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Subchronic Toxicity Evaluation of a Treated Urban Sewage SludgeJoão Francisco Lozano Luvizuttoa; Marize de Lourdes Marzo Solanoa; Daniele Passarelib; Carla Adrieneda Silva Franchia; Gisela de Aragão Umbuzeiroc; João Lauro Viana de Camargoa

a Center for the Evaluation of the Environmental Impact of Human Health (TOXICAM), Departmentof Pathology, Botucatu Medical School, UNESP-São Paulo State University, São Paulo, Brazil b

Department of Clinical Medicine, Faculty of Veterinary Medicine and Zootechnology, São Paulo StateUniversity (UNESP), Botucatu, São Paulo State, Brazil c Faculty of Technology, UNICAMP-CampinasState University, Limeira-SP, Brazil

Online publication date: 17 June 2010

To cite this Article Francisco Lozano Luvizutto, João , de Lourdes Marzo Solano, Marize , Passareli, Daniele , Adriene daSilva Franchi, Carla , de Aragão Umbuzeiro, Gisela and Lauro Viana de Camargo, João(2010) 'Subchronic ToxicityEvaluation of a Treated Urban Sewage Sludge', Journal of Toxicology and Environmental Health, Part A, 73: 13, 916 —925To link to this Article: DOI: 10.1080/15287391003745036URL: http://dx.doi.org/10.1080/15287391003745036

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916

Journal of Toxicology and Environmental Health, Part A, 73:916–925, 2010Copyright © Taylor & Francis Group, LLCISSN: 1528-7394 print / 1087-2620 onlineDOI: 10.1080/15287391003745036

SUBCHRONIC TOXICITY EVALUATION OF A TREATED URBAN SEWAGE SLUDGE

João Francisco Lozano Luvizutto1, Marize de Lourdes Marzo Solano1, Daniele Passareli2, Carla Adriene da Silva Franchi1, Gisela de Aragão Umbuzeiro3, João Lauro Viana de Camargo1

1Center for the Evaluation of the Environmental Impact of Human Health (TOXICAM), Department of Pathology, Botucatu Medical School, UNESP–São Paulo State University, São Paulo, Brazil2Department of Clinical Medicine, Faculty of Veterinary Medicine and Zootechnology, São Paulo State University (UNESP), Botucatu, São Paulo State, Brazil3Faculty of Technology, UNICAMP–Campinas State University, Limeira-SP, Brazil

Disposal of tons of sludge produced daily by sewage treatment plants in large cities is a seri-ous problem. Because recycling and application in agriculture have been proposed, theBrazilian National Environmental Council (CONAMA, 2006) issued a legal norm that regu-lates the use of the sewage sludge (SS) in crops. Due to the complex chemical nature of suchproducts, characterization by analytical methods for health and environmental risk assess-ment has severe limitations. To overcome such limitations, it is necessary to (1) assess thetoxicological potential of SS and (2) identify possible adverse effects in vivo in order to pro-vide critical information for future environmental regulations. The present study was con-ducted to determine the potential toxicity of SS obtained from a representative urbantreatment plant located in the São Paulo State, Brazil. Male and female Wistar rats were fedad libitum a pelleted diet containing varying amounts of SS. No relevant clinical, hematologi-cal, urinary, or gross organ morphological alterations were observed in both genders of ratsorally exposed to SS at up to 3.8 g/kg/d for 90 d. Sewage slude produced increased incidenceof centrilobular hepatocyte hyperplasia at the high dose and significantly increased aspartateaminotransferease (AST) activities at all doses in both genders. Although the present dataindicate some liver involvement, these alterations were considered adaptative and not toxi-cologically relevant, as the responses were relatively mild, not dose dependent, and no otherparameters were markedly affected. The present results may contribute to the establishmentof protocols for potential usage in SS agricultural soil application.

