Subscribe Now Subscribe Today
Research Article

Anomalies in Cyprinus carpio Larvae Exposed to Paper Mill Effluent

Anil K. Tyor, A. Fulia and R.K. Sharma
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail

Water pollution due to urbanization and industrialization are the major cause of decline in aquatic animal populations all over the world. Keeping in view the present study deals with the teratogenic influence of paper mill effluent in Cyprinus carpio larvae under laboratory conditions. Cyprinus carpio larvae were exposed to different concentrations (less than half of the LC50) of paper mill effluent to investigate the effects induced by the effluent. Control was run simultaneously along with all the experimental groups. Larvae were more sensitive after complete yolk sac absorption and increased mortality was observed during this phase. Common morphological abnormalities in hatchlings after exposure to the paper mill effluent include flexure of the larval axis, vertebral dysplasia, abnormalities in eyes such as single eye, microphthalmia, abnormality in the yolk sac and heart tube anomalies. Overall stunted growth was also recorded in the treatment groups depicting hazardous effect of paper mill effluent on larval stages of Cyprinus carpio.

Related Articles in ASCI
Similar Articles in this Journal
Search in Google Scholar
View Citation
Report Citation

  How to cite this article:

Anil K. Tyor, A. Fulia and R.K. Sharma, 2012. Anomalies in Cyprinus carpio Larvae Exposed to Paper Mill Effluent. Journal of Biological Sciences, 12: 321-326.

DOI: 10.3923/jbs.2012.321.326

Received: April 27, 2012; Accepted: June 25, 2012; Published: September 06, 2012


Water pollution due to urbanization and industrialization are the major cause of decline in aquatic animal populations all over the world (Maitland, 1995; Dan’azumi and Bichi, 2010; Singh, 2007; Anbumani and Mohankumar, 2011; Muhamad et al., 2011). The pollutants generated by anthropogenic activities including domestic sewage, pesticides, heavy metals, pulp and paper mill effluents, dye and dye products, pharmaceuticals etc. are known to affect aquatic organisms (Khan and Thulin, 1991; Tamburlini et al., 2002; Sekhar et al., 2003; Prabu and Udayasoorian, 2005; Saxena et al., 2006; Moharram et al., 2011). The pulp and paper mills rank high in terms of water use during paper production on the other hand they are contributing to pollution loads in rivers through effluent discharge (Jayabalakrishnan, 2007). Toxic dyes, bleaching agents, salts, acids and alkalis are present in effluents discharged from pulp and paper industries. Heavy metals such as cadmium, copper, zinc, chromium are present in the pulp and paper mill effluent that ultimately released into aquatic environment (Mathur et al., 2005; Zahrim et al., 2007). Dichloroguicol trichloroguicol, tetrachloroguicol and chlorinated phenols are major contaminants found in the effluent released from pulp and paper mill which are toxic to fish fauna (Leuenberger et al., 1985; Mellanen et al., 1996; Owens, 1991). Various subchronic effects including structural, physiological and biochemical abnormalities in fishes due to paper mill effluent have been documented (Lindstrom-Seppa and Oikari, 1989; Larsson et al., 1988; Pesonen and Andersson, 1992). Oxidative stress induced by effluent resulted in formation of hyperactive metabolites which may lead to mutations in DNA (Hodson et al., 1996; Oakes et al., 2005). Pulp and paper mill industries also alter the macroinvertebrate communities present in rivers (Flinders et al., 2009). Toxicants such as dioxins present in pulp and paper mill effluent, resulted alterations in reproductive performance and development of an organism (Kovacs et al., 1997; Parks et al., 2001). A number of the studies have been conducted on the effect of industrial effluents on fishes. However, very little work has been done on the larval stages of fishes. Therefore, the present study was designed to investigate the effect of paper mill effluent on larval stages of Cyprinus carpio.


