NSave Nature to Survive

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1 ISSN: : Special issue, Vol. 2; ; 2010 NSave Nature to Survive EFFECT OF EFFLUENT WATER DOWNSTREAM TO THE NAGAON PAPER MILL, ASSAM ON OVARIAN FOLLICULAR POPULATION OF IMMATURE FEMALE C3H MICE A. Haque et al. Pulp and paper mill effluent Endocrine disrupting chemicals Estrogenic effects Ovarian follicles Paper presented in International Conference on Environment, Energy and Development (from Stockholm to Copenhagen and beyond) December 10-12, 2010, Sambalpur University 529

2 NSave Nature to Survive A. HAQUE*, J. C. KALITA, D. D. DEKA AND B. K. BARUAH 1 Environmental Endocrine Disruptors Research Lab., Department of Zoology, Gauhati University, Guwahati , Assam, INDIA 1 Environmental Chemistry Lab., Department of Chemistry, Gauhati University, Guwahati , Assam, INDIA E mail: ahaque1282@rediffmail.com ABSTRACT Recent studies confirmed sufficient number of endocrine active estrogenic compounds (known as environmental endocrine disrupting chemicals) present in the water and soil sample contaminated by pulp and paper mill effluents. It is well known that the estrogenic compounds inhibit oocyte nest breakdown and early follicle assembly causing fewer follicular populations in ovary. Therefore, present study was aimed to investigate hormonal effect of the downstream effluent water of Nagaon Paper Mill on ovarian follicular populations in a rodent model. Immature 28-days-old female C3H mice were exposed to effluent in drinking water and sacrificed after 30 days of continuous exposure. 17β-estradiol was used as positive control agent (n = 6 per group). Serial sections of 6 μm thick of each ovary was stained with hematoxylin and eosin and every 12 th and 20 th section was examined for counting smaller (primordial, primary and secondary) and larger (graffian and atretic) follicles respectively to obtain total number of follicles per ovary. The results of the present study showed fewer primary follicle population in the mice exposed to paper mill contaminated water resembling with the estrogen treated animal but not in tap water controls. These data suggest that the chemicals having estrogenic nature probably present in effluent, mimicking estrogens action, may have reduced follicle assembly leading to fewer primary as well as subsequent growing follicles in mice ovary. *Corresponding author 530

3 EFFECT OF EFFLUENT WATER INTRODUCTION Evaluation of the ovary is an important endpoint in toxicological assessments because xenobiotics that cause loss of oogonia, oocytes, or supportive somatic cells may have adverse effects on reproduction (Regan et al., 2005). This may be important, for example in determining the relative roles of gonadotrophic and steroid hormones and local intraovarian growth factors in regulating the survival and maturation of follicles at any stage of their development (Myers et al., 2004). The U.S. Environmental Protection Agency (EPA), U.S. Food and Drug Administration (FDA), and the Organization for Economic Cooperation and Development (OECD) guidelines for microscopic examination of reproductive organs in reproductive toxicity studies recommend qualitative and quantitative evaluation of primordial follicles in the ovary (U.S. EPA, 1998; U.S. FDA, 2000; OECD, 2001). One the most widely used approach for estimating follicle numbers is one based on a histological sampling of the total ovarian mass for the number of primordial, primary and preantral follicles, often followed by the application of a correction factor. This approach essentially entails that an ovary is fixed, paraffin-embedded and serially sectioned, usually at 6-8 μm widths (Tilly, 2003). In recent times, large numbers of estrogenic (Rickwood et al., 2006; Felty, 2007; Chamorro et al., 2010) and androgenic (Cody and Bortone, 1997; Ellis et al., 2001; Wartman et al., 2009) contaminants have been reported in the manufacturing products and discharged effluents of pulp and paper industries. Studies identified a number of reproductive alterations in fish caught downstream water bodies of pulp and paper mill which include reduced gonadal development, increased age of maturation, reduced and altered expression of secondary sex characteristics, reduced fecundity and reduction in circulating levels of biologically active reproductive steroid hormones and many more (McMaster et al., 2003). Estrogens inhibit mouse oocyte nest breakdown and follicle assembly (Chen et al., 2007). Moreover, there are suggestions, that exposure to progesterone may reduce the number of primordial follicles (Kezele and Skinner, 2003). Estrogenic action reduces follicle assembly leading to fewer primary and subsequent developing follicles. Thus, the study of follicular populations provides important information about the function of the ovary, in particular the relationship between folliculogenesis and also environmental factors having estrogenic property that regulate it (Raymond-Whish et al., 2007). Therefore, we aimed to investigate the hormonal effect of the downstream water of a paper mill on ovarian follicular populations in animal model. There are also no studies that have looked at the effect of external agents on primordial follicle formation in utero (Maheshwar and Bhattacharya, 2007). The mill taken for the present study was Nagaon Paper Mill which is situated almost in the midst of the state of Assam in the North Eastern Region of India. This mill is regarded as one of the largest paper mills in Asia. The mill discharges about 90,000 m 3 of effluent water daily excluding its huge volume of semi-solid lime and bleachery waste products causing heavy pollution load in the land, air as well as in the water bodies of downstream area. The entire effluent products are discharged through an open canal into Elenga Beel System which travelling after about 25 km joins ultimately to the mighty river Brahmaputra. The human and animal communities are exposed through different routes of exposure viz., inhalation of polluted air, drinking water, eating contaminated fish, vegetables and many other food items grown in effluent contaminated area. Unfortunately, in spite of having possibility of adverse health effects on reproduction, till date, no study has been carried out regarding the endocrine disrupting activities of the mill discharges on the exposed animal communities as well as on human population. MATERIALS AND METHODS Animals: We executed effluent contaminated water exposure in immature 28-days-old C3H female mice, obtained from the Laboratory Animal House Facility at Gauhati University, India. Animals were housed in wire mesh plastic cages with solid bottom containing sawdust, maintained under uniform condition of 531