Technological and industrial developmentsand continuous increase of population havegenerated a problem that remains with respectto waste residues (Rockefeller, 1998). Urbansewage treatment generates potentially reus-able water and a residue known as sewagesludge (SS) or biosolids, a complex mixture of

inorganic and organic materials, including bio-logic matter of industrial and domestic origin,with a pasty texture, putrid odor, and darkcolor. Tons of SS are produced daily by waste-water treatment plants in large cities, generatingan enormous disposal problem. The com-position of SS depends upon the wastewater

This study was granted by the State of São Paulo Agency for Support of Research (FAPESP), the Center for the Evaluation of theEnvironmental Impact of Human Health (TOXICAM), at the State University of São Paulo (UNESP), and by the National Council forTechnological and Scientific Development (CNPq), Brazil. The authors also thank Paula Regina Pereira Silva for biomedical technicalhelp and CETESB (Companhia de Tecnologia de Saneamento Ambiental) for providing the samples and related data of the sewage sludgesamples tested in this study, as well as information about the sewage treatment process. This article does not necessarily reflect the viewsof CETESB and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorse-ment or recommendation for use.

Address correspondence to João Lauro Viana de Camargo, Botucatu Medical School, São Paulo State University (UNESP),Botucatu, CEP18618-970, São Paulo, Brazil. E-mail: decam@fmb.unesp.br

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SUBCHRONIC TOXICITY OF TREATED SEWAGE SLUDGE 917

treatment process and the original pollutionload of the sewage system, including domestic,industrial, or mixed sources (Schowanek et al.,2004). This complex mixture (heavy metals,pathogenic and not pathogenic organisms,nitrogen, and valuable organic matter) is beingproposed for use in agriculture as an organicfertilizer or soil improver and as raw materialfor pottery manufacture (Baruzzo et al., 2006;Moreira et al., 2001).

In large cities, sewage is composed oforganic residues but also incorporates a widevariety of chemicals produced by roadways,rain runoff, water from offices and industrialfacilities, and residuals from domestic activitiessuch as residues of paintings, drugs, and pesti-cides (Al Zabadi et al., 2008). The potentialhazards of certain heavy metals and diben-zeno-p-dioxins and dibenzofurans that accu-mulate in soils and or in crops from appliedsludge and the subsequent transfer of thesecontaminants through the food chain tohumans are well known (Rideout & Teschle,2004; Stevens et al., 2003; Eljarrat et al.,2007). However, Gaskin et al. (2003) indicatedthat toxic metals have not accumulated insoils after 6 yr or more of SS application.Selivanovskaya and Latypova (2006) also sup-port the use of composted SS as organo-mineral fertilizer for the soil of nursery forestsdue to the absence of any apparent detrimen-tal effects on local microorganisms. When fedwith corn or leaf tissues of corn plants grownon soil containing SS, rats and pigs did not dis-play any significant adverse effects aside fromelevated tissue levels of cadmium (Cd) andnickel (Ni) (Lisk et al., 1982; Miller & Boswell,1981).

Despite the beneficial properties, such asrelatively high levels of organic matter, landapplication and surface disposal of untreatedSS create a risk for human exposure to poten-tially toxic agents and to pathogenic microor-ganisms that produce illnesses through directand indirect contact. Human occupationalexposure to SS may occur during disposal of SSin containment lagoons or application of SS toagricultural soil (U.S. EPA, 1993, 2004). Occu-pational exposure involves contact with multi-

ple potential genotoxins and irritant chemicalsat varying levels and differing routes of expo-sure (by inhalation, dermal exposure, andingestion) (Al Zabadi et al., 2008; Lewis et al.,2002; Harrison & Oakes 2002). Characterizingand quantifying these different chemical expo-sures are extremely difficult, and trying torelate specific adverse outcomes to single SScomponents is inappropriate (Al Zabadi et al.,2008).

Since the beginning of the 1990s the dis-cussion on the use of SS was instigated in theNorthern Europe and gained intensity from1995 onward (Andersen, 2001). Currently,there are several governmental regulations forthe use and disposal of SS, such as those of theEuropean Union (EU), where the use of sludgeis controlled under Directive 86/278/EEC(Schowanek et al., 2004), the United States 40CFR Part 503 (U.S. EPA, 1993), and the Brazil-ian National Environmental Council’s Resolu-tion N° 375 (CONAMA, 2006). All theseregulations define acceptable managementpractices and provide specific numerical limitsfor selected chemical pollutants and pathogensrelated to SS land application.

