RJOAS, 12(96), December 2019

DOI 10.18551/rjoas.2019-12.10

THE EFFECT OF ORGANIC AND INORGANIC PHENOLIC COMPOUNDS

ON HEMOGLOBIN, HEMATOCRIT, AND CORTISOL HORMONE
OF AFRICAN CATFISH (CLARIAS GARIEPINUS)

Saputra Afandi1*, Andayani Sri2, Nusyam Happy2, Rukmono Djumbuh1,

Eny Maria Goreti1, Mulyono Mugi1, Budiani Sri1, Suharyadi1, Marlina Erni1,
Nurhudah M.1, Thaib Effi A.1, Panjaitan Amyda S.1, Rahardjo Sinung1,
Prabowo Guntur1, Ilham1, Hapsyari Fitriska1, Laishevtcev A.3,4
1
Department of Aquaculture, Jakarta Fisheries University, Indonesia
2
Faculty of Fisheries and Marine Science, University of Brawijaya, Malang, Indonesia
3
Federal Scientific Centre VIEV, Moscow, Russia
4
Laboratory of Biological Control and Antimicrobial Resistance,
Orel State University named after I.S. Turgenev, Orel City, Russia

*E-mail: afandi.saputra@yahoo.co.id

ABSTRACT
Phenolic are compounds found in the environment which can cause fish stressed or
xenobiotic. The limited information about toxicology and pathological consequences in the
fish exposed to phenolic compounds needs to be studied. In this research, the African catfish
(Clarias gariepinus) were injected a single dose of phenol (inorganic) and P.crocatum
Polyphenols (organic). The animals were exposed (75% of 96 hours LC50) with phenol
concentration (3 mg/kg) and P.crocatum Polyphenols (1.8 mg/kg). Furthermore, the
hematology was measured on 3 days after injection. The metabolic effects of hemoglobin
and hematocrit decreased significantly (P<0.05) while the cortisol hormone increased
(P<0.05) in a short-term after 3 days of exposure.

KEY WORDS
Phenolic, Clarias gariepinus, P. crocatum polyphenols, cortisol, hemoglobin, hematocrit.

Phenolic compounds are xenobiotic compounds that affect anemia and disrupt the
endocrine system and hormones (Writer et al., 2010; Hori et al., 2006; Awachie and
Ezenwaji, 1998). This compound is classified as toxic chronic and immunotoxic which usually
found in fish tissue (Taysse et al., 1995; Mukherjee et al., 1990). Phenol waste is
accumulated from human activities such as the waste from industry, pharmaceuticals, and
agriculture (Buikema et al., 1979).
The phenol residue comes from industrial and pharmaceutical waste as well as and
natural decomposition (Buikema et al., 1979). Red Betel (P. crocatum) is one of the most
aromatic plants in which the leaves contain phenolic compounds (flavonoids and tannin) and
have a high phenol content (Saputra et al., 2016). Phenolic compounds become a concern
specifically because of its toxic effect in the water environment. However, this compound is
difficult to detect due to the lack of taste and smell (Tilak et al., 2007). A cytotoxic activity of
red betel leaves on Artemia and African catfish was tested in-vivo based on the LC50 and
showed that these plants have a potential of cytotoxic compounds. (Nursyam et al., 2017 and
Emrizal et al., 2014)
The habitat of Clarias gariepinus is in freshwaters which have a risk to be exposed to
chemicals such as phenols (James et al., 2008). Nursyam et al., (2017) and Ibrahem (2012)
reported that in-vivo test with an injection of phenolic compounds in fish C. gariepinus shows
abnormal signs of glucose, BUN, and ALP in the blood and necrosis of liver and kidneys.
Even though there is some research on the in-vivo effect of phenolic compounds towards fish
blood parameters, there is no information about the residues and effects of organic phenolic
compounds from plants to the toxicity on catfish. Therefore, further research is needed to

77
 RJOAS, 12(96), December 2019

determine the in-vivo influence of phenolic compounds of Red Betel Leaf (Piper crocatum) on
Hemoglobin, Hematocrit, and Cortisol Hormones of African Catfish (Clarias gariepinus).

