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International Journal of Indigenous Medicinal Plants, ISSN: 2051-4263, Vol.46, Issue.3 1301
© RECENT SCIENCE PUBLICATIONS ARCHIVES| August 2013|$25.00 | 27702685 |
*This article is authorized for use only by Recent Science Journal Authors, Subscribers and Partnering Institutions*
Toxicity and Effect of Carica Papaya Seed Aqueous
Extract on Liver Biomarkers of Clarias Gariepinus
Joseph E Eyo
Department of Zoology and Environmental Biology, Faculty of Biological Sciences, University of Nigeria, Nsukka,
Enugu State, Nigeria Email: joseph.eyo@unn.edu.ng
Chidinma A Levi
Department of Zoology and Environmental Biology, Faculty of Biological Sciences, University of Nigeria, Nsukka,
Enugu State, Nigeria Email: chidinma.levi@unn.edu.ng
Chinweike N Asogwa
Department of Zoology and Environmental Biology, Faculty of Biological Sciences, University of Nigeria, Nsukka,
Enugu State, Nigeria Email: normi4u@yahoo.com chinweike.asogwa@unn.edu.ng
Elijah C Odii
Department of Zoology and Environmental Biology, Faculty of Biological Sciences, University of Nigeria, Nsukka,
Enugu State, Nigeria Email: elijahrelateswithjesus@yahoo.com
Christian O Chukwuka
Department of Zoology and Environmental Biology, Faculty of Biological Sciences, University of Nigeria, Nsukka,
Enugu State, Nigeria Email: onyichrismac@yahoo.com
Njoku Ivoke
Department of Zoology and Environmental Biology, Faculty of Biological Sciences, University of Nigeria, Nsukka,
Enugu State, Nigeria Email: njoku.ivoke@unn.edu.ng
Uwakwe S Onoja
Department of Home Sciences and Nutrition, Faculty of Agriculture, University of Nigeria, Nsukka, Enugu State,
Nigeria. Email: uwakwe.onoja@unn.edu.ng
Christopher C Onyeke
Department of Plant Science and Biotechnology, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu
State, Nigeria. Email: christopher.onyeke@unn.edu.ng
ABSTRACT
The mesocarp of pawpaw fruits (Carica papaya) is a
delicacy in the tropics. However, the seeds contain toxic
substances such as carpine, papain, etc. The first phase of
the present study determines the acute toxicity of C
.papaya seed extract to Clarias gariepinus juveniles using
static bioassay. Sixty juveniles with mean weight 3.86 ±
1.18g and mean length 9.5 ± 1.52cm were exposed to
triplicate concentrations of 0, 150, 225 and 300mg/l in
twelve 15L plastic tanks, with each replicate having 5 fish.
There were 93.33, 66.67, 46.67 and 0% cumulative
mortalities corresponding to 300, 225, 150 and 0 mg/l of
C. papaya seed aqueous extract within the of hours of
exposure. The 24, 48, 72 and 96 h LC50 values were
1200.78, 426.67, 191.76 and 163.02 mg/l of C. papaya
seed aqueous extract, respectively. These showed that the
effects were dose and time dependent. The second phase
determined the effect of sub-lethal concentrations of the
test substance (0, 50, 75 and 100mg/l) in a renewal
bioassay system on the liver biomarkers. Significant dose
and time dependent changes (p≤0.05) in acid phosphatase
(ACP), aspartate transferase (AST) and alanine transferase
(ALT) activities were noticed. The liver ACP levels were
significantly higher (p≤0.05) at 72 and 144h than at 0h.
Both the AST and ALT levels were significantly higher
(p≤0.05) at 72h when compared with 0h and 144h. The
behavioural responses by Clarias gariepinus in this test
were; erratic movement, air gulping, loss of reflex and
skin discoloration. The maximum admissible toxicant
concentrations ranged from 1.91 to 2.30 log toxicant
concentration (at 95% confidence limit). The results
obtained showed that concentrations of pawpaw seed
extract in excess of 163.02 mg/l can be potentially harmful
to Clarias gariepinus juveniles.