Nevertheless, questions remain regardingthe impact of SS on human and animal healthas well as environmental consequences. Bear-ing this in mind, the toxicological assessment ofSS samples using different types of bioassayswas undertaken (Chenon et al., 2003;Schowanek et al., 2004). The 90-d-long (sub-chronic) study described here aimed to evalu-ate the potential oral toxicity of SS samplesobtained from a selected urban wastewatertreatment plant at the São Paulo State, Brazil,on rats.

MATERIAL AND METHODS

This study design was approved by theEthical Committee on Animal Experimentation(Protocol No. 571, Botucatu Medical School,UNESP).

Animals

One hundred and sixteen 6-wk-oldWistar male and female rats (60 animals each)

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918 J. F. L. LUVIZUTTO ET AL.

were obtained from the MultidisciplinaryCenter for Biological Investigation (CEMIB,UNICAMP, Campinas, SP, Brazil). The ani-mals were maintained at the BotucatuMedical School, UNESP, in an environmen-tally controlled facility: room temperature at22 ± 2°C, humidity at 55 ± 10%, and 12-hlight/dark cycle. Rats were allocated five ani-mals per propylene cage with autoclaved pin-ewood bedding changed three times a week.Prior to experiments, the animals were sub-mitted to a 2-wk acclimatization period, duringwhich they were fed a pelleted diet (Biobase,Bio-tec, Bases Química Ltda., Colombo, PR)and provided filtered water ad libitum.

Sludge Samples

Currently, in Brazil, sanitary galleries col-lect only 32% of the urban sewage, and 20% ofthis amount is treated in wastewater treatmentplants through coagulation, flocculation,decantation, and filtration (IBGE, 2000;Tsutiya, 2001). The SS samples studied in ourlaboratory were repeatedly collected once aweek for 2 mo (May 4 to July 6, 2006) from aselected sewage treatment plant indicated asPCJ1. The samples were collected by CETESB,the Environmental Protection Agency of SãoPaulo State, and kindly provided to this labora-tory. According to the Department of Waterand Electric Energy of the São Paulo State, thePCJ1 plant treats mainly urban effluents andtextile industrial wastewater of an area withapproximately 4.9 million inhabitants, generat-ing about 7 tons of treated sludge per day(DAEE, 2007). The main activities of the popu-lation from the hydrographic basin PCJ (Piraci-caba, Capivari, and Jundiaí) are cultures oforanges and vegetables, as well as sugar canefor production of sugar, alcohol, and cellulose.The sewage treatment consists of a biologicalfilter; the solid phase of the treatment wasanaerobically digested, centrifuged, and beddried. The water content was reduced usingacrylamide polymer. Chemical analysis of fourdifferent samples from the PCJ1 plant showedthat total metal levels in the samples studiedwere all below the Brazilian standards (CET-ESB, 2005) (Table 1).

Experimental Diet

Dried SS samples were maintained at −20°Cuntil mixed with a commercial Nuvilab-CR1powder chow (Nuvital S.A., Curitiba, PR, Bra-zil) at concentrations of 5,000, 10,000, or50,000 ppm. Because there were no previousdata available on sludge mammalian toxicity,the decision on SS concentrations in the dietwas based upon criteria other than toxicity.The highest dietary concentration adopted wasthe maximum recommended level for a testsubstance in the diet of chronic toxicity bioas-say, which is 5% (Chhabra et al., 1990); theother two concentrations were 20% and 10%of the highest one, respectively. The poweredmixture was transformed in pellets using anindustrial mixer (Prensa Peletizadora Cha-vantes, CAF-model M60, PR). The pellets weredried by ventilation at room temperature for24 h. Diets with different sludge concentra-tions were stored in identified plastic bags andmaintained at −20°C for a maximum period of30 d.

Experimental Design

The animals were allocated to 4 groupsof 8 or 10 animals each. The negativecontrol group was fed basal diet. Other threegroups received the experimental diet with5,000, 10,000, or 50,000 ppm SS. The dietswere provided ad libitum. Food and water

TABLE 1. Metal Concentration Range in Sewage Sludge (SS)Samples From the PCJ1 Treatment Plant and the BrazilianStandards

Substances

Range values for 4 different samples of SS from PCJ1 (mg/kg dry weight)

Brazilian official standards for SS agricultural use (mg/kg dry weight)

Arsenic <2.0–2.24 41Barium 63.5–573 1300Cadmium 5.41–10.9 39Lead 102–143 300Cupper 77.9–344 1500Chromium 95.2–724 1000Mercury 1.27–2.16 17Molybdenum < 2.0–2.68 50Nickel 51.7–115 420Selenium 0.22–4.2 100Zinc 1095–1525 2800

Note. Source: CETESB (2005).

