Potential androgenic effects of urban sewage sludge in male rats

Abstract

The processing of urban sewage by waste water treatment plants produces a pasty mixture of complex organic materials (microorganisms, plants, chemical contaminants) and inorganic (minerals, metals, etc.) known as sewage sludge (SS). Facing the possibility of using this mixture for agricultural soil enrichment this study was designed to assess in vivo the potential endocrine disrupting activity of a SS sample using pubertal male Wistar rats or weanling rats in a modified Hershberger assay. Pubertal male rats (42-days old) were exposed ad libitum through adulthood (98-days old) to a diet containing 0, 2500, 5000, 10000 or 20000 ppm of SS. In another study, weanling male rats (21-days old) were treated daily for 10 d by gavage with flutamide (anti-androgenic positive control), testosterone propionate s.c. (androgenic positive control), or SS at 10000 and 20000 ppm in the diet. Despite no alterations in body and reproductive organs weights, diet consumption or sperm count, SS treatment impaired the sperm quality of pubertal rats, as indicated by a decreased proportion of sperm showing progressive movement. No evidence of SS-induced adverse effects was found in weanling rats. The treatments did not induce direct androgenic effects in both life periods studied in rats. However, the observed impaired sperm motility in the SS-treated pubertal rats raises some concern about the use of this material as soil additive, or in other applications where human and animals are potentially exposed.

Keywords:

• endocrine disruption
• male rat development
• sperm count
• sewage sludge

abbreviations

DHT	=	dihydrotestosterone
ED	=	endocrine disruptors
FSH	=	Follicle-Stimulating Hormone
FLU	=	flutamide
LH	=	Luteinizing Hormone
TP	=	testosterone propionate
SS	=	sewage sludge
T	=	Testosterone
WWTP	=	Waste Water Treatment Plant, s.c. subcutaneus injection.

Introduction

In recent years, many studies showed that some environmental contaminants can mimic or antagonize the effects of steroid hormones such as estrogens and androgens, causing what is known as endocrine disruption (ED).¹ Endocrine disrupters are exogenous agents capable of interfering with the synthesis, secretion, transport, binding, action or elimination of natural hormones that are responsible for the maintenance of homeostasis, reproduction, development and normal behavior of the organism.^1,2 Epidemiological investigations indicate increased incidences of reproductive tract malformations and decreased male fertility, as well as increased incidence of testis, prostate, breast and vagina neoplasms.³ These observations may possibly be associated with endocrine disrupting substances occurring in the environment.^4,5

Environmental protection is a goal of most governments in the world and sanitation actions are essential for natural resource conservation and public health protection. The volume of treated waste water in Brazil is still very small, only 20.2% of the municipalities collect and treat, while 32% only collect and 47.8% do not collect.^6,7 The transformation of sewage into usable water by the sewage treatment plants (WWTP) produces as a by-product a complex mixture of inorganic, organic and biological materials, called “WWTP sewage sludge” (SS). In the USA, the term sewage sludge has been replaced by “biosolids”, which indicates that the sludge has been treated to achieve Government standards for reuse.⁸ Basically, SS results from domestic and industrial wastes whose chemical composition may vary according to their origin, region, type of treatment and time of the year,⁸ and therefore, will contain different concentrations of ED, which also may depend on the contaminant regulation policies/monitoring programs of each country, state or city. Agronomic studies indicate SS can serve as a potent fertilizer, with essential plant nutrients, rich in organic matter, acting as a soil conditioner and improving its structure. However, hazardous agents may be present in the SS, including heavy metals, pesticides, drugs and solvents. These compounds pose potential risks to the organisms exposed to them directly or indirectly. Therefore, it is necessary to assess the potential human health impact resulting from application of SS in the soil.⁹ Clarke and Smith (2011) presented a revision of studies quantifying these emerging contaminants in SS samples from different countries, and showed that the concentration of organic pollutants after SS application in agriculture is a priority source of risk to humans and ecosystems.¹⁰ In Brazil, there is no current SS characterization study regarding emerging organic contaminants, except for a report from a project developed by CETESB⁷ and a paper from Da Matta and Umbuzeiro (2014).¹¹