Keywords- Carica papaya, seed aqueous extract,
toxicity, mortality, liver enzymes, Clarias gariepinus
1. INTRODUCTION
Pawpaw (Carica papaya) is a common fruit available
throughout the year in the tropics. The fruits, leaves, seeds
and latex are used as a cure for many tropical diseases
hence the common name “medicine tree” or “melon of
health” [1,2]. The major active ingredients (carpine,
chymopapain, papain, bactericidal aglycone, benzyl
isothiocyanate, aglycoside, sinigrin, the enzyme myrosin
and carpasemine) are present in the seeds [1-3]. The fleshy
part of the fruits (mesocarp) is a delicacy and nutrient-rich
drinks of high demand are produced from them. However,
some of the active substances (e.g. carpine and papain)
from pawpaw are toxic [2]. Carpine are present in traces in
the black seeds of C. papaya. In large quantities, it is used
to lower the pulse rate and depress the nervous system.
International Journal of Indigenous Medicinal Plants, ISSN: 2051-4263, Vol.46, Issue.3 1302
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Externally, the latex is an irritant, dermatogenic and a
vescicant. Internally, it causes severe gastritis. Some
people are allergic to the pollen, the fruit and the latex.
Papain can induce asthma and rhinitis. The acid fresh latex
can cause severe conjunctivitis and vesication. Carpine
and papain also have anti-fertility properties and thus can
be used in birth control [4]. Clarias spp are mostly
freshwater fishes which are distributed throughout the
African and Asian lakes, swamps and rivers. Clarias,
however, can be obtained throughout the year in Nigerian
rivers and are anadromous. The fish is in high demand
because of it rich flesh and good taste. To meet the ever
increasing demand, C. gariepinus is the fish of choice in
Nigerian aquaculture. The aim of this study was to find out
the toxicity effects of C. papaya seed extract on C.
gariepinus by assessing its effect on mortality, liver
enzymes levels and behaviour of C. gariepinus.
2 MATERIALS AND METHODS
2.1 Experimental Fish
Ninety six Clarias gariepinus juveniles with mean length
11.00 ± 3.00 cm and mean weight 6.0 ± 1.5g used in this
study were obtained from Agricultural Holdings, Nsukka.
The fish were transported to the Fisheries and
Hydrobiology Wet Laboratory, Department of Zoology
and Environmental Biology, University of Nigeria,
Nsukka and acclimatized for two weeks. During the
acclimatization, catfish were fed regularly (twice a day)
with the Copens fish feed containing 35% crude protein.
The water was aerated continuously using aquarium
aerators.
2.2 Carica Papaya Seed Aqueous Extract
Twenty five (25) large ripe pawpaw fruits were harvested
from the Agriculture Farm, University of Nigeria,
Nsukka, Enugu State, Nigeria. The mature seeds were
recovered from the fruit, washed and sun dried to constant
weight. 500g of the dry seeds was ground into powder
using Corona grinder (China) and the resulting powder
was soaked in 10 litres of distilled water. The mixture was
allowed to stand for 24 hours with intermittent shaking.
The mixture was filtered using Whatman filter paper
(grade 1: 11 µm) and the filtrate dried into powder using a
rotary evaporator (Stuart, model RE-300, UK) and stored
in seal vials under refrigeration pending use. The aqueous
extract was serially diluted in distilled water to
appropriate concentration before administration.
2.3 Acute Toxicity Test
Sixty catfish juveniles with mean weight 3.86 ± 1.18g and
mean length 9.5 ± 1.52cm were used for this experiment.