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SUBCHRONIC TOXICITY OF TREATED SEWAGE SLUDGE 919

consumptions were estimated once a weekby the differences between the respectiveamounts provided and the residues mea-sured after a 2-d period. The animals wereobserved for clinical signs, including mortalityand moribundity, every other day during thewhole treatment period. Body weights weremeasured twice a week during the wholeperiod. After 28 d of SS exposure, 40 animals(20 males and 20 females) underwent aninterim sacrifice. The other 80 animals(40 males and 40 females) continued thetreatment until d 90. At both times, the ani-mals were sacrificed by exsanguination underanesthesia with 4% sodium pentobarbital(40 mg/kg ip). All animals were submitted tocomplete necropsy.

Urinalysis

At the last week of treatment, freshlyvoided urine was collected individuallybetween 7 and 9 a.m. every morning and priorto necropsy (while under anesthesia). Occultblood, ketone bodies, glucose, protein, pH,urobilinogen, and bilirubin were assayed withreagent strips (Combur10 Test, UX, Roché,Brazil). Sediment (optical microscope), color,odor, cumulative urine, urine volume (mea-sured in graduated cylinders), and specificgravity (clinical reflectometer 301, Biosystems,Brazil) were determined.

Hematology and Serum/Plasma Chemistry

Prior to necropsy, while the animals wereunder anesthesia, blood samples for hematol-ogy and clinical chemistry were collected fromthe left ventricle. In EDTA-2K-treated blood, acomplete blood count was performed on allsamples using an automated hematology ana-lyzer (semi-automatic biochemistry system SB-190, Celm, Brazil). Each counting was con-firmed by systematic analysis of blood smearstained by a Romanowsky stain (Panótico,Laborclin, Brazil). Hematology parametersmeasured were erythrocyte, white blood celland platelet counts, hematocrit, hemoglobin,mean corpuscular volume, and mean corpus-cular hemoglobin concentration.

Blood collected for serum chemistry wasuntreated and centrifuged at 3000 × g forapproximately 10 min. In the serum obtained,measurement of alanine aminotransferase(ALT), aspartate aminotransferase (AST), andcreatinine levels was carried out spectrometri-cally (semi-automatic biochemistry system SB-190, Celm, Brazil) and with commercial kits(Urea Enzymatic Method, Creatinine FastKinetic Method, ALT/GPT UV Kinetic Method,AST/GOT UV Kinetic Method; Laborlab, Bra-zil). For results interpretation, normal valueswere considered according to Harkness andWagner (1994).

Necropsy and Tissue Processing

After exsanguination, the body surface andintracranical, intrathoracic, and intra-abdominalcavities were observed for abnormalities.Organ weights were obtained for the liver, kid-neys, adrenal glands, testes, and spleen. Rela-tive organ weights were calculated based onbody weights measured on the day of sacrifice.The livers of animals fed all doses of SS and thekidneys of 50,000 ppm-fed animals and ofcontrol rats were processed for histologicalanalysis using hematoxylin and eosin (H&E)staining. All the other collected organs—brain,heart, lungs, thymus, trachea, thyroid, pan-creas, bone marrow, ovaries, uterus, urinarybladder, digestive tube, and large and smallintestines—were fixed in 10% buffered forma-lin and stored for later histological processing,if necessary.