To assess the endocrine disrupting potential of chemicals, in vitro tests in combination with short and long term assays with experimental animals have been developed to identify modes and mechanisms of action of those chemicals in specific target organs.^2,12 As an alternative to the standard in vivo Hershberger test (OECD 441)¹³ that requires surgical male castration, Ashby et al. (2004)¹⁴ and Tinwell et al. (2007)¹⁵ proposed a 10-day assay using intact, immature weanling rats, thus avoiding the surgical procedure. With the entire male reproductive system intact, this assay can be used for the detection of compounds with androgenic and antiandrogenic activities. In the present study we evaluated the endocrine disruption potential of a SS sample from a WWTP of the State of São Paulo, Brazil, using 2 experimental male rat models: one used immature weanling animals treated (except for negative control) with testosterone propionate (TP) and flutamide (FLU) or scaled concentrations of SS for 10 days; and the other used intact pubertal animals exposed to different SS doses for 8 weeks, covering an entire period of spermatogenesis (52 days).¹⁶

Results

Immature male rat assay

In-life parameters and organ weights

At termination, there were no differences in body weight gain or water/food consumption between animals exposed to testosterone propionate (TP) + SS treatment and the TP-only group (Table 1). TP significantly increased the relative weights of the epididymis, prostate, and seminal vesicle, and significantly decreased the relative weight of the testes when compared with the negative control group. In the group also treated with flutamide (FLU) there were significant decreases in the relative weights of the testes, epididymis, prostate and seminal vesicles, compared with the group stimulated with TP, demonstrating the anti-androgenic effect of FLU. In the group treated with TP+10000 SS, there was a significant increase in the relative weight of the ventral prostate (Table 2). The other examined organs (liver, kidneys and adrenals) showed no significant differences in weights among all SS-treated groups. Overall, the SS results did not show antiandrogenic activity in this test.

Table 1. Body weight gain, mean water/food consumption and SS intake of immature animals¹ in different groups at the end of the experiment

Groups (ppm)	Number of animals	Body weight gain^2	Water consumption (ml/rat/day)	Food consumption (g/rat/day)	SS consumption (g/kg bw per day)
Negative Control	10	59.4 ± 7.78	17.13 ± 4.87	10.43 ± 3.38	—
TP (androgenic control)	10	71.7 ± 2.95	19.63 ± 5.69	11.41 ± 3.34	—
TP+FLU (anti-androgenic control)	10	66.1 ± 3.35	19.90 ± 5.54	10.56 ± 3.14	—
TP+SS 10000 ppm	10	69.7 ± 5.17	16.75 ± 3.77	10.17 ± 3.26	0.91 ± 0.02
TP+SS 20000 ppm	10	69.2 ± 9.41	17.83 ± 4.25	10.83 ± 3.40	1.85 ± 0.04

¹ Values are mean ± SD.

² Body weight gain = final minus initial body weight.

Note: Animals were 21-day old at the start of treatment. They were dosed on 10 consecutive days with an oral dose of test compound at the appropriate concentration levels and were stimulated by an sc injection of 1mg/kg/day TP; ¹ Values are mean ± SD, and were compared to the final body weight in g for the liver, and in mg for the other organs; Comparisons were performed first between the Negative control and TP-only groups for statistical significance (^a P < 0.05) to ensure that the TP induced the appropriate androgenic effects. A second comparison was performed between TP and TP plus sewage sludge (SS) or FLU (^b P < 0.05); TP: testosterone propionate; FLU: flutamide.

Table 2. Relative organs weights (%) of immature animals in different groups at the end of the experiment

			Relative weights (%)
Groups	Number of animals	Final body weights (g)	Liver	Kidneys	Adrenals	Testes	Epididymes	Ventral prostate	Full seminal vesicle
Negative Control	10	104.8 ± 12.74	5.06 ± 0.28	500.69 ± 43.12	23.2 ± 27.3	364.32 ± 16.82	40.19 ± 3.24	35.56 ± 4.11	14.77 ± 2.73
TP (androgenic control)	10	120.1 ± 7.11	5.28 ± 0.15	496.01 ± 37.34	10.8 ± 1.27	166.97 ± 14.82	52.31 ± 5.16	68.45 ± 9.64	104.32 ± 36.67
TP+FLU (anti-androgenic control)	10	109.3 ± 10.05	5.28 ± 0.29	514.47 ± 42.21	26.8 ± 33.6	126.64 ± 36.19	30.18 ± 4.04	43.79 ± 4.43	56.05 ± 25.75
TP+SS 10000 ppm	10	111.3 ± 8.14	5.22 ± 0.30	503.82 ± 26.49	12.2 ± 3.45	154.65 ± 17.65	52.52 ± 7.28	75.22 ± 13.79	115.84 ± 25.67
TP+SS 20000 ppm	10	117.3 ± 11.84	5.18 ± 0.56	525.25 ± 59.77	11.6 ± 1.89	187.12 ± 32.25	54.37 ± 8.17	64.99 ± 16.87	93.04 ± 36.35