MATERIALS AND METHODS OF RESEARCH

Tested Animal. The African catfish (Clarias gariepinus) used were in the size of 12 to
15 grams. There was 20 fish/tub obtained from the Aquaculture Laboratory of Jakarta
Fisheries University. The commercial feed was given twice a day at a dose of 3%. Before the
experiment was carried out, the fish was adapted in a concrete tub for 1 month. The quality
of DO water conservancy was 3.99-6.53 mg/l with a pH of 7.0-7.6 at 28-30oC.
Isolation and characterization of Organic and Inorganic phenolic compounds. The
organic phenolic compounds used were obtained from the isolation of red betel leaf (P.
crocatum) using maceration, thin layer chromatography, column chromatography, and
characterized by using UV-Vis and Infrared spectrophotometers (Saputra et al. 2016;
Bigoniya and Singh, 2014; Sundang et al. 2012; Vasconcelos et al. 2010). The inorganic
phenolic compounds were obtained from the commercial chemical store.
Sub-lethal toxicity study. The concentration of LC50 from inorganic phenol (3 mg/kg)
and organic phenolic (1.80 mg/kg) is equal to 75% of LC50-96 hours. The exposure to
phenolic compounds was done for 3 days after a single injection (Saputra et al. 2016)
Blood sample. The blood was collected 3 days after exposure to phenolic compounds.
The blood was taken by using a 1 ml syringe on the cardinal vein and then centrifuged at
3000 rpm at 40C for 15 minutes. The serum was separated and stored at a temperature of
15- 200C (Saputra et al. 2016; Varadarajan et al., 2014; Roche and Boge, 2000). The
measurement of hemoglobin level was carried out by the Sahli method (Wedemeyer and
Yasutake, 1977) in Hb/100ml while the hematocrit level was done by the capillary system
(Anderson and Siwicki, 1995). On the other hand, the cortisol concentration plasma (Cort)
was assessed by radioimmunoassay (RIA) (Foster and Dunn, 1974).
Data analysis. The data were analyzed by using the Analysis of Variance (ANOVA)
with Minitab 16 for windows.

RESULTS AND DISCUSSION

The data analysis showed that the hemoglobin after being exposed with phenolic
compounds have a significantly different situation in between the 5% level of treatment, Fhit:
35.58 (P> 0.05). Based on the Figure 1, the hemoglobin rate of African catfish without
treatment (control) is 13.67 Hb/100ml in which it is in line with the research from Alamanda et
al., (2007) that a generally healthy African catfish have a hemoglobin level ranged from 12-
14 Hb/100 ml of blood.
Different conditions indicate that after the hemoglobin is injected, the phenolic
compound was significantly decreased in the treatment of organic phenolic that is 7.67
Hb/100ml. In the inorganic phenolic treatment, it is shown that the hemoglobin was lower
compared to the treatment of organic phenolic that is 6.67 Hb/100ml. According to Nusyam
et al., (2017) and Wahjuningrum et al., (2008), low and high hemoglobin levels indicate that
the fish might be sick or stressed. Roche and Boge (2000) stated that the increase or
decrease in the plasma concentration of hemoglobin is a sign that there is an interference
from the cells. Harikrishnan et al., (2003) also asserted that the damage in red blood cells
causes a decrease in hemoglobin content.
The results of data analysis showed that the hematocrit condition after being exposed
to phenolic compounds was significantly different in between treatments in the level of 5%,
Fhit: 17.35 (P>0.05). Based on the Figure 2 below, the hematocrit in African catfish without
treatment (control) amounts to 37 %. This is in accordance with the opinion of Bastiawan et
al., (1995) that the normal hematocrit of African catfish is ranged from 30.8 to 45.5%.
The inorganic phenolic treatment showed that the hematocrit value was much lower
than the organic treatment which was 23.33%. This value indicates that the treatment of
inorganic phenolic greatly affects the decrease in the African catfish hematocrit. Tsuzuki et

78
 RJOAS, 12(96), December 2019

al., (2001) stated that when the fish hematocrit is less than 22%, anemia will occur whereas if
the hematocrit is greater than 60%, the fish will be in a state of stress. Anderson and Siwicki
(1994) confirmed that low hematocrit can indicate the occurrence of fish contamination.