4 A. HAQUE et al., natural photoperiod (12 hr light/dark cycle), relative humidity (50-85%) and temperature (25 ± 2ºC). All the mice had free access to water and commercially available animal diet with vitamins and minerals supplement (purchased from Agrivet Farm Care Division, Glaxo Smithkline, Chennai, India) and were fed ad-libitum. Treatment Procedures and Tissue Processing: The effluent water sample was collected from a point of Elenga Beel, 5 km away from the industry, filtered and stored at 4ºC for future use. Mice were exposed to paper mill effluent (PME) in drinking water at concentrations of 100% (v/v) and 20% (v/v) where 0.1 mm 17β-estradiol and tap water served as positive and negative control agent respectively (n = 6 mice per group). After 30 days of treatment, mice were sacrificed, ovaries were removed, kept immediately in normal saline and fixed in cold Bouin s fixative. Tissues were processed through different ethanol grades and embedded in paraffin. Sections were cut at 6 μm (6 ovaries from 6 mice per group), deparaffinised in xylene and dehydrated in a series of ethanol baths. Finally, sections were stained with hematoxylin and eosin (H and E), mounted, and observed under microscope (Leitz, Ortholux II, Germany). Morphological classification of follicles: Follicles were classified as primordial if they contained an oocyte surrounded by a partial or complete layer of squamous granulosa cells. Primary follicles showed a single layer of cuboidal granulosa cells. Occasional follicles were observed as intermediate between primordial and primary and had both cuboidal and squamous granulosa cells. If cuboidal cells predominated follicles were classified as the primary type. Follicles were classed as secondary if they possessed more than one layer of granulosa cells with no visible antrum. Early antral follicles possessed generally only one or two small areas of follicular fluid (antrum) whilst antral follicles possessed a single large antral space. Preovulatory follicles had a rim of cumulus cells surrounding the oocyte. Following atresia, the zona pellucida (ZP) remnants (ZPRs) were readily recognized as structures slightly stained (Myers et al., 2004). Quantification of follicles: To determine the total population of different types of follicles per ovary the method used by Pedersen and Peters (1968) and Butcher and Kirkpatric-Keller (1984) were followed. At an average, about 200 serial sections were obtained in each ovary after cutting at the thickness of 6 μm. For each ovary, every 12 th and 20 th section was examined for counting smaller (primordial, primary and secondary) and larger (graffian and atretic) follicles respectively to obtain an overall view of the follicular populations per ovary (Myers et al., 2004). RESULTS AND DISCUSSION The results of different follicular population at 20% and 100% PME and 0.1mM estradiol treated mice are presented in Fig. 1 and 2. As shown in the Figs., there was reduced number of follicles (all types) in estradiol control group in comparison to tap water control. Similar to the estradiol (positive control), number of follicles was reduced in both the doses of PME treated groups except in the primordial follicle number in 100% PME dose ( ± 48.95) (mean ± SEM) where a slight increase was observed in comparison to the tap water ( ± 24.92) as well as estradiol control ( ± 44.47). Almost 2-fold decrease in the total count of secondary, graffian and atretic follicles were observed in 100% PME groups compared to tap water control animals. The difference in the number of primary follicles per ovary in estradiol (179.0 ± 10.3) and 20% PME (207.0 ± 9.2) treated groups were found statistically significant at p< 0.01 compared to tap water control (288.5 ± 29.6) (Fig. 1). Rest of the data, though found a decreasing trend in total follicular count, they did not show any statistical significance with tap water as well as estradiol control groups. Estrogens inhibit mouse oocyte nest breakdown and reduce follicle assembly leading to fewer primary and subsequent developing follicles (Chen et al., 2007). Thus, the study of follicular numbers can provide important information about the function of the ovary in folliculogenesis and also environmental factors having estrogenic property that regulate it (Raymond-Whish et al., 2007). In our experiments, we found a reducing trend of follicular populations in mice exposed to PME (both in diluted and concentrated) and 532