Note: Animals were 21-day old at the start of treatment. They were dosed on 10 consecutive days with an oral dose of test compound at the appropriate concentration levels and were stimulated by an sc injection of 1mg/kg/day TP; ¹ Values are mean ± SD, and were compared to the final body weight in g for the liver, and in mg for the other organs; Comparisons were performed first between the Negative control and TP-only groups for statistical significance (^aP < 0.05) to ensure that the TP induced the appropriate androgenic effects. A second comparison was performed between TP and TP plus sewage sludge (SS) or FLU (^bP < 0.05); TP: testosterone propionate; FLU: flutamide.

Histology of reproductive organs

The prostate and seminal vesicles in animals treated with TP demonstrated large secretory acini, suggestive of a stage of maturation more advanced than the negative controls. Treatment with FLU inhibited the effects of TP, as indicated by the reduction in organ weights and smaller acini with slight secretion.^14,15 There was no effect of all SS-treated groups on the histology of the same collected organs.

Pubertal male rat assay

In-life parameters and organ weights

In this study, there was increased mean food and water intakes by the animals treated with the SS diets, but no significant body weight gain differences were observed at termination (Table 3). The mean relative kidney weight from the 20000 ppm SS animals was significantly increased when compared to the negative control group. In addition, the epididymis of rats treated with SS diets at 5000 and 10000 ppm showed significantly decreased relative weights (Table 4). Considering the absence of histological alterations or a dose-response, and the relatively small variation among the other groups, these results were considered toxicologically relevant.

Table 3. Body weight gain, mean water/food consumption and SS intake of pubertal animals¹ (98-day old) in different groups at the end of the experiment

Groups (ppm)^2	Number of animals	Body weight gain^3	Water consumption (ml/rat/day)	Food consumption (g/rat/day)	SS consumption (g/kg bw per day)
Negative control	10	247.6 ± 27.3	37.09 ± 2.61	23.36 ± 2.16	—
SS (2500)	10	239.4 ± 17.5	38.96 ± 4.88	25.59 ± 2.68^*	0.20 ± 0.004
SS (5000)	10	247.3 ± 19.4	37.29 ± 2.82	25.99 ± 3.88^*	0.40 ± 0.004
SS (10000)	10	257.1 ± 26.2	38.64 ± 2.99^*	27.08 ± 1.48^*	0.84 ± 0.003
SS (20000)	10	232.6 ± 14.9	40.81 ± 2.84^*	26.95 ± 1.93^*	1.67 ± 0.02

¹ Values are mean ±SD.

² Sewage Sludge (SS) in the diet.

³ Body weight gain = final minus initial body weight.

* Means differ from Negative control at P < 0.05.

Table 4. Relative organs weights (%)¹ of pubertal animals (98-day old) in different groups at the end of the experiment

Groups^2 (ppm)	Number of animals	Final body weights(g)	Liver	Kidneys	Adrenals	Testes	Epididymes	Ventral prostate	Full seminal vesicle
Negative control	10	409 ± 30.9	3.48 ± 0.23	0.29 ± 0.02	0.01 ± 0.001	0.36 ± 0.02	0.11 ± 0.01	0.08 ± 0.01	0.26 ± 0.04
SS (2500)	10	421.2 ± 21.2	3.63 ± 0.17	0.29 ± 0.02	0.01 ± 0.001	0.37 ± 0.03	0.11 ± 0.01	0.09 ± 0.01	0.23 ± 0.04
SS (5000)	10	433.3 ± 23.7	3.47 ± 0.42	0.30 ± 0.01	0.01 ± 0.001	0.36 ± 0.03	0.10 ± 0.01^*	0.07 ± 0.03	0.23 ± 0.07
SS (10000)	10	426.6 ± 34.3	3.75 ± 0.30	0.29 ± 0.02	0.01 ± 0.001	0.35 ± 0.02	0.10 ± 0.01^*	0.08 ± 0.01	0.24 ± 0.04
SS (20000)	10	413.1 ± 31.1	3.49 ± 0.29	0.31 ± 0.02^*	0.01 ± 0.001	0.37 ± 0.02	0.11 ± 0.01	0.07 ± 0.01	0.22 ± 0.02