The present study was conducted at Fish and Fisheries Laboratory, Department of Zoology, Kurukshetra University, Kurukshetra (29°58’N Latitude and 76°51’E Longitude) Haryana, India during February 2010.

Two hundred hatchlings of Cyprinus carpio hatched from the eggs which were incubated in the sublethal concentrations of Paper Mill Effluent (PME). These hatchlings were further exposed to similar concentrations till they start feeding orally to study its survival and the abnormalities induced by the PME. Experimental group was divided in three sub groups: Set-A with 1% PME, Set-B having 2% PME and Set-C containing 4% PME. Whole experiment was performed in triplicates. Control was run simultaneously along with the experimental groups. Proper aeration was provided with the help of low pressure aerator. Physicochemical parameters such as DO, temperature, pH were recorded at regular interval during the experiment. No feeding was provided to the hatchlings during this experiment.


The embryos were examined throughout their prehatching development and up to sac fry stage of larvae to record morphological abnormalities and post hatchling development. Cyprinus carpio embryo test organism in control ones began to hatch after 45 h which was preponed by 3 h in 4% of PME and 2 and 1 h in 2 and 1% concentration of PME, respectively. Dose related increase in mortality of embryo was observed during the present study. Healthy normal embryos were observed in control group (Fig. 1). Significant decline in hatching success was observed in PME treated embryos (Tyor et al., 2010). Among the successful hatching embryos, teratism was clearly evident. Five major categories of developmental abnormalities including altered axial curvature, head and deformities, tail malformation were observed. In all 25-37% of the larvae were observed to have one or the other abnormality whereas in 8% of the larvae were found to be malformed. Most commonly encountered defects were flexure in the body axis along the whole length of the larvae or may be easily noticed as bent tails (Scoliotic embryo). In some cases bent was also observed in the pharyngeal region in all the treatment groups (Fig. 2). Malformation of the vertebral axis increased in dose-dependent manner. About 5% of the embryo in control was reported to have anomalies in the vertebral axis whereas in the experimental groups it increased up to 16% (4% PME). Besides bent tail, edema in pericardial region, microphthalmia, fused eye/single eye deformity in the fin region. Gills disorganized head region were also reported in the treated groups.

A slight delay in the yolk absorption was noticed in the treated embryos. In 4% treatment group, the yolk sac was abnormally enlarged and protruded up to the head region which may resulted in death of the larvae. This yolk sac anomaly was observed in 1% of the control group larvae but reached to 5% in the higher concentration (4% PME).

Fig. 1: Normal larvae in control group (50X)

Fig. 2: Flexure of the larval axis, along whole body length (50X)

Malformation of eye included hypoplasia, microphthalmic and anophthalmia. Such abnormalities were observed in all the treatment groups ranging between 3-4% of the larvae (Fig. 3-5). No such abnormality was observed in the control group.

Irregularly shaped heart tube was also observed in the treatment groups. Edema of the pericardial cavity was observed in 2 and 4% effluent concentrations. The abnormality percentage in heart was 2, 1 and 1% in Set-A, B and C, respectively. No abnormality in heart was observed in control group. Red patches near the gill region were observed due to the effect of effluent. Deformities in fin region were recorded in 1% of larvae of control group which increased up to 3% in Set-A and B and 5% in Set-C, respectively. Disorganization in head and caudal region and reduced development of caudal fin was noticed in PME treated larvae (Fig. 4).

Fig. 3: Larva with abnormally large yolk sac extended up to the head region after exposure of 4% concentration of paper mill effluent (50X)

Fig. 4: Microphthalmia or small eye after exposure to 2% concentration of paper mill effluent (50X)

This type of abnormality was not observed in control group (Fig. 1) however, 2-4% of the larvae in treatment groups had abnormal head. Over all stunted appearance was observed in the larvae of the Cyprinus carpio (Fig. 6, 7). Occasionally, treated hatchlings showed twitching and erratic movement as compared with control group. Yolk sac absorption was delayed in PME exposed larvae. In control group yolk was completely absorbed in 4-5 days however, in PME treated group it lasted up to the sixth day. High larval mortality was in the treatment group after complete absorption of yolk sac and the young ones start feeding orally.