5 EFFECT OF EFFLUENT WATER Follicle/ovary Follicle/ovary Treated animal groups Figure 1: Effects of paper mill effluent (PME) contaminated water on primordial and primary follicle populations in C3H mice. Immature 28-days-old female mice were exposed to PME and estradiol in drinking water and sacrificed after 30 days of treatment. Serial histological sections of 6 μm thick (H&E) of each ovary was prepared and every twelfth section was examined to obtain total number of follicular populations per ovary (mean values are shown on the top of each bar; n = 6 per group). *Significantly different from tap water control (p < 0.01). Independent Sample T Test, SPSS 16.0 estradiol (0.1mM) in drinking water for 30 days (Fig. 1 and 2). Similar kind of results were observed in our another experiment on pregnant mice exposed to PME for 30 days before mating and through gestation (data are not shown). We estimated 882 ± 24 and 746 ± 85 primordial, 288 ± 29 and primary, 147 ± 16 and 141 ± 14 secondary, 16 ± 2 and 11 2 graffian and 58 ± 6 and 32 ± 2 atretic follicles per ovary at normal control and PME exposed (20%) mice respectively. This study was designed accordingly with the study carried out by Raymond-Whish and his co-workers (2007) which showed the estrogenic response of uranium contaminated drinking water (below the U.S. EPA Water Standard) in female mice. In the study, they found 65 ± 7 and 37 ± 7 primordial, 38 ± 2 and 25 ± 2 primary, 26 ± 1 and 21 ± 1 secondary, 31 ± 2 and 28 ± 3 graffian and 17 ± 1 and 11 ± 1 atretic follicles per ovary at normal control and 2.5 mg/l uranyl nitrate exposed in 59-days old B63CF 1 mice respectively (n = 6 per group). Myers and his associates (2004) estimated 1930 ± 286 and 2227 ± 101 primordial follicles, and 137 ± 25 and 265 ± 32 primary follicles per ovary at 70 and 100 days old normal wild-type female J129/C57B6 mice respectively. Using every 10th paraffin section of 7 μm thick (with no correction factor), Halpin et al. (1986) reported primordial follicular numbers at 60, 90 and 120 days of age to be 2030, 2220 and 1890 per ovary respectively. In 1991, Smith et al., calculated 50 to 70-days old mice and reported only 371 primordial follicles per ovary. Above data obtained of normal ovary of mice support in broad agreement with our estimation of the follicular count. CONCLUSION In our investigation, we have shown some sort of hormonal activity (estrogenic) of effluent contaminated downstream water of Nagaon Paper Mill on ovarian follicular populations in female mice. The mice exposed to PME had fewer primary follicle populations resembling the similarity with the estrogen treated animal but not in tap water controls. The estrogenic components probably present in PME, mimicking estrogens action, may have reduced follicle assembly leading to fewer primary as well as subsequent follicles in the ovary. Based on these data and in conjunction with other recent reports (Hewitt et al., 2006; Felty, 2007; Wartman et al., 2009; Chamorro et al., 2010), we conclude that the effluent water of Nagaon Paper Mill may have some hormonal agents which ultimately could be the potential risk factor for endocrine and reproductive health with their exposure to animals communities as well as human beings. 533 Treated animal groups Figure 2: Effects of PME contaminated water on secondary, graffian and atretic follicle populations in C3H mice. Immature 28-days-old female mice were exposed to PME and estradiol in drinking water and sacrificed after 30 days of treatment. Serial histological sections of 6 μm thick (H&E) of each ovary was prepared and every twelfth (for secondary follicle) and twentieth (for graffian and atretic follicle) section was examined to obtain an overall view of the follicular populations per ovary (mean values are shown on the top of each bar; n = 6 per group). Independent Sample T Test, SPSS 16.0

