Acute toxicity of textile dye wastewaters (untreated and treated) of Sanganer on male reproductive systems of albino rats and mice

Reproductive Toxicology 19 (2005) 547–556

Acute toxicity of textile dye wastewaters (untreated and treated) of Sanganer on male reproductive systems of albino rats and mice V. Suryavathia , Subhasini Sharmaa,∗ , Shweta Sharmaa , Pratibha Saxenaa , Shipra Pandeya , Ruby Groverb , Suresh Kumarb , K.P. Sharmab a b

Department of Zoology, University of Rajasthan, Jaipur 302004, Rajasthan, India Department of Botany, University of Rajasthan, Jaipur 302004, Rajasthan, India

Received 21 June 2004; received in revised form 1 August 2004; accepted 16 September 2004 Available online 19 January 2005

Abstract This study reports on the toxic effects of 15-days oral administration of untreated (Influent) and treated (Effluent) textile dye wastewaters on male reproductive systems of adult Swiss albino rats (age: 85–90 days) and mice (40–50 days). Textile dye wastewaters decreased body weight (7–25%) and reproductive organ weight (testis, epididymis, prostate gland and seminal vesicle, 2–48%). Similar trends were noted for total protein (14–70%), cholesterol (14–91%) and total lipid (10–30%) content of reproductive organs and spermatozoa, and for fructose levels in seminal vesicle (18–44%). Acid phosphatase activity in prostate however, was increased (11–44%) in the wastewater-exposed animals. Histopathological studies of treated animals revealed altered spermatogenesis, with higher sperm abnormalities, reduction in sperm counts (10–59%), and altered motility (14–56%). The magnitude of these abnormalities was stronger in rats versus mice, while among treatments, it was stronger in the Influent animals. Adverse effects improved when treated rats were allowed to recover 45 days in the control condition. Only recovered Effluent rats were capable of fertilizing normal females indicating that treated wastewater was less toxic; however, in comparison to control rats, litter size and body weight gains of F1 and F2 generations were adversely affected. Thus, the present study has established toxicity of both untreated and treated textile dye wastewater on reproductive biology of male Albino mice and rats. © 2004 Elsevier Inc. All rights reserved. Keywords: Acute toxicity; Untreated and treated textile dye wastewater; Albino rats; Albino mice; Male reproductive organs; Histopathology; Biochemistry; Litter size

1. Introduction The toxicity of azo dyes based on benzidine and its congeners, dimethyl- and dimethoxybenzidine, has been extensively studied insofar as textile, leather and paper industries use a large number of dyes derived from these chemicals. Boeniger [1] reported a higher incidence of urinary bladder tumors among dye industry workers than in the general population. Benzidine causes cancer of the bladder in humans [2]. Sub-chronic exposure (13 week) to benzidinebased dyes resulted in hepatocellular carcinomas and hepatic ∗ Corresponding author. Present address: C-141 A, Malviya Nagar, Jaipur 302017, Rajasthan, India. Tel.: +91 141 2521502. E-mail addresses: subhasini [email protected], subhasini [email protected] (S. Sharma).

0890-6238/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2004.09.011

neoplastic nodules in rats [3] and carcinomas in very short duration [4]. Azo dyes also disrupt rat embryo development [5–10]. Abnormal development of may fly andCulexlarvae has been observed in anaerobically treated (microbial) textile dye wastewater of Sanganer [11]. In mammals, the azo dyes are metabolized to their parent amines by intestinal microflora. These amine derivatives, unlike their parent compounds, are readily absorbed by the gut [12–14]. Their urinary detection has been reported in several exposed species, including humans [15], monkeys [16], rodents and dogs [17]. The amine derivatives may cause mutagenic effects [18–19], which may lead to cancer, as observed in animals repeatedly exposed to aniline through diet [20]. In addition to carcinogenic and teratogenic effects, azo dyes cause dysfunction of reproductive organs in rodents.

