Int. J. Aquat. Biol. (2018) 6(3): 157-161 DOI:
ISSN: 2322-5270; P-ISSN: 2383-0956
Journal homepage: www.ij-aquaticbiology.com
© 2018 Iranian Society of Ichthyology
Original Article
Determination of mancozeb toxicity and biochemical effects in common carp (Cyprinus carpio)
Pooria Simakani1, Mohammad Hadi Abolhasani*2,1Seyyed Morteza Hoseini3
1Department of Environmental Science, Islamic Azad University, Isfahan (Khorasgan) Branch, Isfahan, Iran.
2Waste and Wastewater Research Center, Department of Environmental Science, Islamic Azad University, Isfahan (Khorasgan) Branch, Isfahan, Iran.
3Inland Waters Aquatics Resources Research Center, Iranian Fisheries Sciences Research Institute, Agricultural Research, Education and Extension Organization,
Gorgan, Iran.
Article history:
Received 20 April 2018
Accepted 22 June 2018
Available online 2 5 June 2018
Keywords:
Carp
Mancozeb
Blood
Toxicity
Pesticide
Abstract: The aim of this study was to investigate mancozeb toxicity and its effects on physiological
characteristics of common carp. Fish were reared for one week to acclimatize with the experimental
conditions. For 96h-LC50 determination, the fish were stocked in 16 aquaria at the density of 10 fish
per aquarium. The aquaria were exposed to 8 mancozeb concentrations (two aquaria per
concentration) for 96 h (0, 0.94, 1.87, 3.75, 7.50, 15, 30 and 60 mg/L). 96h-LC50 was calculated
based on the fish mortality, being 8.03 (4.95-13.2) mg/L. For sub-acute test, the fish were exposed
to 0 (control), 1.6, 2.4 and 3.2 mg/L mancozeb (20, 30 and 40% of the 96h-LC50) for one week.
Blood samples were taken from each treatment for determination of plasma glucose, total protein,
albumin, globulin, calcium, alanine aminotransferase (ALT) and aspartate aminotransferase (AST).
Mancozeb exposure led to increase in glucose and AST, and decrease in plasma proteins and ALT.
In conclusion, mancozeb exposure causes stress response, health problem and tissue damage in
common carp.
Introduction
Now a day, input and distribution of pollutants to
ecosystems and their effects are of the main
environmental concerns. Industrial developments and
population growth led to chemical pollutant
accumulation in aquatic ecosystems (Saghali et al.,
2014). The pollutants behaviors may be assessed at
three levels: water column, sediments and biomass of
aquatic organisms (Saghali et al., 2014). One of the
serious threats for human is pollutant entry to waters,
leading to accumulation in aquatic organisms’ body
and moving towards higher levels of food chain (Shaw
and Handy, 2011). Agricultural pesticides are
considered as one of the largest group of
environmental pollutant, which are extensively
studied in aquatic toxicology (Wang et al., 2015).
The effects of the pesticides on fishes are of
important environmental concerns. Mancozeb is a
carbamate fungicide that is used for control of
cucumber mildew. This pesticide has short half-life
but may cause neural and humoral issues in human
*Corresponding author: Mohammad Hadi Abolhasani DOI: https://doi.org/10.22034/ijab.v6i3.494
E-mail address: hadi.mha2001@yahoo.com
(Wang et al., 2014). Today, human consumes aquatic
organisms, as the healthiest sources of food and
protein. Fish are one of the most important aquatic
animals due to high economic value and susceptibility
to pollutants, thus, are used for variety of biological
studies (Hedayati et al., 2016). Different fish vary in
susceptibility to pollutants, thus, it is necessary to
conduct toxicological studies on different fish species
(Mazandarani and Hoseini, 2017). Mancozeb toxicity
has been rarely studied in fish. 96h-LC50 of the
pesticide has been 11.68 mg/L in Oreochromis
mossambicus, which caused behavioral changes in the
fish (Saha et al., 2016). Mancozeb was found to induce
oxidative stress in goldfish (Carassius auratus)
characterized by increased activities of antioxidant
enzymes, and levels of protein carbonyle and lipid
peroxides (Kubrak et al., 2012). Moreover, Bisson and
Hontela (2002) found that mancozeb exposure
interfere with cortisol secretion in response to stress.