6 A. HAQUE et al., ACKNOWLEDGEMENTS We gratefully acknowledge and thank to Ministry of Environment and Forests, Government of India, for the financial support to carry out this work. We express our thanks and deep sense of gratitude to Prof. D. K. Sharma, former Head of the Department of Zoology, Gauhati University for providing laboratory animals and necessary facilities. REFERENCES Butcher, R. L. and Kirkpatric-Keller, D Pattern of follicular growth during the four-day oestrus cycle in the rat. Biol Reprod. 31: Chamorro, S., Hernandez, V., Monsalvez, E., Becerra, E., Mondaca, M. A., Pina, A. and Vidal, G Detection of estrogenic activity from kraft mill effluents by the yeast estrogen screen. Bull. Env.Contam Toxicol. 84: Chen, Y., Jefferson, W. N., Newbold, R. R., Padilla-Banks, E. and Pepling, M. E Estradiol, progesterone, and genistein inhibit oocyte nest breakdown and primordial follicle assembly in the neonatal mouse ovary in vitro and in vivo. Endocrinol. 148: Cody, R. P. and Bortone, S. A Masculinization of mosquitofish as an indicator of exposure to kraft mill effluent. Bull. Env. Contam Toxicol. 58: Ellis, R. J., Van den Heuvel, M. R., Stuthridge, T. R., McCarthy, L. H., Ling, N., Hogg, I. D. And Dietrich, D. R Androgenic responses in two fish species following exposure to a New Zealand pulp and paper mill effluent. Toxicologist. 60: 162. Felty, Q Pulp mill effluent is a source of environmental estrogens on Alabama s Coosa River. J. Agric. Food Env. Sci. 1: 1-7. Halpin, D. M., Jones, A., Fink, G. and Charlton, H. M Postnatal ovarian follicle development in hypogonadal (hpg) and normal mice and associated changes in the hypothalamic-pituitary-ovarian axis. J. Reprod Fertility. 77: Kezele, P. and Skinner, M Regulation of ovarian primordial follicle assembly and development by estrogen and progesterone: endocrine model of follicle assembly. Endocrinol. 144: Maheshwar, A. and Bhattacharya, S Ovarian ageing and fertility-review. Current Women s Health Reviews. 3: McMaster, M. E., Parrot, J. L. and Hewitt, L. M A decade of research on the environmental impacts of pulp mill effluent in Canada ( ). National Water Research Institute, Burlington, Ontario. NWRI Scientific Assessment Report Series No. 4: 84. Myers, M., Britt, K. L., Wreford, N. G. M., Ebling, F. J. P. and Kerr, J. B Methods for quantifying follicular numbers within the mouse ovary. Reprod. 127: Organization for Economic Cooperation and Development Proposal for updating Guideline 416: two generation reproduction toxicity study. In: OECD Guideline for Testing of Chemicals, OECD, Paris. pp Pedersen, R. A. and Peters, H Proposal for a classification of oocytes and follicles in the mouse ovary. J. Reprod Fertil. 83: Raymond-Whish, S., Loretta, P. M., O Neal, T., Martinez, A., Sellers, M. A., Christian, P. J., Marion, S. L., Begay, C., Propper, C. R., Hoyer, P. B. and Dyer, C. A Drinking water with uranium below the U.S. EPA Water Standard causes estrogen receptor-dependent responses in female mice. Env. Health Perspect. 115: Regan, K. S., Cline, J. M., Creasy, D., Davis, D., Foley, G. L., Lanning, L., Latendresse, J. R., Makris, S., Morton, D., Rehm, S. and Stebbins, K STP position paper: ovarian follicular counting in the assessment of rodent reproductive toxicity. Toxicol. Pathol. 33:

7 EFFECT OF EFFLUENT WATER Rickwood, C. J., Dube, M. G., Hewitt, L. M., Kovacs, T. G., Parrott, J. L. and MacLatchy, D. L Use of paired fathead minnow (Pimephales promelas) reproductive test, Part 1. Assessing biological effects of final bleached kraft pulp mill effluent using a mobile bioassay trailer. Env. Toxicol. Chem. 25: Smith, B. J., Plowchalk, D. R., Sipes, I. G. and Mattison, D. R Comparison of random and serial sections in assessment of ovarian toxicity. Reprod Toxicol. 5: Tilly, J. L Ovarian follicle counts - not as simple as 1, 2, 3. Reprod Biol. Endocrinol. 1: U. S. Environmental Protection Agency Health Effects Test Guidelines, OPPTS , Reproduction and Fertility Effects. EPA 712-C U.S. Environmental Protection Agency, Office of Prevention, Pesticides and Toxic Substances, Washington, DC. U. S. Food and Drug Administration Redbook Toxicologial Principles for the Safety Assessment of Food Ingredients. IV.C.9.a. Guidelines for Reproductive Studies. U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Washington, DC. Wartman, C. A., Hogan, N. S., Hewitt, L. M., McMaster, M. E., Landman, M. J., Taylor, S., Kovacs, T. G. and Van den Heuvel, M. R Androgenic effects of a Canadian bleached kraft pulp and paper effluent as assessed using threespine stickleback (Gasterosteus aculeatus). Aquatic Toxicol. 92:

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