548

V. Suryavathi et al. / Reproductive Toxicology 19 (2005) 547–556

For example, prenatal exposure to Congo red permanently reduced the number of germ cells in male and female mice and rats [21–24]. Another study reported adverse effects of exposure on the gonads of male and female offspring, but only the female offspring displayed reduced fertility [25]. Histopathological changes in the testes of textile wastewaterexposed rats (sub-chronic) included a reduction in the number of germ and Leydig cells, resulting in impaired spermatogenesis [26]. Thus, both the dyes and dye-laced wastewater are highly toxic to animals. Sanganer town, a suburb of Jaipur city, is world famous for its textile printing. Presently, azo dyes (rapid fast, direct and reactive) and aniline are used for textile printing in about 400 smaller scale industries. These facilities discharge approximately 10,000–15,000 kL/day of wastewater of which <10% is treated; the remainder is discharged untreated in drains and shallow pools adjoining printing industries, causing a serious pollution problem [11]. A wide-range of animals including cattle drink the contaminated water, either because of the lack of access to safe water or because of the high salt content of the wastewater (2.45 ± 0.9 g/L). Accidental drinking of pool wastewater resulted in calf mortality [27]. There is incomplete knowledge regarding the potential reproductive toxicity of textile dye wastewater in mammals. Therefore, the present study has examined the potential for male reproductive toxicity following short-term exposure of Swiss albino rats and mice to untreated and treated textile dye wastewaters.

Zn = nil; Pb = nil; Ni = nil; optical density values in the visible region of spectrum = 0.20–0.45. Characteristics of the biologically treated Effluent were as follows: pH = 8.1; conductivity = 3.73 M/cm; TDS = 2.27 g/L; TSS = 40 ppm; COD = 328 ppm; Cu = 0.16 ppm; Fe = 0.48 ppm; Mn = nil; Zn = nil; Pb = nil; Ni = nil; color removal = 67–97%. Levels of Cu and Fe for both the Influent and Effluent sources were slightly higher than the limits set by the Indian Council for Medical Research, New Delhi, for drinking water (Cu = 0.05 ppm; Fe = 0.1 ppm) [28]. Thus, azo dyes were the major pollutants in the Influent, whereas their amount decreased markedly following treatment in the Biological Effluent Treatment Plant, as evident by the pronounced reduction in the OD values (67–97%) of Effluent in the visible region of the spectrum. 2.3. Necropsy The initial and final body weights were recorded on the 1st and 15th days, respectively. After 24 h of the experimental period (16th day), all animals (except three rats per group) were sacrificed to expose their reproductive systems. This number was chosen to be most appropriate to the work and to comply with the recommendation of the Institutional Ethical Committee to limit numbers of animals for research. The reproductive organs (testes, epididymides, seminal vesicles and prostate) were carefully removed, washed in normal saline solution (0.9%), blotted and weighed. 2.4. Biochemical analysis

2. Materials and methods 2.1. Animals Healthy, mature Swiss albino male rats (age: 85–90 days) and mice (age: 45–50 days), weighing 265–270 and 35–40 g, respectively, were acclimated 1 week prior to entry into the experimental protocol. Animals were housed in a well-ventilated facility as per guidelines of the Institutional Ethical Committee (temperature = 25 ± 3 ◦ C; humidity = 40–60%; 12 h light:dark cycle) and fed a standard chow (Hindustan Lever Limited, India) and tap water ad libitum. Thereafter, animals were divided into three groups supplied different water treatments for 15 days, including: university tap water (control group), untreated textile dye wastewater (Influent group), and treated textile dye wastewater (Effluent group). Each group had six rats or mice. 2.2. Dye wastewater The textile dye wastewater samples used during the present study were collected from an Effluent Treatment Plant (ETP) at Madrampura, Sanganer, Jaipur. Characteristics of the Influent were as follows: pH = 6.5; conductivity = 5.02 M/cm; TDS = 2.8 g/L; TSS = 260 ppm; COD = 1021 ppm; Cu = 0.16 ppm; Fe = 0.38 ppm; Mn = 0.1 ppm;

Total protein, lipid and cholesterol contents in testes, epididymides, prostate, seminal vesicle and spermatozoa fluid (diluted 1:20 in Krebs Ringer phosphate buffer) from cauda epididymis were estimated by standard methods [29–31]. Acid phosphatase activity in prostate, and fructose levels in the seminal vesicle, were measured according to WHO [32]. 2.5. Sperm analysis Epididymal spermatozoa were separated as per the method of Brooks [33] and their counts and motility were monitored according to Prasad et al. [34]. The smeared slides of spermatozoa were stained with aqueous eosin (5%) and then observed under a microscope (10× × 40×) to assess any morphological abnormalities as described by Feustan et al. [35]. The abnormalities in the head region were examined and noted for signs that this region was flexed, detached and amorphous; for the tail region, the signs recorded were: coiled, bent and twisted flagellum. Results are expressed as the percentage of overall abnormalities in a given treatment. 2.6. Histopathology of testes Testicular tissues fixed in Bouin’s fixative (48 h) were washed in running tap water for 6–7 h to remove excess

