collecting Tank

The tank measures 3 meters long, 3 meters wide and 2.5 meters deep (or a volume capacity of 22.5 cu m equivalent to 22,500 liters). The walls measure 40 cm long and 15



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cm wide and are plastered by a mixture of cement – sand ratio of 1:2 and tiled flooring. It is covered by a pre – fabricated reinforced concrete. A manhole (0.5 m x 0.5 m) was placed neatly at one of the corners.

e. Hand-pump

The hand-pump is used to draw water from the rainwater collecting tank. It is permanently installed between the two houses and about 2.5 meters away from the rainwater collecting tank.

Quality Assessment of Harvested Rainwater

Reflected in Table 1 are results of the microbial and chemical analysis done on the rainwater samples collected from the collecting tank. Table 1. Summary of the results of the microbial and chemical analyses on the rainwater

collected (tank-based) Microbial Parameter Level

Total coliform count < 1.1MPN/100 mL

Escherichia coli ( E. coli ) Count < 1.1 MPN/100 mL

Total hardness 122.0 mg/L

Total dissolved solids 68-238 mg/L

Acidity -68 to -113.0 CaCO3/L Philippine National Standards, 2007: < 1.1 MPN/100 mL (for drinking water); 300 mg/L (for total hardness); 500 mg/L (for total dissolved solids); no standard value for acidity Multiple Tube Fermentation Technique was used in determining the total coliform count of the water samples. The values reflected in the table are indices used to indicate the number of tubes in which the samples were found positive of coliform. Based on standards set for by the Philippine National Standards for Drinking Water (PNSDW) of 2007, drinking water should be negative of coliform, indicated by an index of < 1.1 MPN/100 mL. With this standard value as guide, the harvested rainwater was therefore safe for drinking. Coliform bacteria are indicator organisms which are used in water biological analysis. Coliforms are a group of bacteria which are readily found in soil, decaying vegetation, animal feces and raw surface water. “Total coliform” is the collective name used for all coliform groups. The presence of these coliforms is an indication of contamination of the source of water samples. These indicator organisms may be accompanied by pathogens (i.e., disease causing organisms), but do not normally cause disease in healthy individuals. However, individuals with compromised immune systems should be considered at risk (Buenafe, 2005). Results of the analysis showed that collected rainwater from the tank met the standard of PNSDW for the E. coli count, that is, <1.1 MPN/100mL. Tank water samples did not contain E. coli ably meeting the standards set by PNSDW. Todar (1997) stated that there are harmless



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bacteria that live in digestive tract of animals. These bacteria enter water bodies from human and animal wastes. If a large number of coliform bacteria (over 200 colonies/100 mL of water sample) are found in water, it is possible that pathogenic organisms are also present in water. Coliforms by themselves are not pathogenic; they are an indicator organism, which means they may indicate the presence of pathogenic bacteria (Brown, 1995). In terms of total hardness, ethylenediaminetetraacetic acid (EDTA) Trimetric Method, 2310B was used in determining the properties of water samples. Hardness is a term used to express the properties of highly mineralized water (high TDS concentrations). Water with more than 300 mg/L of hardness is generally considered to be hard, and water with less than 75 mg/L is considered to be soft. Very soft water is undesirable in public supplies because it tends to increase corrosion in metal pipes; also some health officials believe it to be associated with the incidence of heart disease (Nathanson, 1997). The 122 mg/L total hardness of the rainwater harvested and collected was within the standard set by the PNSDW which means that it is safe for drinking. Total dissolved solids dried at 1800C, 2540C were the method used in determining the combined content of all inorganic and organic substances present in the water samples. Total dissolved solids (TDS) are an indication of aesthetic characteristics of drinking water and as an aggregate indicator of the presence of a broad array of chemical contaminants (Wikipedia, 2009). Water samples collected from the storage tank were far below the standards set by PNSDW which is 500 mg/L and, therefore, were acceptable for drinking purposes.

As to acidity, Potentiometric Method, 2310B was the method used in determining the acidity of the water samples. Acidity may pertain to the level of pH between acidity and alkalinity. Palintest Alkaphot test which used a colometric method covers the total alkalinity range 0-500 mg/L CaCO3 to check the suitability of natural drinking water. Results of the test indicate that all water samples met the standard of PNSDW.

Utilization of Harvested Rainwater

Beside its use for drinking purposes, the harvested rainwater was likewise utilized for supplying the water requirements of different vegetable crops (ampalaya, pechay, okra, squash, pole beans, eggplant and tomato), banana and calamansi grown and backyard animals such as cattle (carabao and cow), small ruminants (goat and sheep) as well as native chicken and pigs raised by the households in the project site. With this add-on utilization of harvested rainwater, the more the rooftop rainwater harvesting became highly economically feasible and viable under the village conditions.