However, there are no data about mancozeb lethal
concentration and biochemical effects in common
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Simakani et al. / Determination of mancozeb toxicity and biochemical effects in common carp
carp (Cyprinus carpio). Common carp is an
economically important species and is reared in many
parts of Iran (Hosseini and Hoseini, 2012). It is a
popular fish in Iran north and its population in the
Caspian Sea is supported by stock rehabilitation
activities of Iranian Fisheries Organization, because of
declining the natural population in the sea. As
mancozeb is used in agricultural fields of Caspian
region, it might enter surface water reaching the
Caspian Sea and threatening the organisms. Thus, the
aim of this study was to assess acute toxicity and
biochemical effects of mancozeb on common carp.
Materials and Methods
Fish maintenance conditions: A total number of 280
common carp fingerlings (30 g) were purchased and
transported to laboratory and allowed to acclimatize
under the experimental conditions for one week.
During the acclimation period, the fish were fed twice
a day (1% of biomass) and uneaten feeds were
removed from the aquaria to avoid water pollution.
The fish were randomly distributed into 16 aquaria at
the density of 10 fish per aquarium. The aquaria water
was renewed by 50% per day.
LC50 determination: The fish were exposed to 0, 0.94,
1.87, 3.75, 7.50, 15, 30 and 60 mg mancozeb per liter
(as commercial product with 80% purity) and their
mortality was recorded over a 96-h period. LC50 was
determined according to Hoseini and Nodeh (2011)
using Probit analysis. Two aquaria were assigned for
each concentration and 10 fish were stocked in each
aquarium. The fish were not fed during the
experiment. The aquaria water were daily renewed by
50% and desired amount of mancozeb were added to
the aquaria to maintain the pesticide concentration at
constant levels.
Sub-acute experiment: According to lethal
concentrations of mancozeb, three concentrations of
1.6, 2.4 and 3.2 mg/L (20, 30 and 40% of the 96h-
LC50) along with a control group (totally four
treatments with three replicates). Twelve aquaria
(each containing 10 fish) were used for this
experiment and the fish were allowed to acclimatize
with the experimental conditions for one week. The
fish were fed based on 1 % of biomass and the aquaria
water was daily renewed at 50%. Water dissolved
oxygen, pH, temperature, hardness, nitrite and
ammonia were determined during the experiment
(Hoseini and Nodeh, 2011; Hoseini et al., 2012;
Hoseini and Jafar Nodeh, 2012). The fish were bled
after one-week exposure to mancozeb.
Sampling and blood processing: Six fish were
sampled form each treatment. The fish were
immediately anesthetized in clove oil (100 mg/L) and
bled using heparinized syringe. The blood samples
were centrifuged for plasma separation. The obtained
plasma samples were kept at -20°C until (Taheri
Mirghaed et al., 2017; Taheri Mirghaed et al., 2018).
Plasma glucose (GOD method), total protein
(Biuret method), albumin (Bromocresol green
method), calcium (Cresolphthalein complexone
method), alanine aminotransferase (ALT; conversion
of alanine to lactate) and aspartate aminotransferase
(AST; conversion of aspartate to malate) were
determined using commercial kits (Pars Azmun).
Statistical analyses: Mancozeb LC50 was determined
by Probit regression using Probit Analysis Program V.
1.5. Plasma data normality was checked and then the
data were analyzed by one-way ANOVA and Duncan
test in SAS 9.4 software. P<0.05 was considered as
significance level.
Results
Water quality data are presented in Table 1 and were
within acceptable range.
Fish mortality and mancozeb LC50: The fish mortality
after 24-96 h exposure to different mancozeb
concentrations are presented in Table 2. The results
showed that increase in the pesticide concentrations
and/or exposure time led to increased mortality. Probit
analysis showed that mancozeb LC50 for common
carp was 8.03 (4.95-13.2) mg/L.
Behavioral and morphological alterations: The results
showed that the fish behaviors and morphology
changed markedly. At the early exposure, the fish
were anxious with fast swimming followed by
anorexia. Surface swimming, irregular and imbalance
swimming were observed in the exposed fish.
159
Int. J. Aquat. Biol. (2018) 6(3): 157-161
Morphological changes were mucus hypersecretion,
body and gill discoloration, scale loss and hemorrhage
on body and around operculum.
Biochemical characteristics of fish exposed to
mancozeb: Table 3 shows biochemical data of the fish
exposed to different concentrations of mancozeb.