¹ Values are mean ± SD, and were compared to the final body weight in g for the liver, and in mg for the other organs.

* Means differ from Negative control at P < 0.05.

² Sewage Sludge (SS) in the diet.

Hormone measurements

Serum levels of FSH were significantly decreased in all groups treated with the SS diet (Table 5). Moreover, the SS-fed groups showed diminished LH levels, which varied between 33–65% of the control levels, but this reduction was significant only for the 2500 ppm-fed group. These results may indicate an hypothalamic-hypophysis-gonadal axis alteration. Otherwise, the levels of testosterone (T) and dihydrotestosterone (DHT) did not differ significantly when compared with the control group.

Table 5. Plasma hormones levels¹ of pubertal animals (98-day old) in different groups at the end of the experiment

Groups^2 (ppm)	Number of animals	FSH (ng/ml)	LH (ng/ml)	DHT (pg/ml)	T (ng/ml)
Negative control	10	13.63 ± 2.21	19.84 ± 16.16	136.05 ± 57.99	1.49 ± 1.08
SS (2500)	10	10.31 ± 2.16^*	6.56 ± 2.35^*	145.15 ± 80.84	2.45 ± 2.11
SS (5000)	10	9.58 ± 0.97^*	8.12 ± 1.81	140.38 ± 90.16	1.68 ± 1.34
SS (10000)	10	9.99 ± 1.38^*	12.82 ± 10.86	96.91 ± 42.52	0.88 ± 0.48
SS (20000)	10	9.46 ± 1.24^*	11.44 ± 6.04	146.30 ± 101.32	2.85 ± 2.76

¹ Values are mean ±SD.

² Sewage Sludge (SS) in the diet. FSH: Follicle-Stimulating Hormone; LH: Luteinizing Hormone; DHT: dihydrotestosterone; T: Testosterone.

* Means differ from Negative control at P < 0.05.

Analyses of sperm quality (amount, morphology and motility)

The number of sperm in the testis and epididymis, daily sperm production, and sperm transit time in the epididymis were similar among experimental groups, with no significant influence of dietary SS (Table 6). In the analyses of the dynamics of spermatogenesis, there were also no significant differences between SS-treated and negative control groups (Table 6). There was also no histological alteration of the testes or epididymis of these animals. Otherwise, the proportion of spermatozoa with progressive trajetory (type A) was significantly reduced in the SS-treated groups; the amount of sperm with non-progressive trajectory (type B) or inmotile (type C) were increased (Table 7).

Table 6. Sperm counts¹ of pubertal animals (98-day old) in different groups at the end of the experiment

Groups (ppm)	Number of animals	Sperm number in testis (x106)	Daily sperm production (x106/testis/day)	Relative sperm production (x106/g testis/day)	Sperm number in the caput/corpus epididymis (x106)	Sperm transit time in the caput/corpus epididymis (days)	Sperm number in the cauda (x106)	Sperm transit time in the cauda (days)
Negative control	10	176.57 ± 18.99	28.95 ± 3.11	21.71 ± 2.63	101.92 ± 13.63	3.54 ± 0.51	156.53 ± 18.86	5.43 ± 0.59
SS (2500)	10	180.46 ± 18.07	29.58 ± 2.96	20.73 ± 1.26	111.53 ± 12.41	3.79 ± 0.45	154.35 ± 17.54	5.28 ± 0.86
SS (5000)	10	191.49 ± 21.40	31.39 ± 3.51	22.14 ± 1.91	98.05 ± 14.91	3.15 ± 0.53	139.98 ± 16.91	4.48 ± 0.42
SS (10000)	10	178.87 ± 21.01	29.32 ± 3.44	21.72 ± 1.71	100.92 ± 18.98	3.48 ± 0.76	140.27 ± 15.21	4.85 ± 0.81
SS (20000)	10	180.99 ± 29.07	29.67 ± 4.77	20.79 ± 2.05	101.15 ± 12.92	3.47 ± 0.56	146.07 ± 23.18	5.03 ± 1.09

¹ Values are mean ± SD.