Figure 1 – The Hemoglobin of African Catfish after Organic and Inorganic Phenolic Injection

Figure 2 – The Hematocrit of African Catfish after Organic and Inorganic Phenolic Injection

Figure 3 – Cortisol Hormone of African Catfish after Organic and Inorganic Phenolic Injection

This hormone is the result of endocrine gland secretion. The endocrine glands build the
endocrine system which has the function of regulating the work of the body's physiological

79
 RJOAS, 12(96), December 2019

processes. The value of the cortisol hormone in catfish after being injected with phenolic
compounds can be seen in Figure 3.
The results showed that the cortisol hormone after being exposed to phenolic
compounds was significantly different in between the treatments (5%), Fhit: 3.86 (P>0.05).
Based on the results of the study, the cortisol in African catfish without treatment (control) is
412.76 nmol/L. The increase of cortisol hormone in phenolic treatment was compared with
the control, in which the increase of the hormone was 198.86% in the organic phenolic
treatment and 186.09% in the inorganic phenol treatment. Roche and Boge (2000) reported
that the cortisol hormone in fish treatment was increased by 185.7-218%. According to Zaki
et al., (2012), there was an increase in C.gariepinus fish cortisol after being exposed to
phenol for 3 months at 32.7%.
John et al., (1994) explained that the increased cortisol in phenol treatment can be
associated with fish poisoning and handling. On the one hand, Roche and Boge (2000)
believed that the increase in fish stressors after a single or fraction phenol injection is due to
the disturbances in cells. The damage to endocrine cells in both liver and kidneys causes an
abnormal biochemical blood value that one of which plays a major role in hemoglobin. Low
Hb in tannin injection treatment showed that the spread of oxygen in the body of African
catfish was disrupted. Barton (1987) described that a physiological stress will cause a new
homeostatic condition in fish through a changing metabolism. This response to stress is
controlled by the endocrine system through the release of cortisol hormones and
catecholamine.

CONCLUSION

The results from blood tests show that there is an increase in the cortisol hormone
while the other parameters such as hemoglobin and hematocrit experience a decrease. This
condition explains that the treatment of organic phenolic exposure is comparable to the
inorganic phenol exposure. It can be seen from the changes in the blood hemoglobin and
hematocrit as well as the cortisol hormones of African catfish.

REFERENCES

1. Alamanda, I.E., N.S. Handajani, and A. Budiharjo. 2007. Penggunaan metode hematologi
and pengamatan endoparasit darah untuk penetapan kesehatan Ikan Lele Dumbo
(Clarias gariepinus) di kolam budidaya Desa Mangkubumen Boyolali. Biodiversitas,
(1):34-38.
2. Anderson, D.P., and A.K. Siwicki. 1995. Basic hematology and serology for fish health
programs: Fish Health Section, Asian Fisheries Society. 185-202pp.
3. Anderson, D.P., and A.K. Siwicki. 1994. Simplified assays for measuring nonspecific
defense mechanisms in fish: Fish Health Section, Fisheries Soc. Meeting. 26 pp.
4. Barton, B.A., C.B. Schreck, and L.D. Barton. 1987. Effects of chronic cortisol
administration and daily acute stress on growth, physiological conditions, and stresss
responses in juvenile rainbow trout. Diseases of Aquatic Organisms, (2):173-185.
5. Bastiawan, D., Taukhid, M. Alifuddin, and T.S. Dermawati. 1995. Perubahan hematologi
and jaringan kan Lele Dumbo (Clarias gariepinus) Yang diinfeksi Cendawan
Aphariomyces sp. Jurnal Penelitian Perikanan Indonesia: 106-115.
6. Bigoniya, P., and K. Singh. 2014. Ulcer protective potential of standardized hesperidin, a
citrus flavonoid isolated from Citrus sinensis. Rev Bras Farma,(24):330-340.
7. Buikema, Jr.A.L., M.J. Mc. Ginniss, and Jr.J. Cairns. 1979. Phenolics in aquatic
ecosystems: a selected review of recent literature. Mar Environ Res, (2):87–181.
8. Emrizal., A. Fernando, R. Yuliandri, K. Rullah, Nola, Indrayani, N.R, Reni, A. Susanty, R.
Yerti, F. Ahmad, H.M. Sirat, D. Arbain. 2014. Cytotoxic activities of fractions and two
isolated compounds from Sirih Merah (Indonesian red betel), Piper crocatum Ruiz and
Pav. Procedia Chemistry, (13):79–84.

