The international journal published by the Thai Society of Higher Education Institutes on Environment
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EnvironmentAsia
Available
DOI
9(2) (2016) 48-54
online at www.tshe.org/EA
EnvironmentAsia 2 (2009) 50-54
The aim of the present study was to standardize and to assess the predictive value of the cytogenetic analysis
Abstract
by Micronucleus (MN) test in fish erythrocytes as a biomarker for marine environmental contamination. Micronucleus
frequency
baseline(>
Microplastics in5erythrocytes was evaluated
mm) have gained popularityininand genotoxic
research potential
and the public of
eyea common chemical
in recent years. Thiswas determined
is due to the fact
in
that they contain persistent organic pollutants (POPs) which pose potential risks to the environment and humanfor
fish experimentally exposed in aquarium under controlled conditions. Fish (Therapon jaruba) were exposed 96
health.
hrs to a single heavy metal (mercuric chloride). Chromosomal damage was determined as micronuclei frequency
Bivalves, which are filter feeders, are considered to be good indicators of marine pollution. In this preliminary study, Asian in
fish erythrocytes. Significant increase in MN frequency was observed in erythrocytes of fish exposed to
green mussel (Perna viridis), an example of edible bivalve, cultured in Bacoor Bay, Cavite, Philippines was subjected tomercuric
chloride.
qualitativeConcentration of 0.25 ppm
analysis to determine the induced
presencethe
ofhighest MN frequency
microplastics. Through(2.95 micronucleated
microscopic cells/1000
analysis, cells compared
microplastics were found
to
present in the acid-digested mussel soft tissue. A management program is suggested for policy makers andofstakeholders
1 MNcell/1000 cells in control animals). The study revealed that micronucleus test, as an index cumulativeto
exposure,
reduce the appears
negativeto be a sensitive
impact modelpollution
of microplastic to evaluate genotoxic
to both humanscompounds in fish
and the marine under controlled conditions.
environment.
Keywords:
Keywords: genotoxicity;
microplastics;mercuric chloride;
qualitative micronucleus
assessment; Perna viridis; marine pollution; environmental management
1. Introduction
1. Introduction laboratory
MMT (Million and field conditions.
Metric Tons) perIn 2006
year Soumendra
(Jambeck et al.,
2015). Marine plastic debris primarilybiomarkers
et al., made an attempt to detect genetic come from
In India,are
Plastics about
popular 200due tons of mercury
to several and such
properties its in two fish sources
land-based species,(80%) bata and
Labeothrough Oreochromis
leakage while only
compounds are introduced into the environment
as weight, strength and cost. The rise of consumption mossambica, by MN and binucleate
marine aquaculture and fisheries only contribute (BN)
20%
annually as effluents
over the past few decadesfromhas industries
been a (Saffi, 1981).
huge challenge erythrocytes in the gill and kidney
of these pollutants (Ocean Conservancy, 2015). In erythrocytes
Mercuric
in terms of chloride has been
controlling used in agriculture
environmental pollution.asThe a exposed
recent years, to the
thermal power
emergence of aplant discharge
form plastic at
has been
fungicide, in medicine as a topical
increase in the number of plastics amassed in the antiseptic and Titagarh Thermal Power Plant, Kolkata, India.
studied for its role in pollution - microplastics. Although
disinfectant,
environment and in chemistry
is mainly causedasby an their
intermediate
inert prop- in thereThe haspresent
been no study was conducted
universally accepted to definition
determineat
the
ertyproduction of other mercury
(slow degradation rate) andcompounds.
usage (improper The the
the acute
moment genotoxicity of the heavy
(Van Cauwenberghe et al.,metal compound
2015), individual
contamination of aquatic ecosystems
disposal of plastic waste) (Nor and Obbard, 2014). by heavy HgCl
and group
2
in static systems. Mercuric chloride
researches have contributed their is toxic,
own
metals
Despiteand pesticides
their benefits,hasthe gained increasing
contribution of attention
plastic to solvable in water hence it can penetrate
description of microplastics. In 2004, microplastic the aquatic
in recent decades.
environmental Chronic
degradation has exposure to andin
been significant animals.
size wasMutagenic
defined at studies
aroundwith native fish
20 microns species
(Thompson
accumulation of these chemicals in aquatic
causing damage to marine organisms. Previous studies biota represent
et al., 2004).anInimportant effort
2009, the size in determining
adapted was <5 mm. the This
can result in tissue burdens that produce
have shown that plastics (i.e. microplastics) are ingestedadverse potential effects of toxic agents.
characterization of microplastic size was also This study was
accepted
effects not only
at different trophicin the directly
levels whichexposed organisms,
can potentially cause carried out to
by the Joint evaluate
Group the use
of Experts onofthethe micronucleus
Scientific Aspects
but also in human beings.