V. Suryavathi et al. / Reproductive Toxicology 19 (2005) 547–556

picric acid. After dehydration in a graded series of alcohol and clearing in xylol, these samples were embedded in paraffin (56–58 ◦ C) and sectioned at 5 ␮m thickness using a rotary microtome (Elite Spencer Microtome-820). Sections were collected on clean slides coated with egg albumin. The slides were heated at 48–50 ◦ C for about 5 min, stored at room temperature (23 ± 2 ◦ C) for 24 h, stained with haematoxylin-eosin, and dehydrated in a graded series of alcohol, cleared in xylol, and mounted in DPX [36]. Two perpendicular diameters of 10 seminiferous tubules were measured each in 10 slides, with the aid of an ocular reticule standardized with a stage micrometer. Values were recorded as mean diameter of seminiferous tubule. Leydig cell numbers per microscopic field were counted followed by the measurement of their diameter at 10× × 40× magnification, using a standardized occulometer [37].

549

Fig. 1. Body weight of control (C) and treated rats and mice. ANOVA – rats: F4,10 = 11.46, P < 0.001; mice: F2,6 = 11.13, P < 0.01. bar = SEM; Student’s ‘t-test: significant at * P < 0.05; ** P < 0.01. E = Effluent; I = Inflluent; Er = recovered Effluent; Ir = recovered Influent.

2.7. Fertility test Fertility test of Influent and Effluent exposed males was carried out immediately after treatment. Another test was made in rats after 45 days of their recovery under normal (control) conditions. During this test period, treated males were caged with females (1:3) of proven fertility (estrous phase) for 1 week. Females were then separated and allowed to gestate to term. For females that failed to deliver a litter, this was considered as a sign of male infertility whereas litter delivery indicated male fertility. Litter size, mortality and weight after 7, 14 and 28 days of growth were examined both in F1 and F2 generations. The recovered males were also sacrificed and all parameters discussed above were monitored. 2.8. Statistics The data are expressed as mean ± SEM. Statistical tests (Student’s ‘t’-test; one and two way ANOVA) were applied to find significant difference between values of various parameters recorded for control and treated animals.

Fig. 2. Male reproductive organ weights of control (C) and treated mice. ANOVA: between treatments = F2,6 = 63.09, P < 0.0001; between organs = F3,6 = 71.69, P < 0.0001. Student’s ‘t-test: significant at * P < 0.05; ** P < 0.01.

3.2. Testicular histopathology Histopathological abnormalities were observed in the wastewater-treated animals (Figs. 4 and 5 and Table 1).

3. Results 3.1. Body weight and weight of individual reproductive organs Body weight was significantly lower in treated animals relative to the control group especially for those animals receiving Influent (Fig. 1). The percent reduction in body weight was greater in mice (10–25%) than in rats (7–10%). Nearly similar trends were observed in the weight of reproductive organs (Figs. 2 and 3). Body weight and the weight of individual reproductive organs for the wastewater-exposed rats returned to normal values by 45 days when these animals were switched to drinking normal tap water (Figs. 1 and 3).

Fig. 3. Male reproductive organ weights of control (C) and treated rats. ANOVA: between treatments = F4,12 = 5.78, P < 0.01; between organs = F3,12 = 150.11, P < 0.0001. Student’s ‘t-test: significant at * P < 0.05; ** P < 0.01 *** P < 0.001.

550

V. Suryavathi et al. / Reproductive Toxicology 19 (2005) 547–556

Fig. 4. (a) Transverse section (TS) of control rat testis (200×), (b) TS of Effluent-treated rat testis (200×), (c) TS of Influent treated rat testis (200×), (d) TS of recovered Effluent rat testis (200×), (e) TS of recovered Influent rat testis (200×). ST = seminiferous tubule; L = Leydig cell; S = spermatozoa; IT = interstitial tissue; CD = cellular debris; BV = blood vessel.

These abnormalities were greatest in the Influent group (Figs. 4a–e and 5a–c) and included the following features: reduction in size of seminiferous tubules (Table 1), expansion of interstitial space, loose arrangement of spermatogonia in seminiferous tubules, reduction in the number and size of Leydig cells, and reduction in the amount of sperm. The latter was evident in the Effluent animals but nearly absent in the Influent animals, as evidenced by the presence of debris throughout the lumen of seminiferous tubules. This implies a partial suppression of germ cell division in Effluent-treated animals, and the near complete suppression in the Influent-treated animals. After 45 days of recovery the diameter of seminiferous tubules and Leydig cells (also their number) increased in both Effluent and Influent ani-

mals, being almost equal to control rats in the former case (Table 1). 3.3. Biochemistry of male reproductive organs Wastewater-treated rats and mice showed a significant reduction in several biochemical parameters monitored in the male reproductive organs (testis, epididymis, prostate gland, seminal vesicle and spermatozoa). These effects included reductions in the total protein content, lipid and cholesterol content. The changes were especially evident among Influentexposed animals (Figs. 6–11) where the statistical significance for the reduction in total protein content and cholesterol content was high (alpha level = 1% in Influent animals