Table 7. Sperm motility¹ of pubertal animals (98-day old) in different groups at the end of the experiment

Groups (ppm)	Number of animals	Type A sperm (%)	Type B sperm (%)	Type C sperm (%)
Negative Control	10	63.3 ± 12.4	23.7 ± 9.5	13.9 ± 2.9
SS (2500)	10	48.1 ± 12.1	28.7 ± 11.6	23.7 ± 5.3
SS (5000)	8	61.7 ± 9.1	22.4 ± 8.1	16.4 ± 4.7
SS (10000)	10	30.9 ± 11.1	41.5 ± 13.2	27.9 ± 8.9
SS (20000)	9	26.9 ± 9.3	40.4 ± 7.5	33.0 ± 9.6

¹ Values are mean ±SD. Means differ from negative control at *P < 0.05; Type A sperm:mobile with progressive trajectory; Type B sperm: mobile with non-progressive trajectory; Type C sperm: immotile.

Discussion

Studies to evaluate the toxicity of an environmental mixture are traditionally conducted based on target compounds that are determined in the samples of interest. In this sense, the direct toxicological evaluation of mixtures as SS differs from conventional approaches.^17,18 Although chemical determinations of target compounds are important, such evaluations are not enough for a complete hazard characterization, especially in the case of endocrine disruption. This is because very low amounts of a chemical can cause this effect and in several situations chemical analyses may not be sensitive enough to detect the compounds of interest. Many short term in vitro bioassays have been developed to detect endocrine disrupting activity of environmental samples,^19-21 however, there is insufficient insight concerning the correlation between these short-term bioassays and the prediction of long-term in vivo adverse effects on the reproductive system.²² To provide more comprehensive information about the endocrine disrupting potential of an actual complex environmental mixture an SS sample was selected and tested in 2 rat in vivo studies based on 2 important mammalian life stages (immature and pubertal).

In our study with immature male rats, changes in testes, epididymis, seminal vesicle, and prostate weights in the groups treated with TP and TP + FLU were consistent and provided confidence regarding the androgenic/antiandrogenic procedures used. The SS dietary treatments did not induce adverse effects in male rats in any of the tested parameters. In the group treated with 10000 ppm SS, there was a significant increase of the ventral prostate mean weight compared to the negative control and to the group only exposed to TP. Because this change was an isolated event with no dose-response pattern, it was considered of no biological relevance.

Hormonal homeostasis is modulated by the life stage of the mammalian organism and, therefore, susceptible to endocrine disrupting action in puberty, with implications for adulthood.²³ FSH plays a major role on Sertoli cells during development, but in pubertal rats the effects are more subtle and also connected to the function of Sertoli and germ cells.^24-26 LH stimulates Leydig cells to secrete androgens - particularly testosterone, which is important for the maintenance of the reproductive organs, development of male secondary sexual characteristics, maintenance of spermatogenesis, including sperm maturation, and consequently for fertility.²⁷ The results with 98-day old rats indicated significantly decreased plasma concentrations of FSH in all groups treated with SS diet, and LH (between 33–65%, which was significant only at the lowest dose). Although not clearly correlated to the observations of the present study, these hormone levels results may indicate a treatment-related hypothalamic-hypophysis-gonadal interaction. This can possibly induce reproductive impairment following a chronic exposure and/or exposure at the windows of susceptibility periods.

Otherwise, the daily sperm production and sperm transit time through the epididymis did not differ significantly among the pubertal SS-treated groups. There were also no histological alterations of the testes or epididyms of these animals. The histology of the testes was evaluated to detect possible impairment of the spermatogenesis process, which is considered a sensitive parameter of testicular toxicity. Histomorphometry of the testes is also a quantitative, sensitive and supportive analysis for ED detection and will be considered in future studies. No morphological changes were observed in these 98-day old animals' seminiferous tubules, suggesting that the SS treatment did not induce damage to the process of spermatogenesis. However, the reduction in the number of spermatozoa with progressive trajectory, along with a corresponding increase of non-progressive and inmotile sperm in the groups exposed to 10000 ppm or 20000 ppm SS could represent impaired sperm quality, with potential for decreased fertility of rats exposed to those 2 highest concentrations of the sewage sludge.