Fig. 5: Larvae with fused eye as well as abnormal caudal fin (50X)

Fig. 6: Larvae showing overall stunted growth after exposure of 4% paper mill effluent (50X)


The paper mill effluent exposure induced abnormalities including conditions wherein, the yolk sac get protruded up to the head region, deformities in eyes, spinal curvature, abnormal head and overall stunted growth were observed during the present study. Woodworth and Munday (2000) observed similar abnormalities in eyes and yolk sac due to the effect of paper mill effluent in larval stages of Tasmanian blennies (Parablennius tasmanianus). According to Van Leeuwen et al. (1985), accumulation of environmental toxicants in protein which may result in enhancement of toxicant concentration as well as the accessibility of the toxic compound to larvae during yolk sac absorption.

Fig. 7: Stunted growth and disorganized head region due to exposure of 4% concentration of paper mill effluent (50X)

2,3,7,8-tetrachlorodioxin (TCDD) is a component of paper mill effluent which have been reported to induce anomalies in yolk sac and head region in larvae (Walker et al., 1994). The observation of the present investigation showed that paper mill effluent induced abnormalities in body structure and function which may possibly result in death of deformed larvae. In the present study mortality was also observed during yolk sac absorption strongly support the observations of Helder (1980) in which yolk sac absorption process has the major contribution in redistribution of toxicants such as TCDD which may result in alteration in physiological functions that ultimately lead to death of aquatic organisms Helder (1980). The paper mill effluent induced the morphological alterations such as single eye, microphthalmia, abnormality in fin region, abnormality in heart in Cyprinus carpio larvae observed during the present study are similar to the morphological abnormalities observed in fish exposed with effluents released from other plants such as sugar-beet processing plant (Hegrenes, 1999). Curvature in the body axis as observed in the present study was also documented by Boudreau et al. (2005) in the larvae of the estuarine mummichog, Fundulus heteroclitus, induced by androgenic and anti-androgenic compounds present in the environment. Boudreau et al. (2005) observed that gross morphological abnormalities in embryonic, larval and juvenile stages of fish are the sensitive indicator of exposure of toxicant. Bleached Kraft mill effluent have been documented to induce the alteration in Gonadosomatic Index (GSI) and the decline in number of germ cells which may lead to complete voiding of germ cells in the organism with the passage of time (Jobling et al., 1996; Leatherland, 1992). Pryce-Hobby et al. (2003) observed that chemical constituents present within pulp mill effluent bind to steroid binding proteins, hence alter the properties of steroid binding proteins. Effluent exposure induced masculinization of females in fish populations (Howell et al., 1980). Earlier studies have been demonstrated that discharges of wastewater from papers mills have various substances with androgenic or anti-estrogenic activities (Karels et al., 1999; Hegrenes, 1999; Larsson and Forlin, 2002). Fathead minnows exposed with paper mill effluent over a full life cycle induced depression in sex steroid production, delay in sexual maturity, reduced egg production as well as changes in the secondary sex characteristics (Munkittrick et al., 1998).


From the present study it becomes clear that pulp and paper mill effluent have deleterious effects in larval stages of Cyprinus carpio. Despite lower doses of exposure, prolonged duration paper mill effluent lashed environment leads to serious development problems associated with the survival of aquatic organisms. Further research is needed to better understand of mechanism involved in toxicity caused by paper mill effluent. The present investigation will help in formulating policies and strategies to circumvent the toxic effects of paper mill effluent.