V. Suryavathi et al. / Reproductive Toxicology 19 (2005) 547–556

551

Table 1 Diameter (␮m) of seminiferous tubules (ST) and Leydig cells (LC) (also number) in the control and treated animals Animals

Control Treatments Influent Effluent Post-treatments Influent Effluent

Rats

Mice

ST

LC

Diameter

Diameter

280 ± 4

6.9 ± 0.2

129 ± 4*** 240 ± 7***

3.0 ± 0.2*** 5.0 ± 0.2***

170 ± 8*** 262 ± 3**

3.6 ± 0.3*** 6.0 ± 0.2**

ST

LC

Number

Diameter

Diameter

Number

10 ± 0.7

271 ± 2

6.7 ± 0.01

9 ± 0.9

4 ± 0.6*** 9 ± 0.6

118 ± 2*** 239 ± 2***

2 ± 0.03*** 6.1 ± 0.02***

4 ± 0.6*** 8 ± 0.9

5 ± 0.5*** 9 ± 0.6

NA NA

NA NA

NA NA

NA = not available; ± = S.E. ANOVA – rats: ST-diameter = F4,45 = 136.34, P < 0.0001; LC-diameter = F4,45 = 62.33, P < 0.0001; LC-number = F4,45 = 18.22, P < 0.0001; mice: ST-diameter = F2,27 = 2921.11, P < 0.0001; LC-diameter = F2,27 = 58810.36, P < 0.0001; LC-number = F2,27 = 10.41, P < 0.001. ∗∗ Significant at P < 0.01. ∗∗∗ Significant at P < 0.001.

Fig. 6. Total protein content in male reproductive organs and sperm of control (C) and treated mice. ANOVA: between treatments = F2,8 = 17.38, P < 0.05; between organs = F4,8 = 19.75, P < 0.05. Student’s ‘t-test: significant at * P < 0.05; ** P < 0.01 *** P < 0.001.

Fig. 5. (a) TS of control mouse testis (100×), (b) TS of Effluent-treated mouse testis (100×), (c) TS of Influent treated mouse testis (100×). ST = seminiferous tubule; L = Leydig cell; S = spermatozoa; IT = interstitial tissue; CD = cellular debris; BV = blood vessel.

Fig. 7. Total lipid content in male reproductive organs and sperm of control (C) and treated mice. ANOVA: between treatments = F2,8 = 104.45, P < 0.0001; between organs = F4,8 = 16.62, P < 0.001. Student’s ‘t-test: significant at ** P < 0.01 *** P < 0.001.

552

V. Suryavathi et al. / Reproductive Toxicology 19 (2005) 547–556

Fig. 8. Total cholesterol content in male reproductive organs and sperm of control (C) and treated mice. ANOVA: between treatments = F2,8 = 214.29, P < 0.001; between organs = F4,8 = 19.81, P < 0.05. Student’s ‘t-test: significant at * P < 0.05; ** P < 0.01 *** P < 0.001.

Fig. 9. Total protein content in male reproductive organs and sperm of control (C) and treated rats. ANOVA: between treatments = F4,16 = 62.23, P < 0.0001; between organs = F4,16 = 170.52, P < 0.0001. Student’s ‘t-test: significant at * P < 0.05; ** P < 0.01 *** P < 0.001.

versus 5% in Effluent animals). A nearly similar trend was observed for the fructose content of seminal vesicles, which was reduced in the treated animals (Fig. 12). This effect in seminal vesicles was in contrast to the effect on acid

Fig. 10. Total lipid content in male reproductive organs and sperm of control (C) and treated rats. ANOVA: between treatments = F4,16 = 25.38, P < 0.0001; between organs = F4,16 = 69.32, P < 0.0001. Student’s ‘t-test: significant at * P < 0.05; ** P < 0.01.

Fig. 11. Total cholesterol content in male reproductive organs and sperm of control (C) and treated rats. ANOVA: between treatments = F4,16 = 50.31, P < 0.0001; between organs = F4,16 = 86.55, P < 0.0001. Student’s ‘t-test: significant at * P < 0.05; ** P < 0.01.