The acute toxicity test was conducted to determine the
level of toxicity of pawpaw seed aqueous extract. C.
gariepinus juveniles were batch-weighed and distributed
randomly to twelve (15 litres) plastic tanks. Each container
was covered with nylon mesh tied firmly with rubber strap
to prevent the fish from jumping out. Each treatment
group were dosed with 0 (control), 150, 225 and 300 mg/l
of C. papaya seed aqueous extract [5] and replicated thrice
with each replicate containing five fish. The toxicity
testing was done using static bioassay whereby there was
no aeration, no water change nor feeding throughout the
test period [5]. The mortality and behavioural changes of
the catfish during the acute toxicity test was monitored for
four days and the 96 hour-LC50 determined graphically
using probit transformation. The inability of fish to
respond to external stimuli was used as an index of death.
The temperature, pH, DO and total hardness were 27.0 ±
2.6˚C, 7.50 ± 1.02mg/l, 6.7 ± 0.52mg/l and 110 ± 2.28mg/l
equivalent of CaCO3, respectively during the study.
2.4 Sub-Lethal Toxicity Test
Thirty six juveniles of 3.86 ± 1.18g and 9.5 ± 1.517cm
were used for this experiment. Each treatment group was
replicated thrice with each replicate containing three fish.
The pawpaw seed extract was administered at sub-lethal
concentrations (1/3 of the LC50) of 0 (control), 50, 75 and
100mg/l in a renewal bioassay system [6]. The water and
the tested compound were changed daily without reducing
or changing the toxicant concentration. The liver from
different treatments groups were assayed for enzyme
activities at 0, 72 and 144h. The fish were dissected and
their various livers collected. The liver enzymes studied
were acid phosphatase (ACP) [7], aspartate
aminotransferase (AST) and alanine aminotransferase
(ALT) [8]. Apart from monitoring and recording fish
mortality, behavioural and dermatological changes such
as: erratic swimming, air gulping, loss of reflex, skin
discoloration and haemorrhage were monitored.
2.5 Statistical Analysis
Mean values were analysed for significant differences
(p≤0.05) using the analysis of variance (ANOVA).
Differences between means were partitioned using the
Duncan new multiple range test. The statistical package
for social sciences (SPSS), version 17, was used for all
analysis. The probit value was determined from the probit
model developed by Finney [9].
3. RESULTS
3.1 Behavioural Responses
The catfish juveniles showed behavioural responses to C.
papaya seed aqueous extract. The behavioural responses
were both extract concentration and time dependent with
0mg/l inducing lesser behavioural responses than 300mg/l
for both acute and sub-lethal toxicity phases and 24h and
48h producing lesser behavioural changes than 96h and
168h for the acute and sub-lethal toxicity phases,
respectively. The observed behavioural responses were
loss of reflex, air gulping, erratic swimming, discoloration
of skin and haemorrhage (Table 1).
3.2 Mortality
Percentage mortality at 24 h increased with increase in
toxicant concentration. Catfish juveniles exposed to 150,
225 and 300mg/l had 6.67, 13.33 and 20.00% mortalities,
respectively (Table 2). The 24 h LC50 at 95% confidence
International Journal of Indigenous Medicinal Plants, ISSN: 2051-4263, Vol.46, Issue.3 1303
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limit was estimated as 1200.78mg/l (Fig. 1). The
percentage mortality at 48h increased with the toxicant
concentration. Catfish juveniles exposed to 150, 225 and
300mg/l had 13.33, 13.33 and 20.00% mortalities,
respectively (Table 2). The 48h LC50 at 95% confidence
limit for toxicant concentration was estimated as
426.67mg/l (Fig. 2). The percentage mortality at 72h
increased with the toxicant concentration. Catfish
juveniles exposed to 150, 225 and 300mg/l had 20.00,
26.67 and 33.33% mortalities, respectively (Table 2). The
72h LC50 at 95% confidence limit for toxicant
concentration was estimated as 191.76mg/l (Fig. 3).
