International Journal of Marine Science 2015, Vol.5, No.27: 1-11
9
of sediment in situ, and replacing it with clean sandy
sediment from Nusakambangan Island, some of
which had been treated (polluted) with oil. 50 x 50 x
15 cm of the oil-polluted sediment was inserted into
the enclosure mesocosm boxes which lay on a 100 x
100 x 40 cm uncontaminated sand base from
Nusakambangan Island.
In the middle of each plot a pore water sampler was
constructed. It was made from a hose protected by
PVC paralon with small holes in 5 – 10 cm from the
end which allowed filtered pore seawater from treated
sediment to be collected. It was immersed to a depth
of 10 cm during the experiment.
4.7 Sampling Strategy
Periodical sampling was conducted 0, 3, 8, 16, 23, 60
and 90 days after treatments. Sampling were
conducted before high tide (water height was about 5
cm inside each plot). The samples were sediment and
pore water. Composite sediment samples for oil and
microbial analysis were collected from 5 sampling
points in each plots. The samples were then separated
into an oil-free glass (100 gram) for oil analysis and a
50 mL sterile falcon tube for microbial analysis. All
samples were transported to the laboratory in a cool
box with ice inside.
Pore water samples for environment analysis were
collected carefully by syringe to avoid oxygen
contamination and transferred into 50 mL airtight
bottles. All samples were transported to the laboratory
in a cool box with ice inside.
4.8 Sample Analysis
4.8.1 Oil Analysis
Oil concentration was determined gravimetrically (US
EPA, 1999). Samples were prepared by putting a 2 g
subsample of homogenized sediment into a glass tube.
Extraction was conducted by using the maceration
method with a mixture of Dichloromethane : n-hexana
(1:1) in proanalysis grade as a solvent. Na
2
SO
4
was
used to absorb the water remained in samples. The
performance of oil biodegradation expected on an oiled
shoreline can be estimated based on the first-order
kinetic models. The first order relationship was
expressed as: C
t
= C
0
e
(-kt)
where C
0
is initial oil
concentration at day 0, C
t
is oil concentration in time t,
k is the biodegradation rate coefficient. In this case,
we used as depletion (decay) rate coefficient.
4.8.2 Microbial analyses
Subsample sediments from each plot were processed
for enumeration of total cell bacteria. They were
prepared by mixing 1 g wet weight of sediment into
dilution water containing 9 mL seawater sterile. Then,
this solution was placed in vortex at 300 rpm for
15 minutes to detach the bacteria from the sediment.
Dilution of this subsample sediment was transferred
into Acridine Orange solution, then filtered by
polycarbonate membrane (0.22 µm) that submerged
previously on sudan black solution. Direct counting
under epifluorescent microscope was used for
enumerating total cell bacteria (Hobbie et al.
,
1977).
4.8.3 Environmental parameters
To monitor changes in the sediment’s environmental
conditions during the experiment, measurements of
salinity, pH, Dissolved Oxygen (DO) and
Oxida tion-
reduction potential (ORP) were carried out immediately
after sampling by using a hand refractometer, pH
meter (Horiba, Navi D-54), DO meter (Horiba, YSI
55)and ORP meter (Horiba, Navi D-52), respectively.
4.9 Statistical analysis of data
The experiment was conducted using three independent
replicates. Data were subjected to analysis of variance
and the averages were compared by Duncan multiple
range test at p< 0.05. All statistical analysis was
performed using SPSS 16. The rate of oil degradation
and relation with other parameters measured were
assessed by simple regression and correlation analysis.
Acknowledgments
The authors thank the following for funding and
supporting
this project: Research Center for Oceanography-Indonesian
Institute of Science
, Pertamina Refinery Unit IV, Cilacap and
BPLHD Cilacap Regency (Mr. Indarto). Additionally, we wish
to thank to the field crew for the long hours and hard work and
without whom this project would never
succeeded. We also
grateful to Mari Rhydwen who has
providing us invaluable
assistance in the manuscript preparation.
Author’s contributions
YD carried out the design and execution of the study,
performed the statistical analysis and drafted the manuscript.
HSS conceived of the study, participated in the design and
coordination, and helped to refine the manuscript. TP and DAS
participated in the design of the study, supervised the
implementation of study and helped to refine the manuscript
participated in the design of the study, supervised and