bioaccumulation. This phenomenon can affect test (MN) for the estimation of aquatic
of Marine Environmental Protection (GESAMP) (Nor pollution
Fish provides
biological processesa of suitable
marinemodel for monitoring
organisms. In addition, using marine 2014).
and Obbard, edible Other
fish under lab conditions.
researchers have presented
aquatic
food safety and human health are also at risk,quality
genotoxicity and wastewater if these microplastics as particles with <1 mm size which
because of its ability to metabolize
affected organisms are consumed (Vandermeersch xenobiotics and 2. Materials
is argued to beand
moremethods
instinctive since micro refers
accumulated
et al., 2015). pollutants. A micronucleus assay has to the micrometer range (Van Cauwenberghe et al.,
been
used successfully in several species (De
It has been estimated that up to about 12.7 million Flora, 2.1.
2015).Sample Collection
As a marine pollutant, microplastics exist in two
et al.,of1993,
tons plasticAl-Sabti
still ended andup Metcalfe, 1995).
in the ocean in The
2010 forms - primary and secondary. Primary microplas-
micronucleus (MN) test has been
(Jambeck et al., 2015; Andrady and Neal, 2009; Van developed tics,The fish species
directly move intoselected
water for the through
bodies present study
ground
together
Cauwenberghe withet DNA-unwinding
al., 2015). The Philippines assays was as was collected from Pudhumadam coast
runoff. These are mainly composed of virgin of Gulf of
plastic
perspective methods for mass
ranked No. 3 by mass of mismanaged plastic wastemonitoring of Mannar, Southeast Coast of India. Therapon
pellets, scrubbers, and microbeads. These plastic forms
clastogenicity
following China and and
genotoxicity
Indonesia. in fish and mussels
In 2010, plastic jarbua belongs
are abundant to the order
in cosmetic Perciformes
products of theas
like exfoliants
(Dailianis et al., 2003).
marine debris in the Philippines ranged from 0.28-0.75 family Theraponidae. The fish species, Therapon
well as industrial abrasives (Andrady, 2011). On the
The MN tests have been successfully used as jarbua (6-6.3 cm in length and 4-4.25 g in weight)
a measure of genotoxic stress in fish, under both was selected for the detection of genotoxic effect
C. Ryan Argamino et al. / EnvironmentAsia 9(2) (2016) 48-54
other hand, secondary microplastics are degradation habitat includes hard surfaces (rocks, pilings and
products of larger plastics (mesoplastic/macroplastic) floating docks, as well as sandy or muddy bottoms about
which are already in the ocean or seashore. Microplastics a foot below the low tide mark) (McGuire and Stevely,
in this form are produced via several pathways such 2009). Their diet includes microscopic phytoplankton,
as mechanical, photo (oxidative) and/ or biological zooplankton and suspended organic detritus in the water
degradation (Masura et al., 2015). Majority of (Rajagopal et al., 2006). In the Philippines, P. viridis
microplastics in the marine environment are or tahong is considered as one of the seven major
secondary microplastics formed through the weathering aquaculture species (Food and Agricultural
of plastic in the seashore. Plastics on beaches have a Organization, 2006). Aquaculture of tahong can
faster degradation rate due to the higher temperature be traced back to 1955 which was initiated by the
(of sand) as compared to both plastics deep in the Philippine Bureau of Fisheries and Aquatic
ocean or floating on the water surface (Andrady, 2011). Resources in Binakayan, Cavite (Yap, 1999). Al-
Mussels, locally known as tahong, are generally though it is a source of food locally, discretion is
defined as mollusk bivalves which grow in the wild advised for consumption due to their tendency for
or through aquaculture. These suspension feeders are bioaccumulation of toxins substances detrimental to
considered to be one of the best biological indicators human health (McGuire and Stevely, 2009). Thus,
of marine pollution because of various characteristics determining the presence of microplastics in these
including their geographical distribution. Their tissues marine species will not only be instrumental to
accumulate pollutants due to their feeding mechanism profiling marine pollution but will also serve as a tool
and their tendency to stay attached to surfaces which in improving guidelines for food safety.
make them relatively inactive in terms of mobility In this study, we present our findings regarding the
thus rendering them as good pollution gauges. They presence of microplastics in cultured bivalves available
can also be easily sampled and thus, allow for frequent for human consumption. These particles were positively
experiments and monitoring (Vasanthi et al., 2012; identified through microscopic analysis and other
Figueiras et al., 2002; Chase et al., 2001). The species qualitative criteria. In addition, management procedures
of mussel used in this experiment is Perna viridis according to best practices were also discussed which
(P.vidiridis). P. viridis is a large species of mussel will aid policy makers in reducing the risk posed by
ranging from 8-16 cm (Rajagopal et al., 2006). Their these pollutants to the environment and human health.