Table 1
Behavioural and dermatological changes of Clarias gariepinus juveniles exposed to varied concentrations of Carica
papaya seed extract during acute and sub-lethal phases
ACUTE TEST
Exposure
time (hour)
24
48
72
96
Concentration
(mg/L)
0
150
225
300
0
150
225
300
0
150
225
300
0
150
225
300
Behavioural
changes
Loss of reflex
-
+
+
+
-
++
++
++
-
++
++
++
-
+++
+++
+++
Air gulping
-
-
+
+
-
++
++
++
-
++
++
++
-
+++
+++
+++
Erratic
swimming
-
-
-
+
-
-
+
+
-
+
+
++
-
++
++
+++
Dermatological changes
Discoloration
-
+
+
+
-
++
++
++
-
++
++
++
-
+++
+++
+++
Haemorrhage
-
-
-
-
-
-
-
-
+
+
+
-
++
++
++
Sub-lethal test
Exposure
time (hour)
48
96
144
168
Behavioural
changes
Loss of reflex
-
-
-
-
-
-
-
-
-
-
+
-
-
-
+
Air gulping
-
-
+
+
-
-
+
+
-
+
+
+
-
+
++
+++
Erratic
swimming
-
-
-
-
-
-
-
-
-
-
-
-
-
-
++
++
Dermatological changes
Discoloration
-
+
+
+
-
+
+
+
-
+
+
++
-
+
++
+++
Haemorrhage
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Where - = no significant sign, + = low severity, ++ = moderate severity and +++ = high severity.
Table 2
Percentage mortality of Clarias gariepinus juveniles exposed to varied concentrations of Carica papaya seed extract
during acute phases
TOXICANT
CONCENTRATION
(MG/L)
PERCENTAGE MORTALITY
24h
48h
72h
96h
Cumulative
mortality (%)
0
0.00
0.00
0.00
0.00
0.00
150
6.67
13.33
20.00
6.67
46.67
225
13.33
13.33
26.67
13.33
66.67
300
20.00
20.00
33.33
20.00
93.33
LC50 (mg/l)
1200.78
426.67
191.76
163.02
-
Log (concentration)
3.08
2.63
2.28
2.21
-
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Fig. 1 Probit transformed responses for 24h exposure of
Clarias gariepinus exposed to graded concentrations of
Carica papaya seed aqueous extract
Fig. 2 Probit transformed responses for 48h exposure of
Clarias gariepinus juveniles exposed to graded
concentrations of Carica papaya seed aqueous extract
Fig. 3 Probit transformed responses for 72h exposure of
Clarias gariepinus exposed to graded concentrations of
Carica papaya seed aqueous extract
The percentage mortality at 96h increased with the
toxicant concentration. Catfish juveniles exposed to 150,
225 and 300mg/l had 6.67, 13.33 and 20.00% mortalities,
respectively (Table 2). The LC50 at 95% confidence limit
was estimated as 163.02mg/l (Fig. 4). The models explain
the data were highly efficient at 97.6%.
3.3 Liver Enzymes
The ACP levels in the liver tissues were significant higher
(P≤0.05) at 72 and 144h when compared to 0h.
Fig. 4 Probit transformed responses for 96h exposure of
Clarias gariepinus juveniles exposed to graded
concentrations of Carica papaya seed aqueous extract
The AST activity in the liver of C. gariepinus juveniles
exposed to C. papaya seed extract increased from 0 to 72
hours was highest at 144h in 75 and 100mg/l C. papaya
seed extract treatments. For exposure period of 72h, AST
activity in the liver was highest for 50, 100 and 75mg/l C.
papaya seed extract treatments. The ALT enzyme activity
in the liver of C. gariepinus exposed to C. papaya seed
extract was dose and time dependent. The liver ALT
activity in all the treatments increased from 0 to 72 and
144h. Furthermore, the ALT activity increased
progressively with increase in toxicant concentration. The
mean values were statistically significant (p≤0.05) (Table
3).