Mancozeb concentrations had significant effects on
plasma glucose and the highest glucose levels were
observed ta the concentrations of 3.2 mg/L (P<0.05).
Mancozeb concentrations significantly affected
plasma total protein and globulin levels and the
highest and lowest levels were observed in the control
and 3.2 mg/L mancozeb groups, respectively
(P<0.05). Mancozeb had significant effects on plasma
albumin levels and 3.2 mg/L mancozeb led to
significant decrease in plasma albumin compared to
the other treatments. There was no significant
difference in plasma calcium levels among the
treatments. The highest and lowest plasma albumin
levels were observed in the control and 3.2 mg/L
mancozeb treatments, respectively (P<0.05).
Mancozeb exposure significantly affected ALT
activities and the highest and lowest activities were
observed in the control and 3.2 mg/L mancozeb
treatments, respectively (P<0.05). The lowest plasma
AST were observed in the control group that was
significantly different compared to the other groups.
1.6 and 2.4 mg/L mancozeb groups had similar
enzyme activities and significantly lower than 3.2
mg/L mancozeb group (P<0.05).
Discussion
In aquatic toxicology, LC50 lower than 1000 ppb says
“very toxic” substance, between 1000 and 10000 ppb
says “moderately toxic” substance, and higher than
10000 ppb says “less toxic” substance. Accordingly,
mancozeb is moderately toxic for common carp
juveniles. The resulted LC50 in the present study was
slightly lower than that reported in O. mossambicus
(Saha et al., 2016). Fish behaviors under toxicant
exposure are ideal tools to assess the effects of aquatic
pollutants on fish, because behavioral indicators reflex
the physiological states. Toxicant exposure may
induce a complete behavioral change that affects fish
survival in natural ecosystems, particularly in toxicant
concentrations lower than lethal concentration.
Table 1. Water physicochemical parameters during the experiment.
Parameters Hardness (mg/L
CaCo3)
Temperature
(°C)
pH Dissolved oxygen
(mg/L)
Nitrite
(mg/L)
Ammonia
(mg/L)
Content 318 ± 21.12 18.25 ± 0.21 7.3 ± 0.35 7.3 ± 0.4 1.1 ± 0.12 0.67 ± 0.1
Table 2. The fish mortality after 24-96 h exposure to different mancozeb concentrations.
Concentration (mg/L) Number of fish 24h 48h 72h 96h
0.96 10 0 0 0 0
1.87 10 0 0 1 2
3.75 10 0 0 1 4
7.5 10 0 0 1 5
15 10 0 1 3 7
30 10 0 1 4 8
60 10 0 2 5 10
Table 3. Plasma biochemical characteristics of common carp exposed to different concentrations of mancozeb for one week (Different letters show
significant difference).
Parameters Control 1.6 mg/L 2.4 mg/L 3.2 mg/L
Glucose (mg /dL) 67.3±5.43c 117±21.07b 140.45±23.12ab 153.44±12.34a
Total protein (g /dL) 4.33±0.21a 3.56±0.43b 2.43±0.44b 2.23±0.47b
Albumin (g /dL) 1.50±0.07a 1.45±0.06a 1.40±0.02a 1.32±0.03b
Globulin (g/dL) 2.80±0.11a 2.11±0.12b 1.02±0.08c 0.91±0.05c
Calcium (mg /dL) 7.32±0.5a 6.56±0.54a 6.95±0.32a 6.55±0.4a
ALT (U/L) 73.31±9.27a 66.23±11.26b 57.57±3.46c 54.13±3.21c
AST (U/L) 23.11±3.66c 45.01±6.17b 46.69±5.83b 52.12±12.03a
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Simakani et al. / Determination of mancozeb toxicity and biochemical effects in common carp
Behavioral changes are mainly related to
cholinesterase inhibition, alternation of brain
neurotransmitter levels, sensory deficiency, and
impaired gonadal or thyroid hormone levels (Taheri
Mirghaed and Ghelichpour, 2015). Scott and Sloman
(2004) monitored the relation between behavioral and
physiological indicators of toxicity in fish. In addition,
a positive correlation was observed between brain
acetyl cholinesterase activity and swimming speed of
Gambusia affinis after lethal exposure to
monocrotophos (Kavitha and Rao, 2007).
Plasma glucose increased in the mancozeb-treated
fish suggesting stress induction due to the toxicant
exposure. Under stressful conditions and toxicant
exposure, fish need more energy supply to cope with
toxicant-induced adverse effects; thus, circulating
levels of glucose increases (Hoseini et al., 2018).