Financial assistance provided by University Grants Commission (New Delhi) in the form of Rajiv Gandhi National Fellowship to Anju Fulia is highly acknowledged. The authors are thankful to the National Fish Seed Farm, Jyotisar (Kurukshetra), Sultan Fish Seed Farm, Butana (Karnal) and Fish Seed Farm situated in village Mandheri (Kurukshetra) for providing fish eggs.

1:  Maitland, P.S., 1995. The conservation of freshwater fish: Past and present experience. Biol. Conserv., 72: 259-270.
CrossRef  |  Direct Link  |  

2:  Azumi, S.D. and M.H. Bichi, 2010. Industrial pollution and heavy metals profile of challawa river in Kano, Nigeria. J. Applied Sci. Environ. Sanitation, 5: 23-29.
Direct Link  |  

3:  Singh, S.K., 2007. Effect of irrigation with paper mill effluent on the nutrient status of soil. Int. J. Soil Sci., 2: 74-77.
CrossRef  |  Direct Link  |  

4:  Anbumani, S. and M.N. Mohankumar, 2011. Nuclear and cytoplasmic abnormalities in the fish Catla catla (Hamilton) exposed to chemicals and ionizing radiation. Res. J. Environ. Sci., 5: 867-877.
CrossRef  |  Direct Link  |  

5:  Muhamad, M.H., S.R.S. Abdullah, A.B. Mohamad, R.A. Rahman and A.A.H. Khadum, 2011. Treatment of adsorbable organic halides from recycled paper industry wastewater using a GAC-SBBR pilot plant system. J. Applied Sci., 11: 2388-2393.
CrossRef  |  Direct Link  |  

6:  Khan, R.A. and J. Thulin, 1991. Influence of pollution on parasites of aquatic animals. Adv. Parasitol., 30: 201-238.
PubMed  |  

7:  Tamburlini, G., O.V. Ehrenstein and R. Bertollini, 2002. Children's health and environment: A review of evidence. Environmental Issue Report No. 129, WHO/European Environment Agency, WHO Geneva, pp: 223.

8:  Sekhar, K.C., N.S. Chary, C.T. Kamala, J.V. Rao, V. Balaram and Y. Anjaneyulu, 2003. Risk assessment and pathway study of arsenic in industrially contaminated sites of hyderabad: A case study. Environ. Int., 29: 601-611.
CrossRef  |  Direct Link  |  

9:  Prabu, P.C. and C. Udayasoorian, 2005. Decolorization and degradation of phenolic paper mill effluent by native white rot fungus Phanerochaete chrysosporium. Asian J. Plant Sci., 4: 60-63.
CrossRef  |  Direct Link  |  

10:  Saxena, M.P., P. Kaur, H.M. Saxena and J. Kapur-Ghai, 2006. Antibiotic resistant bacteria isolated from fish died on exposure to chromium. J. Fish. Aquatic Sci., 1: 209-212.
CrossRef  |  Direct Link  |  

11:  Moharram, S.G., O.M. Wahbi and Z.A. El-Greisy, 2011. Effect of polluted water from the Egyptian Eastern Mediterranean coast on reproductive, toxicological and hematological characteristics of Siganus rivulatus. Pak. J. Biol. Sci., 14: 668-681.
CrossRef  |  Direct Link  |  

12:  Jayabalakrishnan, R.M., 2007. Effect of vermiculite as an ameliorant for paper mill effluent irrigated soil and on the productivity of sunflower. J. Agron., 6: 175-178.
CrossRef  |  Direct Link  |  

13:  Mathur, N., P. Bhatnagar, P. Nagar and M.K. Bijarnia, 2005. Mutagenicity assessment of effluents from textile/dye industries of Sanganer, Jaipur (India): A case study. Ecotoxicol. Environ. Safe., 61: 105-113.
CrossRef  |  Direct Link  |  

14:  Zahrim, A.Y., M.L. Gilbert and J. Janaun, 2007. Treatment of pulp and paper mill effluent using photo-fenton's process. J. Applied Sci., 7: 2164-2167.
CrossRef  |  Direct Link  |  