There are reports of a progressive decrease of sperm quality in humans during the last 50 y^28,29 and this has been associated with environmental factors and lifestyle.^30-032 Unlike the genetic causes, environmental causes of male infertility are of particular interest because they are susceptible to preventive or mitigation measures. A significant and correctable factor is the change in nutrition and lifestyle. Experimental animal studies have demonstrated the importance of nutrition on spermatogenesis.^33-35

Rodent fertility is not a very sensitive parameter for toxicological studies, because these animals have an excessive production of sperm. In humans, however, the sperm production is much less abundant.³⁶ Thus, the potential for a toxic agent to affect male reproduction is probably greater for humans, since in rats a reduction of 50 to 90% in the number of fertile sperm does not decrease fertility.³⁷ The number of sperm present in the testes and its total daily production are also important indicators of male fertility.¹⁴ Although no effect was seen in daily sperm production in 98-day old rats, impaired motility is dependent on changes in the maturation of spermatozoa in the epididymis,^38,39 suggesting that this organ, more than the testis, could be sensitive to possible SS toxicity. The present results on sperm count in the testis and epididymis, as well as the kinetic analysis of spermatogenesis in pubertal rats, showed that the this process was occurring normally and was not altered by SS treatment. However, further analysis of other comprehensive male reproductive parameters, including fertility and reproductive performance, are needed to confirm the gametes functional integrity after SS exposure, especially because this sperm quality impairment could be secondary to an anti-androgenic effect.

The differences between the sensitivities of immature and pubertal animals to SS also need further studies, but could also be explained by the design of each experiment. In the immature animals, the chemical castration could reduce the detection of SS endocrine low dose effects. There are also age-dependent differences among the germ cells complement (related to proteins secreted by seminiferous tubules cells)⁴⁰ and expression/levels of induction of nuclear receptors, for example (e.g., ER-β induction increases after puberty).⁴¹

The present studies indicated that the SS sample used has no androgenic activities, but caused sperm quality impairment in pubertal male rats exposed for 8 weeks to 2500, 5000, 10000 and 20000 ppm of SS in the diet. Even though there is weak evidence that human health has been adversely affected by exposure to endocrine-active chemicals, there is sufficient evidence and laboratory supportive studies to conclude that adverse endocrine‐mediated effects have occurred in some wildlife species.⁴² The results of the present study must be taken into account in decisions about disposal of SS material in soil to reduce the reintroduction of endocrine-disrupting substances into the environment through agricultural use, for example, or other applications where human and animals are potentially exposed. The amount of SS ingested by the rats in our study was relatively small, even though endocrine disruption effect was observed. The characterization of SS samples is also important to verify which and in what concentrations ED substances are present in tons of this material that are daily produced. If SS is going to be considered as a commercial product, as it is now in Brazil, it should be regulated appropriately including the possible risks of its use in agricultural soils. A proper evaluation of the risks involved in the manipulation of SS by Waste-Water Treatment Plants operators and agricultural applicators should be performed, even when the main objective is to amend the soil properties to enhance its productivity. This is not in current practice in Brazil right know, as far as we know, but in any case, other studies such as dermal absorption, inhalation, skin irritation, etc are necessary to calculate the risk of non-dietary exposure as well. At the moment our study can offer some information on hazard characterization of this SS sample.

Material and Methods

Chemicals

Testosterone propionate (TP) (CAS: 57–85–2, 98% purity) and flutamide (FLU) (CAS: 13311–84–7, >99% purity) were purchased from the Sigma-Aldrich (Saint Quentin Fallavier, France) and were suspended in corn oil. The suppliers of all additional compounds are mentioned in the text.