80
 RJOAS, 12(96), December 2019

9. Foster, L.B., and R.T. Dunn. 1974. Single antibody technique for radioimmunoassay of
cortisol in unextracted serum or plasma. Clin Chem, (20):365–368.
10. Harikrishnan, R., M.N. Rani, and C. Balasundaram. 2003. Haematological and
biochemical parameters incommon carp, Cyprinus carpio, following herbal treatment for
Aeromonas hydrophilla infection. Aquaculture,221:41-50.
11. Ibrahem. M.D. 2012. Experimental exposure of African catfish Clarias gariepinus
(Burchell, 1822) to phenol: Clinical evaluation, tissue alterations and residue assessment.
Journal of Advanced Research (3):177–183.
12. James, M.O., L.D. Stuchal, B.A. Nyagode. 2008. Glucuronidation and sulfonation in vitro
of the major endocrine-active metabolites of methoxychlor in the channel catfish,
Ictalurus punctatus and induction following treatment with 3-methylcholanthrene. Aquatic
Toxicology, (2):227–238.
13. John, F., Carragler, and M.R. Christine. 1994. Primary and secondary stress responses
in golden pereh Macquoria ambigua. J. comp. Biochem. Physiol, 107A (1):40-56.
14. Mukherjee, D., S. Bhattacharya, V. Kumar, and J. Moitra. 1990. Biological significance of
[14C] phenol accumulation in different organs of a murrel, Channa punctatus, and the
common carp, Cyprinus carpio. Biomed. Environ. Sci, (3):337–342.
15. Nursyam, H., S. Andayani and A. Saputra. 2017. The effect of tannin from red betel
(Piper crocatum) leaves towards blood biochemistry and histology of North African catfish
(Clarias gariepinus). AACL Bioflux., Volume 10, Issue 5, pp 1386-1393.
16. Roche, H., and G. Boge. 2000. Fish blood parameters as a potential tool for identification
of stress caused by environmental factors or chemical intoxication. Mar. Environ. Res.
Part C 125:27–44.
17. Saputra, A., S. Andayani and H. Nusyam. 2016. Total quantity of phenol and isolation
methanol tannin extract of red betel leaf (Piper crocatum). International Journal of
PharmTech Research. Vol.9, No.7, pp 146-153.
18. Sundang, M, S.N.S. Nasir, C.S. Sipaut, and H. Othman. 2012. Antioxidant activity,
phenolic, flavonoid and tannin content of Piper betle and Leucosyke capitella. Malaysian
Journal of Fundamental and Applied Sciences. (1):1-6.
19. Taysse, L., D. Troutaud, N.A. Khan, P. Deschaux. 1995. Structure activity relationship of
phenolic compounds (phenol, pyrocatechol and hydroquinone) on natural
lymphocytotoxicity of carp (Cyprinus carpio). Toxicology, (1-3):207–214.
20. Tilak, K.S., K. Veeraiah, M.S. Butchiram. 2007. Effect of phenol on haematological
components of Indian major carps Catla catla, Labeo rohita and Cirrhinus mrigala. J
Environ Biol, (2):177–179.
21. Tsuzuki, M.Y., K. Ogawa, C.A. Strussmann. 2001. Physiological responses after stress
and subsequent recovery at different salinities in adults pejerrey Odontesthes
bonariensis. Aquaculture, 200:349-362.
22. Vasconcelos, P.C.P, M.A. Andreo, W. Vilegas, C.A. Hiruma-Lima, C.H. Pellizzon. 2010.
Effect of Mouriri pusa tannins and flavonoids on prevention and treatment against
experimental gastric ulcer. Journal of Ethnopharmacology, 131:146–153.
23. Varadarajan, R, H. Sankar H.S, J. Jose and B. Philip. 2014. Sublethal effects of phenolic
compounds on biochemical, histological and ionoregulatory parameters in a tropical
teleost fish Oreochromis mossambicus (Peters). International Journal of Scientific and
Research Publications, (3):2250-3153.
24. Wahjuningrum, D., N. Ashry and S. Suryati. 2008. Pemanfaatan ekstrak daun Ketapang
Terminalia Cattapa untuk pencagahan and pengobatan Ikan Patin (Pangasionodon
hypophthalmus) yang Terinfeksi (Aeromonas hydrophila). Jurnal Akuakultur Indonesia
7(1):82-88.
25. Wedemeyer G.A., and W.T. Yasutake. 1977. Clinical methods for the assessment of the
effects of environmental stress on fish health. Technical Paper of the U.S. Fish and
Wildlife Service. Vol. 89. U.S. Department of the Interior Fish and Wildlife Service,
Washington, D.C., USA. 18p.
26. Zaki, M.S., N. El-Batrawy, and N. M. Taha. 2012. Dose phenol toxicity affected endocrine
status in African Catfish (Clarias gariepinus). Life Science Journal, 9(1):636-639.

81