The ACP enzyme activity in the liver of Clarias
gariepinus decreased gradually from with exposure time
except for the control. The mean values of ACP enzyme
activity for the duration of exposure (hours) were
statistically significant (p<0.05). Furthermore, the AST
enzyme activity increased sharply across the exposure
periods. Also, the mean value of AST enzyme activity at
0h was statistically significant to 72h and 144h (p≤0.05),
whereas, the mean value of AST enzyme activity at 72h
and 144h were not statistically significant (p>0.05).
However, the ALT enzyme activity in the liver increased
progressively with exposure time. Also, the mean values
of ALT enzyme activity is statistically significant to 72h
and 144h (p<0.06), whereas, the mean value at 72h and
144h of ALT enzyme activity are not statistically
significant (p≥0.05).
4. DISCUSSION
Bioassay of toxicant occupies a central place in aquatic
ecotoxicology. The aim of such test is to determine the
critical amount of toxicants or their mixtures that can be
tolerated by the aquatic organisms and to predict the
influence of the toxicant. The present study was not the
first toxicological test of pawpaw powder extracts to
aquatic animals. Data obtained from this study showed
that percentage mortality of C.gariepinus juveniles
International Journal of Indigenous Medicinal Plants, ISSN: 2051-4263, Vol.46, Issue.3 1305
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Table 3
Mean value of enzymes activity in liver of Clarias gariepinus exposed to Carica papaya seed aqueous extract
DOSAGE (MG/L)
EXPOSURE DURATION (HOURS)
0
72
144
Acid phosphatase (ACP)
0
2.142351 ± 0.27a*
1.236254 ± 0.508a*
1.44767 ± .034a*
50
3.40200 ± 0.34b**
3.13000 ± 0.577c**
1.24933 ± .092a*
75
4.51633 ± 0.86c**
1.07667 ± 0.033a*
1.28300 ± .044ab*
100
3.49067 ± 0.87b***
2.01967 ± 0.058b**
1.48467 ± .039ab*
Aspartate transferase (AST)
0
12.91 ± 1.00c*
12.95 ± 1.00a*
13.03 ± 1.73a*
50
10.00 ± 1.01b*
23.00 ± 0.00c***
17.00 ± 1.00b**
75
10.53 ± 0.57b*
19.00 ± 0.00b**
18.00 ± 1.00b**
100
9.70 ± 0.39a*
23.00 ± 0.00c**
36.00 ± 0.00c***
Alanine transferase (ALT)
0
5.72 ± 0.00a*
5.83 ± 1.33a*
5.72 ± 0.00a*
50
7.00 ± 0.31c**
5.33 ± 1.33a*
8.00 ± 2.31b***
75
6.33 ± 0.38b*
6.67 ± 1.33b*
10.67 ± 1.33c**
100
6.40 ± 0.60b*
10.67 ± 1.33c**
11.00 ± 1.00c**
Mean values having the same alphabets as superscripts along the column do not show significant difference (p≥0.05).
Mean values having the same asterisk as superscripts along the row do not show significant difference (p≥0.05).
increased with increase in concentration of C. papaya and
was dose dependent. The observed values for catfish
juveniles mortality was in agreement with those of
Ayotunde and Offem [5,6] for Nile tilapia. Acute toxicity
occurred at concentrations comparable to those of lead
[10], diazinon [11], phenol [12] and tetrachloromethane
[13] but lower than those of benzene [14], methanol [15]
and acetonitrile [16]. However, pawpaw powder was less
toxic than chlorine [17] and ammonia [18]. For such
comparison to be meaningful, species variability and
possible differences in water quality needs to be accounted
for. The latter is important, since hardness, alkalinity and
pH of the medium can all influence the species and the
extent of toxicity [19,20]. However, because the same
medium was used, changes in the effective toxicant
concentration due to possible interaction with the medium
were ruled out. In this experiment, the 96 h LC50 value of
aqueous extracts of pawpaw seed powder to C. gariepinus
juveniles was higher than the value obtained Nile tilapia
fingerlings exposed to similar concentrations of pawpaw
seed aqueous extract [5,6]. The difference in toxicity may
be species and size specific. In a similar experiment with
organochlorine substances, Albaiges et al. [21] revealed
that the levels of chemicals in the gonads and liver of fish
were similar in both adults and young fishes, which
indicated that the age and thus size of fish was a
significant factor in the accumulation of toxicants.