Sludge sample

Treated sludge sample from a selected sewage treatment plant, herein named PCJ1, was collected on January 21th, 2008. The PCJ1 plant treats mainly urban effluents and textile industrial wastewater from an area with approximately 5 million people. The WWTP generates around 7 tons/day of treated SS (DAEE, 2004–2007).^43,44 The sewage is treated by biological filters, the solid phase is anaerobically digested, centrifuged and bed dried; and the water content is reduced using acrylamide polymer. The treated SS sample was transported to the laboratory and stored at −20°C until processed. Chemical analysis of this PCJ1 sample has been presented elsewhere,⁷ and was in compliance with the standards issued by Brazil (Resolution CONAMA 357/2006),⁴⁵ EUA (USEPA 1993)⁴⁶ and Europe (Directiva 1986/278 EEC).⁴⁷ Chlorinated benzenes, persistent organic pollutants, phthalate esters, phenolic and aromatic hydrocarbons, which are not regulated, were all below the detection limits of the method (0.005 mg/kg).⁷ Nevertheless, this PCJ1 sample was mutagenic for Salmonella typhimurium (Ames test) with 710 revertants/g (TA98+S9) and 1,500 rev/g (TA100+S9).⁷ Samples from the same WWTP (PCJ1) were also genotoxic in the Tradescantia micronucleus assay and water elutriates from this sample were acutely toxic to Daphnia similis and Vibrio fischeri.^7,48

Experimental diet

Sewage sludge samples were maintained at −20°C until mixed with a commercial powder chow (Purina-Labina Evialis, Paulínia, SP/Brazil) at concentrations of 2500, 5000, 10000 and 50000 ppm. Doses selection was based on previously published data with another SS sample but generated under the same treatment conditions.^49,50 The powdered mixture was pelleted using an industrial mixer (Prensa Peletizadora Chavantes, CAF-model M60, PR). The pellets were dried by ventilation at room temperature for 24 h. Diets with different sludge concentrations were stored in labeled plastic bags and maintained in a −20°C freezer for a maximum period of 30 d

Animals and housing

The study design was approved by the Ethical Committee in Animal Experimentation (UNESP Medical School, SP, Brazil, Protocol No. 775). Newborn male Wistar rats (Multidisciplinary Center for Biological Investigation - CEMIB, Brazil) were weaned, weighed and allocated to treatment groups at postnatal day (PND) 21. The animals were maintained in an environmentally controlled facility: room temperature at 22 ± 2°C, humidity at 55 ± 10%, and 12-h light/dark cycle. Rats were allocated 3–5 animals per propylene cage with autoclaved pinewood bedding changed 3 times a week. They were fed a balanced laboratory diet (Purina, Paulínia - SP, Brazil) and filtered water was provided ad libitum through glass bottles.

Experimental design

Immature male rat assay

Fifty weanling 21-day old male rats were separated into 5 groups of 10 animals each, treated daily for 10 d with flutamide (FLU) by oral gavage (anti-androgenic control; 3 mg/kg/day), testosterone propionate s.c. (androgenic control; 1 mg/kg/day s.c.), or SS at 10000 and 20000 ppm in the diet (Fig. 1). Saline solution (0.9% NaCl) was used as vehicle in s.c. injections in a 2 ml/kg b.w. volume; corn oil was the vehicle in the gavage administrations (5 ml/kg b.w.). A negative control group received basal diet, saline solution s.c. and corn oil by gavage. The animals fed the SS diet also received testosterone propionate (TP) s.c. and corn oil by gavage. The TP dose (1 mg/kg/day) was the same as used by Ashby et al. (2004) in young weanling not-castrated rats in their proposal to modify the Hersberger test.¹⁵ The flutamide (FLU) dose (3 mg/kg/day; oral gavage) was recommended for the Hershberger test validation.⁵¹

Figure 1. Experimental design of the study.

Animals were observed every day during the treatment period for clinical signs, including morbidity and mortality. The animals were sacrificed by exsanguination under deep anesthesia in a CO₂ chamber. Blood samples from the left ventricle were collected and stored at −20°C until analyzed for sex hormone concentrations. At necropsy, the reproductive organs (testis, epididymis, seminal vesicles, prostate and coagulating glands) and also liver and kidneys were excised, trimmed free of fat and connective tissues, and weighed. Then, they were immersed in 10% buffered formalin for 48 hours, except the right testicles and epididymis which were fixed in Alfac (5% acetic acid, 10% formaldehyde 100%, 85% alcohol 80%).

