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Plant Archives Vol. 19, Supplement 2, 2019 pp. 721-724 e-ISSN:2581-6063 (online), ISSN:0972-5210
STUDY OF SOME MOLECULAR BIOMARKER (COMET ASSAY)
IN ONE SPECIES SNAIL UNDER EFFECT THE PESTICIDES
Shaimaa satae M. Ali, Lubna A.M.** and Ayad M.J. Al-Mamoori
*
Center for Environmental Research and Studies , University of Babylon, Iraq
*
**
College of science , University of Babylon, Iraq
Email : ayadmj77@gmail.com
Abstract
In this study, the molecular biomarker (comet assay) was used as a technique in order to study the effect of the pesticides on some aquatic
organism, since the exposure to the pesticides in the environment are very widely. (Cerithium tenellum) was selected as one species of snail
in al-Hilla river, collected from al-Hilla river and exposed to three concentration from insect pesticides (0.001 ppm, 0.015ppm, 0.02ppm).
After 24 hours, the observation was conduct for the selected concentration and it was found that the first concentration of the pesticide
caused slow movement in species but the second and three concentration caused death of the species. During the reading of the (DNA)
through technique (comet assay), depending on the comet length and tail moment, the first concentration the ratio damage of (DNA) was
(36), while the second concentration (47) and third concentration was (92), these results refer to the first concentration which has a slight
effect, while second and third concentration its effect was deadly. From the results, the main conclusion is that the concentrations (0.015
ppm, 0.02 ppm) highly recommend not to use in agriculture or aquatic environment, because of its effect on organism sand aquatic
environment specially if the exposure was daily to the pesticides.
Keywords : Molecular biomarker (comet assay), Cerithium tenellum
Introduction
There are many groups of organisms such as fish,
amphibians, invertebrates, plants or microorganisms in the
fresh water represent as a community in it and pesticides can
have harmful effects on these organisms. The effects caused
by the pesticide are dangerous on the organism and the
community described by interaction between all organism in
environment such as competition or predation and indirect
effects (Preston, 2002)
The comet assay has been used as an important tool for
monitoring genotoxicity in aquatic environments (Lemos et
al., 2005) there are large amounts of pesticides application
globally and given the fact that they are prepared to be harm
to all organism, there is a high probability for counteractive
environmental effects also on non-target pollutant (Oerke EC
et al, 2004)
Among the tests for genotoxicity, the micronucleus test
has been widely utilized in fish to determine exposure to
water pollutants, in environment as well as under
experimental laboratory conditions (Minissi et al., 1996).
Farmers are using a variety of pesticides indiscriminately and
a sharp increase was observed during the last decades
(Rahman, 2013). The application of pesticides may lead to
contamination of the aquatic environment through several
ways including: spray drift, runoff, and leaching (van den
Brink et al., 2009). A chemical and biological monitoring
will be accomplished to assess the exposure concentrations
and the ecological risks of these pesticide concentrations in
the field using (Bollmohr et al., 2009; Preston, 2002).
The aim of this study: To knowledge the harmful which it
happens the pesticide in aquatic organism, and therefore
aquatic environment.
Methods and Materials
1. Sampling Sites and Collection: Collected samples from
species snail: (Cerithium tenellum) from al hilla river and
exposure to three concentration from insect pesticides in
three baskets respectively (0.001 ppm, 0.015 ppm, 0.02
ppm).
Phylum : Mollusca,
Class : Gastropoda. (cuvier 1795),
Sup class : Gaenogastropoda.
Super family : cerithioidea,
Family : cerithiidae.
Genus : Cerithium.
species : C. tenellium (G.B. Souerbyll 1855)
Fig. 1: Snail (Cerithium tenellum)
2- Single Cell Gel Electrophsoresis:
By comet assay kit: OxiSelect
TM
Comet Assay Kilt (3-Well
Slides, 75 Tests)
CELL BIOLABS, INC. STA-351. Using this kit by alkalin
conditions order producer in the kit
Tissue Preparation: from body fluid (Mussel Hemolymph)
After ending all steps of kit, it possible determination
Tail length= length of tail measured by ocular micrometer
(µm)
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DNA Tail Moment (%) = the product of distance and
normalized intensity integrated over the tail length,
Ʃ
(Lx.
%DNAx)
Comet Assay Index = (width of head / length of tail)
Classes of damage = according to comet assay index :1.2 – 2
(Low damage), 2.1-3 (Medium damage), up to 3 (High
damage).
Fluorescence microscope determines the exact shape of the
glow in the DNA, and determination comet length and tail
moment according to comet assay program on computer.