However, these results disagreed with the size-specific
sensitivity to acute chemical toxicity observed in some
aquatic animals with the smallest individuals showing the
highest sensitivity [22,23]. The size-specific and
interspecific difference in lethal level will allow the
effective usage of pawpaw seed as anti-fertility agent in
tilapia polyculture with catfish [5,6]. Clarias sp is
ecologically adapted to muddy environments in which
temporary changes in water chemistry are more rapid and
the contaminant concentration are usually higher. Such an
environmental stress may facilitate tolerance to increased
concentrations of contaminants [24]. This view was
supported by the observation, which revealed that 96 h
may not to be sufficient time to determine the asymptotic
LC50 for the pawpaw seed powder concentration to Clarias
fingerlings since mortality would have continued if
exposure time was extended. Three factors for the
selective toxicity of toxicants for various fish species such
as: different inhibition of acetyl cholinesterase, different
detoxification and absorption has been suggested [25]. The
above factors may probably be responsible for the
different reactions showed by catfish fingerlings in
response to varying concentration of aqueous extracts of
C. papaya seed. The reactions were more pronounced at
higher concentration due to increased inhibition of
acetylcholinesterase which eventually results in the death
of the fish [11,26-29]. In toxicological experiments, the
time of exposure has large effect on biological response.
The general rule of thumb is that the longer the exposure
time, the lesser the LC50 value and the greater the toxicity.
Results of this study showed similar pattern having lesser
96h LC50 than 48h LC50 and so on, with increasing ratios
of 24:48h, 24:72h and 24:96h LC50 as 2.81, 6.26 and 7.37
indicating delayed acute toxicity response. Dose-response
approach in estimating the lethal effects of toxicants on
organisms have been criticized for lacking real ecological
meaning [30,31]. Nonetheless, regulatory norms have been
built around LC50 values that can be compared across
toxicants and organisms [13]. Thus, LC50 values from
dose-response bioassays have become the starting points
for ecologically relevant studies of toxicant effects on
animal populations [32].
Liver ACP levels were significantly decreased in C.
papaya treated fish at various concentrations. The ACP is
an inducible enzyme because its activity goes up when
there is a toxic impact and the enzyme begins to
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counteract the toxic effect [33]. Subsequently, the enzyme
may begin to drop either as a result of having partly or
fully encountered the toxin or as a result of cell damage.
Alteration in the membrane permeability can have severe
consequences such as leakage of hydrolytic enzyme
including ACP, which would have detrimental effect on
the cell. However, if due to toxicity of a substance, there is
increased ACP activity, then it means that the substance
interacted with the lysosome and caused an increase in the
lysosomal activity in the liver [33].
Liver AST and ALT were significantly increased in C.
papaya treated fish, though not all exposure days were
statistically significant. This indicated that C. papaya
stimulates glutamate transaminase activity in the liver
which could be due to injury caused by C. papaya, which
may stimulate tissue repair through protein turn over and
increased respiration.
5. CONCLUSIONS
The pawpaw seed powder had a positive toxicity effect
correlating with exposure time from 24 to 96h on C.
gariepinus. From the toxicity tests pawpaw seed powder
concentration as low as 163.02mg/l can be potentially
hazardous to some freshwater fish. Therefore, acute
toxicity data of the present study provide baseline
information needed to develop models on the use of
pawpaw seed powder as piscidal agent.
ACKNOWLEDGEMENT
We are indebted to the Department of Zoology and
Environmental Biology, University of Nigeria, Nsukka for
providing laboratory space and facilities for the study. We
are also thankful to Mr. and Mrs. Levi Echefu who funded
the study. There is no conflict of interest among the
authors.
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