Acceptability of the Rainwater Harvester

Barangay Sapilang is an identified area that lacks supply of water. Based on the information gathered from the community people, this area is seriously experiencing water scarcity most of the time throughout the year due to poor source of water supply. The residents rely largely on a reservoir that was constructed in the school campus. Considering the population in the area, the water supply coming from this reservoir could not completely provide the required volume of water for their daily personal and farm needs. They even organized themselves to plan out other ways and means for another possible source of water. They constructed a deep well somewhere



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in their area as a supplemental source of water. Hence, the establishment of the Rainwater Harvesting Project in the area was very timely and a welcome relief in the community. The community found the rainwater harvesting system as convenient in the sense that it provides water at the point of consumption, and family members have full control of their system, thereby greatly reducing the operation and maintenance problems. Aside from knowing that rainwater harvested is suitable for drinking, the community noted that it can be used for other purposes such as those mentioned above, that is, for irrigating vegetable and fruit crops as well as supplying the water needs of the animals tended in the backyard. This alone, according to the barangay respondents, justifies the putting up of the rainwater harvesting project in their locality. Political Acceptability Various levels of governmental and community involvement in the development of rainwater harvesting technologies in different parts of Asia were noted. In the Philippines, both governmental and household-based initiatives played key roles in expanding the use of this technology, especially in water scarce areas like barangay Sapilang. Upon showing the advantages and benefits that could be derived from the rainwater harvesting system, the local officials agreed to include the system as priority project of the barangay. The same project was planned to be indorsed to the Local Government Unit of Bacnotan for possible integration to its five-year development plan so that the greater number of barangays in the municipality could benefit from the project.

Conclusions and Recommendations Conclusions Based on the findings of the project, the following conclusions were derived: a. The rainwater harvesting system harnessing house rooftops is technically feasible,

environmentally sound, economically viable, socially and politically acceptable. b. Harvested rainwater is safe for drinking and could be utilized to augment the water

requirement of different crops grown and animals raised in the backyard. Recommendations Taking into account the above findings, the following recommendations are forwarded: 1. Initiative of piloting the house rooftop rainwater harvesting system be intensified and

expanded to far- flung barangays especially to those areas experiencing water quality problems for drinking purposes.

2. The rainwater harvesting system be part of the initiative or part of the ordinance of the local government units under their water for all programs so that regular budget allocation be given to the project.

3. Wide dissemination of the project be done at the LGUs level as an adaptation strategy to address water scarcity attributed to climate change and El Niňo.



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References Coombes, P., Kuczera, G., Kalma, J. (2000). Rainwater Quality from Roofs Tanks and Hot Water Systems at Fig Tree Place. Proceedings of the 3rd International Hydrology and Water Resources Symposium, Perth. Every Water Drop Counts: Rooftops as Potential Rainwater Harvesting System, (Nillo, R.G, and MADRID, A.E., 2009) Macomber, P.S.H. (2001). Guidelines on rainwater catchment systems for Hawaii. College of tropical agriculture and human resources, University of Hawaii, Manoa. Publication no. RM-12. (http://www2.ctahr.hawaii.edu/oc/freepubs/pdf/RM-12.pdf) Michaelides, G. (1987). Laboratory Experiments on Efficiency of Foul Flush Diversion Systems; 3rd International Conference on Rainwater Cistern Systems, Khon Kaen. (http://www.eng.warwick.ac.uk/ircsa/abs/3rd/b1.html) UNEP (1998). Sourcebook of Alternative Technologies for Freshwater Augmentation, United Nations Environment Programme, Nairobi. (http://www.unep.or.jp/ietc/Publications/TechPublications/) Vasudevan, P., Tandon, M., Krishnan, C., Thomas, T. (2001). Bacteriological Quality of Water in DRWH. 10th Conference of the International Rainwater Catchment Systems Association, International Rainwater Catchment Systems Association. WHO (1996). Guidelines for Drinking Water Quality, 2nd Edition, Vol. 2. World Health Organization, Geneva. Websites http://en.wikipedia.org/wiki/Water http://www.rainharvesting.com.au/rainwater_research.asp http://waterwiki.net/index.php/Rainwater_harvesting http://www.http://www.eng.warwick.ac.uk/ircsa/abs/10th/3_05.html http://rambler.newcastle.edu.au/%7Ecegak/Coombes/Hydro20003.htm http://www.gdrc.org/uem/water/rainwater/introduction.html

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