Previous studies on common carp have shown that
exposure to toxicants led to hyperglycemia in the fish
(Hoseini and Tarkhani, 2013; Hoseini et al., 2014;
Hoseini et al., 2016a).
Plasma proteins are suitable indicators of fish
health. They are responsible for many vital functions
in body including immune response and antioxidant
system (Silva et al., 2010). Decreased total protein,
albumin and globulin may suggest liver damage as
most of the proteins, including albumin, are
synthesized in fish liver (Hoseini and Tarkhani, 2013).
The present results are in agreement with those
reported previously (Shariff et al., 2001; Hoseini and
Tarkhani, 2013; Hoseini et al., 2016a).
ALT and AST are non-functional enzymes in
circulation, but indicators of tissue damage. They are
found at high concentrations in liver and kidney, thus
damages to this organs lead to increased enzymes’
activity in blood (Haschek et al., 2009). Accordingly,
the present results show that mancozeb had
detrimental effects on carp liver and kidney. Similar
results was found in common carp exposed to other
toxicants (Hoseini et al., 2016b; Hoseini et al., 2016a;
Ghelichpour et al., 2017; Hoseini et al., 2018).
In conclusion, mancozeb exposure causes stress
response, health problem and tissue damage in
common carp. Such adverse effects may decrease the
fish well-being and survivorship in natural
environment.
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Int. J. Aquat. Biol. (2018) 6(3): 157-161
E-ISSN: 2322-5270; P-ISSN: 2383-0956
Journal homepage: www.ij-aquaticbiology.com
© 2018 Iranian Society of Ichthyology
چکیده فارسی
(Cyprinus carpio) معمولی کپور ماهی در مانکوزب بیوشیمیایی آثار و مسمومیت تعیین
3حسینی مرتضی سید، 2*ابوالحسنی هادی محمد ،1پوریا سیمکانی
.ایران، اصفهان ،(خوراسگان) اصفهان واحد اسالمی آزاد دانشگاه زیست، محیط علوم گروه1
.ایران ،اصفهان ،(خوراسگان) اصفهان واحد اسالمی آزاد دانشگاه زیست،محیط علوم گروه پسآب، و پسماند تحقیقات مرکز2
.ایران ،ترویج کشاورزی، گرگانتو تحقیقات علوم شیالتی ایران، سازمان تحقیقات، آموزش و یهای داخلی، انستمرکز تحقیقات ذخایر آبزیان آب3
چکیده:
مدت یک هفته با ها بهبود. ماهی ماهی کپور معمولیهای فیزیولوژیکی در هدف از این تحقیق بررسی مسمومیت مانکوزب و اثرات آن بر شاخص
سازی شدند. ماهی در هر آکواریوم ذخیره 10آکواریوم با تراکم 16ساعته، ماهی ها در LC50 96شرایط آزمایشگاهی سازگار شدند. جهت تعیین
گرم بر لیتر( قرار گرفتند میلی 60و 30، 18، 50/7، 75/3، 87/1، 94/0، 0غلظت مختلف مانکوزب ) 8ساعت در معرض 96مدت ها بهسپس آکواریوم
گرم بر لیتر بود. برای انجام ( میلی95/4-2/13) 03/8ساعت محاسبه شد و برابر با 96بر اساس تلفات در خالل LC50آکواریوم برای هر غلظت(. 2)
درصد 40و 30، 20گرم بر لیتر مانکوزب قرار گرفتند )میلی 2/3و 4/2، 6/1)شاهد(، 0مدت یک هفته در معرض ها بهآزمایش تحت حاد، ماهی
LC50 ،گلبولین، کلسیم، آالنین آمینوترنسفراز و آسپارتات (. نمونه خون از همه تیمارها برای تعیین مقدار گلوکز، توتال پروتئین، آلبومین
ز در پالسما ها و آالنین آمینوترنسفراآمینوترنسفراز در پالسما گرفته شد. مانکوزب باعث افزایش گلوکز و آسپارتات آمینوترنسفراز و کاهش پروتئین
شود. آسیب بافتی در ماهی کپور معمولی می شود که مسمومیت با مانکوزب باعث بروز استرس، افت سالمت وگیری میشد. نتیجه
.کش آفت مسمومیت، خون، مانکوزب، کپور، :کلمات کلیدی