3- Statistical A
nalysis
The significance of the differences was evaluated using the
oneway ANOVA by comparing comet %, tail length and tail
moment of samples.
Fig. 2: Three concentration of pesticides
Table 1: The criteria of DNA damage in the species (Cerithium tenellum) according to Comet Assay in the different
concentration.
Concentration (3) Concentration (2) Concentration (1) pesticides
350.8±105 Comet L. 2.70±121.8 Comet L. 1.20± 70.1 Comet L.
46.2±40 Tail L µm 15.5±25.5 Tail Lµm 36.9±
22 Tail L µm.
32.9±15.2 Mome nt%
Damage
92
36.8±17.4 Moment%
Damage
47
1.98±14.69 Moment %.
Damage
36
756±2 Comet L. 42.3± 2 comet L. 56.2±3 Comet L.
7.8±3 Tail Lµm 9 ±1 Tail Lµ m
30±
Tail Lµm.
1.2±2 Moment%
Control
7
0 Moment%
8
0
±
0 Moment%
Control
9
(Cerithium tenellum
Fig.3 : The damage of DNA in the comet assay in snail (Cerithium tenellium) in (1) concentration
Fig. 4 : The damage of DNA in the comet assay in snail (Cerithium tenellium) in (2) concentration
Study of some molecular biomarker (comet assay) in one species snail under effect the pesticides
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Fig. 5 : The damage of DNA in the comet assay in snail (Cerithium tenellum) in (3) concentration
Fig. 6 : The of DNA in the normal cell ( control ) by comet assay in snail (Cerithium tenellium)
Discussion
In recent years, there has been an increasing interest in
the effects of toxicants on aquatic organism due to the
importance of this organism in rivers, ponds and estuaries,
exposed to wastes of the productive activity. Chemical
contamination of water may affect molecular (genetic
alterations) that can be used as markers of DNA alterations in
environmental pollution. (Jha, 2008).
The snail one from species that lives in fresh water and
sensitivity to any change in characteristics of water, because
chemical and physical pollutant or any toxins for example
(pesticides), which can be genotoxicity (Anderson, 1989).
These results cnfirmed the pesticides the effect is
obvious, in first concentration was ratio damage of DNA
(36) : comet length (70.1), tail length (22.1µ m), but the snail
in this concentration stayed alive, only movement slow. This
refer to the dose of pesticides Non-lethal, the average dose
refers to the amount of pesticide to which an animal is
subjected (orally, dermally, or through inhalation). When the
dose is low, but Its toxicity is higher chemical may be more
harmful, and sometimes a large dose its toxicity is lower
chemical. Dosages can be weighted as the weight of toxicant
per unit (kilogram) of body weight (expressed as mg
pesticide/kg of body weight) or as the focus of toxicant in the
water or food supply (usually expressed as parts per million,
ppm or parts per billion, ppb) (Johnson et al., 1980). Many
pollutants present in the aquatic environment not only
endanger the survival and physiology of the organism’s
present.
It also induces genetic alterations which may lead to
mutations and cancer (Russo et al., 2004) Either in second
concentration: the ratio damage of DNA (47), comet length
(121.8), tail length (25.5µ m), and after period 24 hour, these
snail died. this refer to the dose of pesticides was lethal.
The third concentration was more harmful
concentrations because the ratio of damage very high of
DNA (92), comet length (105.1), tail length (40 µ m), and
after period 24 hour, these snails died. This refer to the dose
of pesticides was lethal. A Killer dose is the quantity of
pesticide required to cause death. Because not all organisms
of a species die at the same amount (some are more tolerant
than others), Perfect quantity a toxicity amount measurement,
called a Lethal Concentration 50 (LC
50
), is used. This is the
amount of a pesticide that kills 50% of a test community of
organism within a
set period, usually 24 to 96 hours.
Dosage can be measured as the weight of toxicant per
unit (kilogram) of body weight (expressed as mg pesticide/kg
of body weight) or as the concentration of toxicant in the
water or food supply (usually expressed as parts per million,
ppm or parts per billion, ppb) (Rosenberg, 1975).
All these results to Indicate that all pesticides are
chemicals contaminated with the aquatic environment, it has
the effect of either a simple or deadly poison to living
organisms. Where the effect reaches the level of deadly
genetic contamination on another organism in the aquatic
environmental.
The best concentration possible to use is less than
(0.001 ppm), that it no any effect on another organism.
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Study of some molecular biomarker (comet assay) in one species snail under effect the pesticides
