IJERD – International Journal of Environmental and Rural Development (2012) 3-2

Research article
erdChanges in Escherichia coli Efflux from Farmland by
Surface Runoff and Percolation under Different
Application Methods of Manure
YUTA ISHIKAWA
Graduate School of Agriculture, Tokyo University of Agriculture, Japan
Email: y_i4kawa@yahoo.co.jp

MACHITO MIHARA*
Faculty of Regional Environment Science, Tokyo University of Agriculture, Japan
Email: m-mihara@nodai.ac.jp
Received 15 February 2012

Accepted 18 April 2012(* : Corresponding author)

Abstract The application of cattle manure in farmlands was considered as a proper
treatment from a viewpoint of organic agriculture. However, pathogenic bacteria known as
E. coli in immature fermented manure happen to spread out from farmlands. So, theobjective of this study is to investigate E. coli efflux under different application methodsof cattle manure. Slope modeling experiment was conducted under an artificial rainfall
simulator. Slope plots were filled with soil then fresh cow dung and 2 weeks fermented
manures were applied into the plots. Cow dung and manure were applied with 2application methods: the broadcasting method and the incorporating method. Based on theexperimental results, it was found that E. coli efflux increased with soil loss throughsurface runoff. Moreover, it showed that the amounts of E. coli efflux were higher thanthat of input in either plot broadcasted or incorporated with cow dung. In addition, there
were tendencies that E. coli had been transported by percolation although it was smaller innumber than that by surface runoff.
Keywords Escherichia coli, total suspended solid, soil erosion, cow dung, manure

INTRODUCTION
Large amount of dung has been produced from cattle farms. So, making manure was considered as
one of the proper treatments for utilizing cattle manure from a viewpoint of organic agriculture.
However, most of manures produced from cattle farms may include immature fermented manure.
The pathogenic bacteria known as E. coli may survive and remain in immature fermented manure(Chun-Ming et al., 2005). It was reported by Islam et al. (2005) that E. coli applied through immature fermented manure survived in longer days at farmlands. Also, some reports mentioned that theefflux of E. coli occurs through surface runoff from grazed land and farmland where cattle manures
were applied (Mishra et al., 2007; Yagura et al., 2006) and large amounts of E. coli were transported along with soil erosion from farmland where fresh dung was applied (Ishikawa and Mihara,2010). In addition, E. coli efflux remarkably influences environmental pollution in watersheds aswell as contamination of potable water source affecting human health (Tamura et al., 2006; Mishina et al., 2007). However, only few studies has been carried out concerning the efflux of E. colifrom farmlands where immature fermented manures were applied.
In this study, the objectives were to investigate the relationship between the efflux of E. coli
and total suspended solids under broadcasting and incorporating methods of manures, to evaluate
the difference between E. coli efflux through surface runoff and that through percolation, and tocompare the efflux of E. coli with the input amounts of E. coli.

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METHODOLOGY
Cow dung and manure used in this experiment were collected from Fuji Farm, a cattle farm of Tokyo University of Agriculture located in Shizuoka Prefecture. There were two types of cow dungcollected for the experiments, the fresh cow dung (Fig. 1a) and the 2 weeks fermented manure (Fig.
1b) with water contents at 84 and 72%, respectively. For sanitary reasons, collected cow dungs
were kept in a close container under anaerobic condition. Organic matters were 89% in fresh cow
dung and 87% in 2 weeks fermented manure as shown in Table 1. The colony forming unit (cfu) ofE. coli was 19×106 cfu/g in fresh cow dung and 21×103 cfu/g in 2 weeks fermented manure.

(a)(b)

Fig. 1 Piles of fresh cow dung (a) and 2 weeks fermented manure (b) at Fuji Farm
Table 1 Properties of cow dung and fermented manure
Period of fermentation

Escherichia coli
(cfu/g)Water content(%)

Organic matter(%)

0 days

19×10684

89

2 weeks

37287

Cow dung
Manure21×10

Stainless slope model plots of 130 cm long and 11 cm wide were filled up with light clay soil,andosol, at 5 cm deep (Table 2). The plots were set up at 8 degrees in slope then cow dung and 2
weeks fermented manure were applied. The amounts of cow dung and manure applied into eachplot were 1000 g/m2 (10 t/ha) in dry mass. Both of fresh cow dung and fermented manure were applied by broadcasting method and incorporating method into each designated slope model plot.
Broadcasting method is spreading fresh cow dung or fermented manure on soil surfaces, while incorporating method is mixing fresh cow dung or fermented manure with soil. The prepared slopemodel plots were kept to make soil the 24 hour field capacity moisture before starting the rainfall
simulation.
Table 2 Physical properties of soil
Specificgravity
2.68

Particle size distribution(%)

Soil texture

GravelCoarse sandFine sandSiltClay

2.0

12.3

27.8

30.2

27.7

LiC

Table 3 Chemical and biological properties of soil
Ignition loss(%)

Total nitrogen(10-5 kg/kg)

Total phosphorus(10-5 kg/kg)

Escherichia coli
(cfu/g)13.41

226.3130.36

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Artificial rainfall simulation was carried out at rainfall intensity of 60 mm/hr for 2 hours. Suspended water samples through surface runoff were collected from each plot (Fig. 2). Suspended
water samplings were done every 15 minutes for the first one hour and every 30 minutes for thesecond one hour of the simulation. Percolation water was sampled only once after 24 hours passed
from the end of simulation.
Artificial rainfall at 60 mm/h8°Percolation water

Fresh or fermented cow dung

Surface water discharge

Fig. 2 Slope model plot and suspended watercollected by surface runoff andpercolation water