Fig. 12. Fructose content in seminal vesicle of control (C) and treated rats and mice. ANOVA – rats: F4,10 = 15.72, P < 0.05; mice: F2,6 = 15.13, P < 0.01. Student’s ‘t-test: significant at * P < 0.05; ** P < 0.01 *** P < 0.001.

phosphatase activity in the prostate gland, which showed the opposite trend (Fig. 13). Most of these changes disappeared when Influent and Effluent rats were reverted to control conditions for 45 days. This led to substantial recov-

Fig. 13. Acid phosphatase level in prostate gland of control (C) and treated rats and mice. ANOVA – rats: F4,10 = 6.25, P <0.01; mice: F2,6 = 130.55, P < 0.0001. significant at * P < 0.05; ** P < 0.01.

V. Suryavathi et al. / Reproductive Toxicology 19 (2005) 547–556

553

Table 2 Sperm counts (×106 /ml) and their motility (%) in the control and treated animals Animals

Control Treatments Influent Effluent Post-treatments Influent Effluent

Sperm (rats)

Sperm (mice)

Count

Motility

Count

Motility

67 ± 3

74 ± 2

58 ± 2

78 ± 1

29 ± 1 (57)*** 42 ± 1 (37)**

21 ± 1*** 52 ± 1***

24 ± 1 (59)*** 42 ± 1 (28)**

14 ± 2*** 56 ± 3**

40 ± 1.2 (40) 60 ± 1.2 (10)

62 ± 1.2 70 ± 1.2

NA NA

NA NA

Data in parenthesis indicate percent reduction in values in comparison to control. NA = not available; ± = S.E. ANOVA – rats: sperm count: F4,10 = 77.21, P < 0.0001; sperm motility: F4,10 = 237.55, P < 0.0001; mice: sperm count: F2,6 = 169.17, P < 0.0001; sperm motility: F2,6 = 259.40, P < 0.0001. ∗∗ Significant at P < 0.01. ∗∗∗ Significant at P < 0.001.

ery of total protein content, lipid, cholesterol and fructose content, although acid phosphatase activity remained lower (Figs. 9–13). 3.4. Sperm analysis Animals drinking the textile wastewaters showed adverse effects on sperm (morphology, numbers, motility and biochemistry) that were more severe in the Influent-treated group for both species (Tables 2 and 3 ; Figs. 6–11). Morphological abnormalities involved the sperm head (headless, amorphous, or bent at a right angle relative to the tail) and the sperm tail (bent, twisted, coiled). Abnormal sperm were more frequent in the Influent animals, and less frequent in animals after the 45 day recovery period (Table 3).

males were allowed to recover for 45 days on a normal tap water supply (Table 4). Litter sizes were, however, decreased in comparison to litters sired from control males (Table 4). Matings between F1 generation animals was also successful although their litter sizes were again smaller than normal. Mortality was similar to the control level in the F1 and F2 generation matings; however, these litters showed slower weight gain than normal, especially in the F2 generation. Monitoring of body weight provides an index of general health status of the animals and such information may also be important for the interpretation of reproductive health. Therefore, some adverse effects of the wastewater exposure continued into the F1 and F2 generations to influence litter size and general health status (as reflected in the body weight) despite normal reproductive behavior.

3.5. Fertility Males exposed to the Influent- and Effluent-treated wastewater failed to sire a litter when these animals were mated with normal fertile females for 1 week immediately after the exposure phase of the experiment. This negative effect on fertility was also evident in the Influent-treated rats after the 45-day recovery period despite the finding that these animals showed normal sperm (Table 3). On the other hand, the Effluent-treated rats sired litters after these

4. Discussion Findings in the present study with acute (15 day) exposure of male rats and mice to drinking water from treated (Effluent) and untreated (Influent) textile dye wastewater of Sanganer, India showed adverse effects on reproductive parameters. These findings are generally consistent with the studies by Gray and Ostby [24], who reported a reduction

Table 3 Percentage of normal and abnormal sperms in the control and treated rats and mice Animals

Control Treatments Influent Effluent Post-treatments Influent Effluent

Sperm (rats)

Sperm (mice)

Normal

Abnormal

Normal

Abnormal

93 ± 0.6

7 ± 0.6

92 ± 1.5

8 ± 1.5

38 ± 3.5*** 54 ± 1.8***

62 ± 3.5*** 46 ± 1.8***

33 ± 2.6*** 46 ± 0.3***

67 ± 2.6*** 54 ± 0.3***

44 ± 1.5*** 72 ± 1.2***

56 ± 1.5*** 28 ± 1.2***

NA NA

NA NA

NA = not available; ± = S.E. ANOVA – rats: F4,10 = 129.89, P < 0.0001; mice: F2,6 = 307.66, P < 0.0001. ∗∗∗ Significant at P < 0.001.