Bacoor Bay-cultured samples (P.viridis) were Previous studies confirmed the presence of
obtained from Sineguelasan Seafood Terminal in microplastics from marine samples using different
Bacoor, Cavite, Philippines. The mussel samples were methods. Raman Spectroscopy (Van Cauwenberghe
harvested on January 2016. Fig. 1 shows a map of and Janssen, 2014), Fourier Transform Infrared
Metro Manila and its adjacent provinces. The drop pin Spectroscopy coupled with either a Microscope or
marks the location of Bacoor Bay where the samples an Attenuated Total Reflectance attachment (Nor and
were cultured and harvested. All glassware were Obbard, 2014), as well as the Fluorescence Microscope
thoroughly cleaned and rinsed with filtered deionized (Noren, 2007) were utilized by various studies. The
water (Elga PURELAB Flex) to avoid contamination absence of these instruments should not be a hindrance
dilution (Van Cauwenberghe and Janssen, 2014). in the analysis of this important class of pollutants.
Wet digestion using acid was used to extract Thus, the following criteria were used in determining
the microplastics for the samples (Vandermeersch the presence of microplastics (Noren, 2007):
et al., 2015). Twenty milliliters (20 mL) of 70% HNO3 a) No cellular or organic structures are visible in
(UNIVAR) were added to three mussels in an the plastic particle/fibre.
Erlenmeyer flask. Five replicates were prepared. b) Clear and homogeneously colored particles
Mussel tissues in acid were left in in the hood for 40 (blue, red, black and yellow)
hours to achieve optimum digestion. The samples were c) If the particle transparent or whitish, it shall be
then heated until boiling for 15-20 minutes using a hot examined with extra care in a microscope under high
plate to evaporate the acid and dry the sample. Twenty magnification.
milliliters of warm deionized water (~80 degrees Figs. 2 and 3 show examples of microplastics
Celsius) were added to each vessel for dilution (Van observed in each of the five samples.
Cauwenberghe and Janssen, 2014). Digested samples Microplastics observed in the samples were all
were then subjected to vacuum filtration using a found to be < 1mm. Transparent whitish and reddish
Buchner funnel and Whatman Filter Paper Grade 1 particles measuring at around 10 to 30 microns (0.1
(11-micron pore size). Filters were dried for 2 hours to 0.3 mm) were observed in Samples 1, 2, and 5.
in an oven at 40 degrees Celsius. Dried filters were On the other hand, blue fibers (~0.5 mm in length)
analyzed for the presence of microplastics using a Nikon were observed in Sample 3 and 4. These observations
SMZ 745T Stereomicroscope (Van Cauwenberghe and qualify in the abovementioned criteria (Noren, 2007)
Janssen, 2014; Song et al., 2015).
Figure 5. Comprehen
nsive waste generation
g maanagement plan
p
Figure 5. Comprehen
nsive waste generation
g maanagement plan
p
Another dim mension of the propossed management schem me is obtainning feedbaack f
Another stakehol
dim
mension
lders ofon the
theiirpropossed nmanage
perception of thement mebyismicroplastic
riskks schem
posed obtainningc feedbaack from
contaminat ion (Risk Peercep
stakehollders on theiir perception n
and Mannagement) (F of the risk
ks 51
posed by microplastic c contaminat ion (Risk
Fig. 6). It sttarts with proomoting awaareness on thhe impactsPeerception
oof these polllutant
and Mannagement)humans
(F
Fig. 6).
andIt the
sttarts with proomoting
envvironment aliike. awaareness
Educatin on thhe impacts
ng stakeholdders andoofmak
these
kingpoll
lutants
them to
unnderstand thee haz
of comm municating thet understo
risks ood
invvolved
by theis to convert com
stakeholders s. mplex terms
Developin ng ato manage
a languaage that
ement plancanforbethis
fully
soccial, behavio
oral
understoood by the stakeholders s. Developin ng a manage ement
psycholoogical aspecct of microplastic mannagement invplan for this soccial, behavio oral,
volves surveys, psycho andological studdies
psycholoogical aspecct acknowl
of micro plasticdem
ledging man nagement
ographic inv
volves Th
diffferences. surv
heseeys, psycho
include r ologicalcul
regional, studdies,econom
ltural, and mic, educatioona
acknowlledging demographic diffferences. Thhese include regional,
r cul
ltural,
other vaariances in beehaviour andd perceptions (GESAMP, 2015). econom mic, educatio onal, and
other vaariances in beehaviour anddArgamino
C. Ryan perceptions
et al. /(GESAMP,
EnvironmentAsia2015).
9(2) (2016) 48-54
sed
cts ofor
o non-
ngthen
hysical
gation
s from
ntities
restrial
ational
s,ogram
and
ntation
nicipal
being
mental
Figure 7. Microplastic Management
t Framework
Figure 7. Microplastic Managementt Framework
4. Conclusions 4. Conclusions
ects of
ngthen
igation 53
entities
ational
C. Ryan Argamino et al. / EnvironmentAsia 9(2) (2016) 48-54
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