15:  Leuenberger, C., W. Giger, R. Coney, J.W. Graydon and E. Molnar-Kubica, 1985. Persistent chemicals in pul pmill effluents: Occurrence and behaviour in an activated sludge treatment plant. Water Res., 19: 885-894.
CrossRef  |  Direct Link  |  

16:  Mellanen, P., T. Petanen, J. Lehtimaki, S. Makela and G. Bylund et al., 1996. Wood-derived estrogens: Studies in vitro with breast cancer cell lines and in vivo in trout. Toxicol. Applied Pharmacol., 136: 381-388.
CrossRef  |  PubMed  |  Direct Link  |  

17:  Owens, J.W., 1991. The hazard assessment of pulp and paper effluents in the aquatic environment: A review. Environ. Toxicol. Chem., 10: 1511-1540.
CrossRef  |  

18:  Lindstrom-Seppa, P. and A. Oikari, 1989. Biotransformation and other physiological responses in whitefish caged in a lake receiving pulp and paper mill effluents. Ecotoxicol. Environ. Saf., 18: 191-203.
PubMed  |  

19:  Larsson, A., T. Andersson, L. Forlin and J. Hardig, 1988. Physiological disturbances in fish exposed to bleached kraft mill effluents. Water Sci. Technol., 20: 67-76.
Direct Link  |  

20:  Pesonen, M. and T. Andersson, 1992. Toxic effects of bleached and unbleached papermill effluents in primary cultures of rainbow trout hepatocytes. Ecotoxicol. Environ. Saf., 24: 63-71.
CrossRef  |  Direct Link  |  

21:  Hodson, P.V., S. Efler, J.Y. Wilson, A. El-Shaarawi, M. Maj and T.G. Williams, 1996. Measuring the potency of pulp mill effluents for induction of hepatic mixed-function oxygenase activity in fish. J. Toxicol. Environ. Health, 49: 83-110.
PubMed  |  

22:  Oakes, K.D., L.M. Hewitt, M.E. McMaster, C. Wood, K.R. Munkittrick and G.J. van der Kraak, 2005. Oxidative stress and sex steroid levels in fish following short-term exposure to Pulp-Mill effluents. J. Toxicol. Environ. Health, 68: 267-286.
CrossRef  |  Direct Link  |  

23:  Flinders, C.A., G.W. Minshall, R.L. Ragsdale and T.J. Hall, 2009. Patterns of macroinvertebrate assemblages in a long-term watershed-scale study to address the effects of pulp and paper mill discharges in four US receiving streams. Integr. Environ. Assess. Manag., 5: 248-258.
CrossRef  |  Direct Link  |  

24:  Kovacs, T.G., R.H. Voss, S.R. Megraw and P.H. Martel, 1997. Perspectives on Canadian field studies examining the potential of pulp and paper mill effluent to affect fish reproduction. J. Toxicol. Environ. Health., 51: 305-352.
PubMed  |  

25:  Parks, L.G., C.S. Lambright, E.F. Orlando, L.J. Guillette Jr., G.T. Ankley and L.E. Gray Jr., 2001. Masculinization of female mosquitofish in kraft mill effluent-contaminated fenholloway river water is associated with androgen receptor agonist activity. Toxicol. Sci., 62: 257-267.
CrossRef  |  PubMed  |  

26:  Tyor, A.K., A. Fulia and R.K. Sharma, 2010. Impact of paper mill effluent on the survival and hatchability of eggs of Cyprinus carpio. Res. J. Environ. Toxicol.,

27:  Woodworth, J.G. and B.L. Munday, 2000. Sublethal effects of combined parental and embryo exposure of the Tasmanian Blenny (Parablennius tasmanianus) to euclypt-based pulp mill effluent. Aust. J. Ecotoxicol., 6: 103-108.
Direct Link  |  