554

V. Suryavathi et al. / Reproductive Toxicology 19 (2005) 547–556

Table 4 Litter size, their mortality and body weights after 7, 14 and 28 days Parameters

Control

Effluent reversal F1 generation

Litter size Weight (g) 7 days 14 days 28 days Mortality (%)

11 ± 0.88 21.4 ± 0.93 27 ± 1.28 45.4 ± 1.20 Nil

5 ± 0.33

(55)**

12.5 ± 0.83 (42)** 22.8 ± 1.23 (16) 42.6 ± 0.69 (6) Nil

F2 generation 7 ± 0.88 (36)* 10.9 ± 1.12 (49)** 17 ± 1.5 (37)** 31.1 ± 1.42 (31)** Nil

± = S.E. Data in parenthesis indicate percent reduction in values in comparison to control. ∗ Significant at P < 0.05. ∗∗ Significant at P < 0.01.

in testicular weight of mice and rats exposed directly to dyes such as congo red, diamine blue and chlorazol Black E, which may be ascribed to a widespread testicular damage [38]. A reduction in testicular protein content has been linked to testicular dysfunction [39]. The reduction in cholesterol content may be linked to altered androgen synthesis [40] and its impaired secretion due to the possible effect of exposure on the size and number of Leydig cells [41]. The effect on testicular protein content and steroid hormone synthesis are likely related, since the latter is sensitive to protein synthesis inhibition [42]. Thus, the decrease in testicular protein content and cholesterol content observed in the wastewater-treated animals might be linked with the suppression of synthesis and secretion of androgen hormone and, in turn could explain the reduction in reproductive organ weights [43] and perhaps impaired spermatogenesis as well. Gray and Ostby [24] also reported reduction in number of germ cells in mice and rats exposed to congo red dye. Copper, a gonadotoxic pollutant present in textile wastewater, has been reported to damage germ cells at specific developmental stages in rams [44] and to induce oligospermia in experimental animals [45]. Upon arriving in the epididymis sperm undergo progressive morphological and physiological maturation, which empowers their motility and competence for fertilization [46,47]. Both parameters were affected in animals exposed to textile dye wastewaters although it is not clear if the toxicity was inherent to the epididymis. Other factors that might affect these parameters include the altered protein, lipid and cholesterol content of sperm. Furthermore, alterations in the seminal fluids could be involved as indicated by the associated decline in fructose content of the seminal vesicles, and the increased acid phosphatase activity in the prostate gland. The latter might reflect enhanced lysosomal hydrolase activity, thereby interfering with important reproductive processes such as capacitation [48] and the acrosome reaction [49] that could contribute to male infertility. The reduced ability of wastewater-treated males to sire a litter when bred to normal fertile females would reasonably be explained by the alterations observed in sperm parameters, including altered sperm morphology, reduced sperm counts, altered sperm motility, and other changes that affect

the competency or capacitance for fertilization. This notion is supported by the fact that exposed male rats recovering for 45 days on normal drinking water were able to mate successfully with fertile females, and that these males showed a recovery of normal sperm morphology, sperm counts, and sperm motility. It is important to note that first generation rats sired by the Effluent-treated males were also fertile, bearing viable litters (F2 generation). Similarly, male mice exposed acutely to congo red for 8–12 days, also displayed normal fertility when mated with untreated females for over 10 months [50]. It is thus evident that the reproductive behavior of recovered Effluent rats, as well as their F1 generation, was normal. On the other hand, the adverse effects on litter size and general health status (in terms of body weight gain) persisted into the F2 generation. Although we have not examined in any detail the physiology of F1 and F2 generations, the limited data on litter size and body weight gain suggests the transmission of a permanent physiological disorder that should be investigated further. In conclusion, the present study indicates that textile dye wastewaters of Sanganer have severe toxic effects on general health states and reproductive systems of male albino rats and mice. The toxicity is, however, relatively mild amongst those animals receiving treated (Effluent) wastewater versus those exposed to the untreated (Influent) wastewater. Although the untreated wastewater caused complete sterility in albino rats, this effect was reversible in the case of treated wastewater. We speculate this toxicity was mediated by the two specific pollutants in the textile wastewater used here, e.g. dyes (major) and copper (minor). These findings demonstrate clearly the importance of proper treatment of textile wastewaters prior to discharge.

Acknowledgements We are thankful to the Head, Department of Zoology for providing laboratory facilities and the Coordinator, Special Assistance Program, Department of Zoology, University of Rajasthan, Jaipur and the Department of Biotechnology, New Delhi for extending financial assistance to the present study.