Pubertal male rat assay

Fifty pubertal male rats (42-days old) allocated into 5 groups of 10 animals were exposed through adulthood (98-days old) to a diet (ad libitum) containing 0, 2500, 5000, 10,000 or 20,000 ppm of SS (Fig. 1). Body weights were measured twice a week. Food and water consumption measurements were taken once a week. Systemic serum and organ collection and procedures were the same as for the immature rat assay.

Hormone measurements

This analysis was conducted in the pubertal male rats assay only. Plasma follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were assayed by a double-antibody radioimmunoassay (RIA) method with specific kits provided by the National Hormone and Peptide Program (Harbor-University of California at Los Angeles). The FSH primary antibody was anti-rat FSH-S11, and the standard was FSH-RP2. The antiserum for LH was LH-S10 using RP3 as reference. Serum concentrations of testosterone (T) and dihydrotestosterone (DHT) were determined using specific RIA kits provided by MP Biomedicals (Orangeburgh, NY, USA) and Diasource - Genese (SP, BR), respectively. The lower limit of detection for FSH, LH and T was 0.2, 0.04, 0.07 ng/mL and 20 pg/mL for DHT. The intra-assay coefficients of variation were 3%, 4%, 10.1% and 5.2%, for FSH, LH, T and DHT, respectively. In order to avoid inter-assay variation, all samples were assayed in the same RIA. The number of animals evaluated per each group was 10, 10, 8, 10 and 9 for the 0, 2500, 5000, 10000 or 20000 ppm of SS diet, respectively.

Sperm counts, daily sperm production, and sperm transit time through the epididymis

The process of tissue preparation is described in Perobelli et al. (2010).⁵² Spermatids and spermatozoa present in the caput/corpus and cauda epididymidis were enumerated as described by Robb et al. (1978),⁵³ with modifications by Fernandes et al. (2007).⁵⁴ The analytic methods are also described in Bellentani et al. (2011),⁵⁵ and were only conducted in the pubertal male rat assay.

Histological analyses of testis and epididymis

This analysis was conducted in both the immature and pubertal male rat assays. The left testis and epididymis were embedded in paraplast and subjected to histological cuts of 5 µm thickness, and stained with hematoxylin and eosin (H&E). Histological evaluations were carried out with the use of transverse sections of the testis and the entire length of the longitudinal section of the epididymis (initial segment, head, body and tail). The epididymis was evaluated for the presence of sperm in the ductal lumen and the structure and appearance of the epithelium and the interstitium.

Sperm morphology and motility

This analysis was conducted only in the pubertal male rat assay. Immediately after euthanasia, spermatozoa were removed from the left vas deferens with the aid of a syringe and a needle, and thoroughly rinsed with 1.0 mL of formol-saline. The slides and material preparation were described elsewhere⁵⁵ and the analysis of sperm motility was performed as described by Perobelli et al. (2010).⁵²

Statistical analyses

Statistical analyses were performed using Jandel Sigma Stat software (Jandel Corporation, San Rafael, CA). All data were analyzed by analysis of variance (ANOVA), followed by the Dunn's test to determine significance. The criterion for significance was set at P < 0.05, compared to the negative control group (basal diet) of each assay.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Disclosure

This article does not necessarily reflect the views of CETESB and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

Acknowledgments

The Companhia Ambiental do Estado de São Paulo (CETESB) provided the samples and related data of the sewage sludge samples tested in this study, as well as information about the sewage treatment process. The authors also thank Wilma de Grava Kempinas (UNESP, Botucatu, Brazil) for project cooperation, Janete Aparecida Anselmo-Franci and Ruither de Oliveira Gomes Carolino (USP) for radioimmunoassay procedures, and Paulo Roberto Cardoso, Paulo César Georgette and Maria Luísa Ardanaz (UNESP) for technical support.

Funding

This study was supported by the Center for the Evaluation of the Environmental Impact of Human Health (TOXICAM), at UNESP Medical School, the Institute of Chemistry, at UNICAMP, the School of Dentistry, at USP, the State of São Paulo Agency for Support of Research (FAPESP), by the National Council for Technological and Scientific Development (CNPq), Brazil.