28:  Van Leeuwen, C.J., P.S. Griffioen, W.H.A. Vergouw and J.L Maas-Diepeveen, 1985. Differences in susceptibility of early life stages of rainbow trout (Salmo gairdneri) to environmental pollutants. Aqual. Toxicol., 7: 59-78.
CrossRef  |  Direct Link  |  

29:  Walker, M.K., P.M. Cook, A.R. Batterman, B.C. Butterworth and C. Berini et al., 1994. Translocation of 2,3,7,8-tetrachlorodibenzo-p-dioxin from adult female lake trout (Salvelinus namaycush) to oocytes: Effects on early life stage development and sac fry survival. Can. J. Fish. Aquatic. Sci., 51: 1410-1419.
CrossRef  |  Direct Link  |  

30:  Helder, T., 1980. Effects of 2,3,7,8-Tetrachlorodiben20-p-dioxin (TCDD) on early life stages of the pike (Esox lucius L). Sci. Total Environ., 14: 255-264.

31:  Hegrenes, S.G., 1999. Masculinization of spawning channel catfish in the Red River of the North. Copeia, 2: 491-494.
Direct Link  |  

32:  Boudreau, M., S.C. Courtenay, D.L. MacLatchy, C.H. Berube, L.M. Hewitt and G.J. Van Der Kraak, 2005. Morphological abnormalities during early-life development of the estuarine mummichog, Fundulus heteroclitus, as an indicator of androgenic and anti-androgenic endocrine disruption. Aquatic Toxicol., 71: 357-369.
CrossRef  |  Direct Link  |  

33:  Jobling, S., J.P. Sumpter, D. Sheahan, J.A. Osborne and P. Matthiessen, 1996. Inhibition of testicular growth in rainbow trout (Oncorhynchus mykiss) exposed to estro genic alkylphenolic chemicals. Environ. Toxicol. Chem., 15: 194-202.
CrossRef  |  Direct Link  |  

34:  Leatherland, J., 1992. Endocrine and Reproductive Functions in Great Lakes Salmon. In: Chemically Induced Alterations in Sexual and Functional Development: The wildlife/Human Connection, Colborn, T. and C. Clement (Eds.). Princeton Scientific Publishing Inc., Princeton, NJ, USA., pp: 129-145.

35:  Pryce-Hobby, A.C., M.E. McMaster, L.M. Hewitt and G. Van Der Kraak, 2003. The effects of pulp mill effluent on the sex steroid binding protein in white sucker (Catostomus commersoni) and longnose sucker (C. catostomus). Comp. Biochem. Physiol. Part C, 134: 241-250.
CrossRef  |  PubMed  |  

36:  Howell, W.M., D.A. Black and S.A. Bortone, 1980. Abnormal expression of secondary sex characters in a population of mosquitofish, Gambusia affinis holbrooki: Evidence for environmentally-induced Masculinization. Copeia, 4: 676-681.
Direct Link  |  

37:  Karels, A., M. Soimasuo and A. Oikari, 1999. Effects of pulp and paper mill effluents on reproduction, bile conjugates and liver mfo (mixed function oxygenase) activity in fish at Southern Lake Saimaa, Finland. Water Sci. Technol., 40: 109-114.
CrossRef  |  Direct Link  |  

38:  Larsson, D.G.J. and L. Forlin, 2002. Male-biased sex ratios of fish embryos near a pulp mill: Temporary recovery after a short-term shutdown. Environ. Health Perspect., 110: 739-742.
Direct Link  |  

39:  Munkittrick, K.R., M.E. McMaster, L.H. McCarthy, M.R. Servos and G.J. van der 1998. An overview of recent studies on the potential of pulp‐mill effluents to alter reproductive parameters in fish. J. Toxicol. Environ. Health Part B: Crit. Rev., 1: 347-371.
CrossRef  |  PubMed  |  

©  2021 Science Alert. All Rights Reserved