V. Suryavathi et al. / Reproductive Toxicology 19 (2005) 547–556

References [1] Boeniger M. Carcinogenecity and metabolism of azo dyes, especially those derived from benzidine. USDHHS (NIOSH) Tech Rep 1980:80–119. [2] Haley TJ. Benzidine revisited: a review of the literature and problems associated with the use of benzidine and its congeners. Clin Toxicol 1975;1:13–42. [3] National Cancer Institute, Carcinogenesis Testing Program. 13-week subchronic toxicity studies of Direct Blue 6, Direct Black 38 and Direct Brown 95 dyes. Carcinogenesis Technical Report US Government Printing Office, Washington, DC; 1978, p. 108. [4] National Institute for Occupational Safety, Health Center for Disease Control. Special hazard review of benzidine-based dyes. Cincinnati, OH: DHHS (NIOSH) Publication; 1980. p. 80–109. [5] Wilson JG. Teratogenic activity of several azo dyes chemically related to trypan blue. Anat Rec 1955;123:313–34. [6] Beaudoin AR, Pickering MJ. Teratogenic activity of several synthetic compounds structurally related to trypan blue. Anat Rec 1960;137:297–305. [7] Beaudoin AR. The teratogenicity of congo red in rats. Proc Soc Exp Biol Med 1964;117:176–9. [8] Lloyd JB, Beck F. The relationship of chemical structure to teratogenic activity among bisazo dyes: a re-evaluation. J Embryol Exp Morphol 1966;16:29–39. [9] Beck F, Lloyd JB. The teratogenic effects of azo dyes. Adv Teratol 1966;1:131–93. [10] Beck F. Induced cell injury and cell death as a cause for congenital malformation in rats. Histochem J 1981;13:667–79. [11] Sharma KP, Sharma K, Bhardwaj SM, Chaturvedi RK, Sharma S. Environment impact assessment of textile printing industries in Sanganer, Jaipur: a case study. J Indian Bot Soc 1999;78:71–85. [12] Bowman MC, Oller WL, Nony CR, Rowland KL, Billedeau SM, Lowry LK. Metabolism and distribution of two 14 C benzidinecongener-based dyes in rats as determined by GC, HPLC and radioassays. J Anal Toxicol 1982;6:164–74. [13] Bowman MC, Nony CR, Billedeau SM, Martin JL, Thompson Jr HC, Lowry LK. Metabolism of nine benzidine-congener-based azo dyes in rats based on gas chromatographic assays of the urine for potentially carcinogenic metabolites. J Anal Toxicol 1983;7:55–60. [14] Bos RP, Krieken W, Seijsters L, Koopman JP, Dejonge HR, Theuws JLG, et al. Internal exposure of rats to benzidine-based dyes after intestinal azo reduction. Toxicology 1986;40:207–13. [15] Lowry LK, Tools WP, Boeniger MF, Nony CR, Bowman MC. Chemical monitoring of urine from workers potentially exposed to benzidine-derived azo dyes. Toxicol Lett 1980;7:29–36. [16] Rinde E, Troll W. Metabolic reduction of benzidine azo dyes to benzidine in the rhesus monkey. J Natl Cancer Inst 1975;55:181–2. [17] Lynn RK, Danielson DW, Ilias AM, Wong K, Kennish JM, Mathews HB. Metabolism of bisazobiphenyl dyes derived from benzidine, 3,3 -dimethylbenzidine or 3,3 -dimethoxybenzidine to carcinogenic aromatic amines in dog and rat. Toxicol Appl Pharmacol 1980;56:248–58. [18] Miller JA, Miller EC. The carcinogenic azo dye. Adv Cancer Res 1953;1:340–90. [19] Chung KT, Fulk GE, Andrews AW. Mutagenicity testing of some commonly used dyes. App Environ Microbiol 1981;42:641–8. [20] USEPA. Aniline fact sheet, pollution prevention and toxics. 1985; 749:F-95-002. [21] Gray Jr LE. An extended evaluation of an in vivo teratology screen utilizing postnatal growth and viability in the mouse. Terato Carcino Muta 1984;4:403–26. [22] Gray Jr LE, Ostby J, Ferrell J, Sigmon R, Cooper R, Linder R, et al. Correlation of sperm and endocrine measures with reproductive success in rodents. In: Sperm measures and reproductive success. Liss, NY: Institute for Health Policy Analysis Forum on Science, Health and Environmental Risk Assessment; 1989. p. 193–209.