Fig. 3 Propagated E. coli by culture
media in petri dishThe colonies of E. coli in the collected suspended water samples were evaluated through laboratory analysis. The analysis was carried out by bacteria culture medium XM-G as shown in Fig.
3. The mass of solids in surface runoff and in percolation water were also measured by oven drying
method.
RESULTS AND DISCUSSION
The relationship between E. coli efflux and total suspended solids loss from the plot applied freshcow dung was summarized in Fig.4. It was observed that E. coli efflux significantly increased withtotal suspended solids in surface runoff from both application methods. A confidence interval at
99% was also observed in surface runoff from the plots broadcasted and incorporated with cow
dung. However, E. coli efflux tended to be very low in correlation with suspended solid losses forpercolation water.
The relationship between E. coli efflux and suspended solids loss in the plots applied 2 weeks
fermented manure was shown in Fig. 5. There was a tendency that E. coli efflux increased withsuspended solids losses in surface runoff. It was also observed that E. coli efflux was very low inbroadcasted plots comparing to that in incorporated plots. The tendency of E. coli efflux with suspended solids loss was very low in percolation water. Therefore, it was considered that E. coli efflux had a remarkable tendency to be transported along with suspended solids loss for surface runoff, but that tendency was not clear for percolation water.
Additionally, Fig. 6 shows the total input and efflux of E. coli through surface runoff or percolation from the plots applied cow dung. The experimental results of fresh cow dung indicated thatthe number of E. coli efflux was higher than that of input in both broadcasting and incorporating
methods. However, the significant difference was not observed between the input and the efflux ofE. coli. In the plots applied 2 weeks fermented manure, it was observed that E. coli efflux was
higher than that of input in incorporation methods as shown in Fig. 7. Although a significant difference was not observed, the number of E. coli efflux from incorporated plot was higher than thatfrom broadcasted plot. The tendency of E. coli efflux being higher than that of E. coli input wasconsidered that E. coli may be facultative anaerobic pathogens which may propagate for a certain
period after applying into an open land. Hence the condition before applying E. coli was an anaerobic then it changed to aerobic condition after applying.

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2E+10 920×10

Runoff frombroadcasted plot
R = 0.8888 **1.5E+10 915×10

Runoff fromincorporated plot
R = 0.9311 **1E+10 910×105E+09 95×10Percolation frombroadcasted plot
R = 0.674400

Percolation fromincorporated plot
R = 0.5493

0200400600800

1000

100000000
10×107

Cumulative E.coli efflux (cfu/m2)

Cumulative E.coli efflux (cfu/m2)

IJERD – International Journal of Environmental and Rural Development (2012) 3-2

Runoff frombroadcasted plot
R = 0.9759**
Runoff fromincorporated plot
R = 0.9264**
Percolation frombroadcasted plot
R = 0.6968
Percolation fromincorporated plot
R = 0.9999**

80000000
8×107
60000000
6×107
40000000
4×107

20000000
2×10700

0200400600800** Counfidence interval at 99%** Counfidence interval at 99%

Fig. 4 Relationship between E. coli efflux andsuspended solids loss from plots where
fresh cow dung was applied

1.E+121012
1.E+101010

10
1E+1010applied

AA

A

Fig. 5 Relationship between E. coli efflux
and suspended solids loss from plots
where 2 weeks fermented
manure was applied
Number of E.coli (cfu/m2)

Number of E.coli (cfu/m2)

1.E+141014

1000

Cumulative suspended solids loss (g/m2)

Cumulative suspended solids loss (g/m2)

100000000
108

AAA

1000000
106

1.E+081081.E+06106

10000
104

1.E+04104
1.E+02102
1.E+00100

Input1001021010

Efflux from
Efflux frombroadcasted plot incorporated plot
Surface runoff

Input

Efflux from
Efflux frombroadcasted plot incorporated plot

Percolation

Surface runoff

Fig. 6 Number of E. coli input in fresh cow
dung and E. coli efflux through surface runoff and percolation

Percolation

Fig. 7 Number of E. coli input in 2 weeks
fermented manure and E. coli efflux through surface runoff andpercolation

1E+121012

E.coli efflux (cfu/m2)

AA

a*b*1E+08108

AaaA

1E+04
104

1E+00
100

Cow dung
broadcasted

Cow dung
incorporated

2 weeks manure
broadcasted

2 weeks manure
incorporated

Surface runoff

Percolation

Significant difference at *95%

Fig. 8 Total efflux of E. coli from plots applied fresh cow dung or 2 weeks
fermented manure by broadcasting or incorporating
methods
The total efflux of E. coli through surface runoff and percolation are shown in Fig. 8. It wasindicated that the efflux of E. coli decreased with the fermentation. The tendency of E. coli efflux
through surface runoff to be larger than that through percolation was also observed. It was clearlyindicated that E. coli efflux occurred not only through surface runoff but also through percolation.
A significant difference at 95% was observed in only the case fresh cow dung applied by incorporating method.

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CONCLUSION
Based on the experimental results, it was clearly observed that E. coli efflux significantly increased
with the increase in total suspended solids in surface runoff from the plots applied fresh cow dungand from the plots applied 2 weeks fermented manure. Also, the same tendency that E. coli efflux
increased with the increase in total suspended solids by broadcasting and incorporating methods.
The efflux of E. coli through percolation was significantly lower than that of E. coli efflux
through surface runoff. It was also observed that the number of E. coli efflux was higher than thatof E. coli input from both plots applied fresh cow dung and 2 weeks fermented manure.
The existence of E. coli were found even in the 2 weeks fermented manure indicating a potential of E. coli efflux after applying into farmlands particularly by incorporating application method.
Therefore, fermentation of cow dung for more than two weeks before applying into farmlands ishighly suggested.
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