555

[23] Gray Jr LE, Ostby JS, Ferrell J, Rehnberg G, Linder R, Cooper R, et al. A dose-response analysis of methoxychlor-induced alterations of reproductive development and function in the rat. Fundam Appl Toxicol 1989;12:92–108. [24] Gray Jr LE, Ostby JS. The effects of prenatal administration of azo dyes on testicular development in the mouse: a structure activity profile of dyes derived from benzidine, dimethyl benzidine or dimethoxy benzidine. Fund Appl Toxicol 1993;20:177–83. [25] Gray Jr LE, Marshall R, Setzer RW. Correlation of ejaculated sperm numbers with fertility in the rat. Toxicologist 1992;12:443. [26] Mathur N, Krishnatrey R, Sharma S, Sharma KP. Toxic effects of textile printing industry effluents on liver and testes of albino rats. Bull Environ Contam Toxicol 2003;71:453–7. [27] Sharma K. Environmental impact assessment of textile industry wastewaters in Sanganer environment. Ph.D. Thesis. University of Rajasthan, Jaipur, India; 2000. [28] Goel PK, Sharma KP. Environmental guidelines and standards in India. Jaipur: Technoscience Publications; 1996. [29] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with folin phenol reagent. J Biochem 1951;193:265–75. [30] Frings CS, Fendley JW, Dunn RT, Queen CA. Improved determination of serum lipids by the sulphophospho vanillin reaction. Clin Chem 1972;18:673–4. [31] Francy RJ, Amador E. Serum cholesterol measurement on ethanol extraction and ferric chloride sulphonic acid. Clin Chem Acta 1968;2:255. [32] WHO. Laboratory manual for the examination of the human semen and sperm-cervical mucus interaction. New York: Cambridge University Press; 1999. [33] Brooks DE. Activity of androgenic control of glycolytic enzymes in the epididymal spermatozoa of the rats. Biochem J 1976;156: 527–37. [34] Prasad MRN, Chinoy NJ, Kadam KM. Changes in succinic dehydrogenase levels in rat epididymis under normal and altered physiological conditions. Fertil Steril 1972;23:186–90. [35] Feustan MH, Bodnai KR, Kerstette SL. Reproductive toxicity of 2-methoxy ethanol applied dermally to occluded and non-occluded sides in male rats. Toxicol Appl Pharmacol 1989;100:145–65. [36] Humason GL. Animal tissue techniques. San Francisco: Freeman; 1972. [37] Russel LD, Ettlin RA, Hikim APS, Clegg ED. Histological and histopathological evaluation of the testes. Clearwater, FL: Cache River Press; 1990. [38] Keel AB, Abhey TO. Influence of bilateral cryptorchidism in the mature rat: alteration in testicular function and serum hormonal levels. Endocrinology 1980;107:1226–33. [39] Robaire B, Hermo L. Efferent ducts, epididymis and vas deferens; structure, functions and their regulation. In: Knobil E, Neil J, editors. The physiology of reproduction, vol. 1. New York: Raven Press; 1988. p. 999–1080. [40] Perlman PL. The functional significance of testis cholesterol in the rat: histochemical observation on testis following hypophysectomy and experimental cryptorchidism. Endocrinology 1950;46: 341–6. [41] Guyton AC, Hall JE. Reproductive and hormonal functions of the male (and the pineal gland). In: Text book of medical physiology. 9th ed; 1998. p. 1003–16. [42] Stocco D. StAR protein and regulation of steroid hormone biosynthesis. Ann Rev Physiol 2001;63:193–213. [43] Mukherjee M, Chattopadhyay S, Mathur PP. Effect of flutamide on the physiological status of epididymis and epididymal spermatozoa. Andrology 1992;24:113–6. [44] Vrzgulova M. Histological and submicroscopical findings on the seminiferous parenchyma in rams after copper oxide intoxication from industrial emissions. Funct Dev Morphol 1993;3:115–9. [45] Chowdhury AR, Naha N. Heavy metal induced toxicity in male reproductive system. Ind J Toxicol 2002;9:61–7.

556

V. Suryavathi et al. / Reproductive Toxicology 19 (2005) 547–556

[46] Bedford JM. Development of the fertilizing ability of spermatozoa in the epididymis of the rabbit. J Exp Zool 1966;163:319. [47] Cooper TG, Waites GMH, Nieschlag E. The epididymis and male fertility. Int J Androl 1986;9:81–90. [48] Gwatkin RBL, Hutchinson CF. Capacitation of hamster spermatozoa by ␤-glucuronidase. Nature 1971;229:343–4.

[49] Mc Rorie RA, Williams WL. Biochemistry of mammalian fertilization. Ann Rev Biochem 1974;43:777–803. [50] Gray Jr LE, Ostby JS, Kavlock RJ, Marshall R. Gonadal effects of fetal exposure to the azo dye congo red in mice: infertility in female but not male offspring. Fund Appl Toxicol 1992;19:411– 22.