Liver sections of animals fed 50,000 ppmwere also immunohistochemically stained forthe detection of hepatocytes expressing theenzyme gluthatione S-transferase, placentalform (GST-P). These GST-P-positive cells arealtered hepatocytes that may constitute cellfoci of varied numbers and sizes, regarded asputative preneoplastic lesions (Ito et al.,1998, 2003). Briefly, for GST-P analysis liversections were sequentially treated with 3%H2O2 in phosphate-buffered saline (PBS) for10 min, nonfat milk for 60 min, polyclonalantibody rabbit anti-rat GSTP (Medical andBiological Laboratories Co., Japan) (dilution1:1000) overnight, biotinylated goat anti-rabbit

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920 J. F. L. LUVIZUTTO ET AL.

immunoglobulin (Ig) G (dilution 1:200) for60 min, and avidin-biotin-peroxidase solution(dilution 1:50) for 45 min (EliteABCkit, VectorLaboratory, EUA). Chromogen color develop-ment was done with 3,3-diaminobenzidinetetrahydrochloride (DAB, Sigma Chemical Co.,USA). Liver sections were counterstained withHarris’s hematoxylin. GST-P-positive (GST-P+)cells and foci were counted, and liver sectionareas were measured using a Nikon photomi-croscope (Microphot-FXA) connected to a KS-300 apparatus (Kontron Elektronic, Germany).In each group only GST-P-positive rare smallaltered cell foci were present.

Statistical Analysis

Statistical analyses were performed usingJandel Sigma Stat software (Jandel Corporation,San Rafael, CA). Values of final body weightsand body weight gain, relative and absoluteorgan weights, food consumption, and bio-chemical and hematological data were ana-lyzed by analysis of variance (ANOVA), anddata were subjected to the Kruskal–Wallis testto determine significance. The criterion for sig-nificance was set at p < .05.

RESULTS

The final mean sewage sludge consump-tion after the 90-d exposure period is pre-sented in Table 2. For each dietary level of SS,animals of both genders ingested about thesame amount of sludge daily, which was onaverage 0.4, 0.8, and 3.8 mg/kg.d. After 28and 90 d, SS treatment did not markedly affectmortality, clinical signs, responses to stimuli, orfood and water consumption when comparedwith the control. During the entire experimen-tal period all groups presented progressive gainof body weight, irrespective of the levels of SSexposure.

Hematology analyses did indicate few sig-nificant alterations after 90 d of study (Table 2)in both genders but not after 28 d (data notshown). These differences were not consideredtoxicologically relevant as they were not dosedependent and occurred only in the lower

dose groups. No significant alterations weredetected in urine (data not shown). In addi-tion, no significant alterations were detected inclinical biochemistry parameters when com-pared to controls, except for significantlyincreased AST activity in all SS-fed females at90 d (Table 3). The activities of AST in maleswere quantitative but not significantly increased.

No alterations in the absolute (not shown)or relative weights of any organ were observedat d 28 and 90 (data not shown).

Histological examination of the liverrevealed mild centrilobular hypertrophy ofhepatocytes in 9 out of 10 male rats fed50,000 ppm SS (Figure 1B). A single male ani-mal also developed one small liver clear cellfocus (not shown). In 10,000 ppm-fed malecentrilobular hypertrophy was minimal andoccurred rarely. No liver alterations were seenin male rats fed 5,000 ppm or in females ani-mals given any SS dietary concentration levels.The numbers of GST-P-positive cells (Figure1C) were similar in male and female animalsand were not influenced by exposure to50,000 ppm SS. Rare and small GST-P-positivefoci were also observed in control and treatedgroups (Figure 1 D).

DISCUSSION

The present findings indicate that this spe-cific SS composite sample, obtained from aselected wastewater treatment station at theSão Paulo State, Brazil, did not induce sys-temic toxicity in both genders of Wistar ratswhen administered via the diet at concentra-tions up to 3.8 g/kg/d for 90 d.

Male and female animals grew progres-sively during the experiment, with no apparentdifferences among the groups. In spite of therelatively high levels of SS in the diet, no alter-ations were noted in animals sacrificed at d 28and 90 with respect to clinical status or foodand water consumption, or to urinary andhematology analyses. No marked alterationswere detected in mean absolute or relativeweights of liver, kidneys, spleen, adrenals, andtestes. Since organ weight changes have beenaccepted as a sensitive indicator of chemically

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921

TABL

E 2.

Hem

atol

ogy

Val

ues

in t

he D

iffer

ent

Expe

rimen

tal W

istar

Rat

Gro

ups

Afte

r a

90-d

Per

iod

of D

ieta

ry E

xpos

ure

to S

ewag

e Sl

udge

Sam

ples

Fro

m t

he P

CJ1

Trea

tmen

t Pla

nt

SS in

the

diet

(p

pm)

nH

b (g

/dl)

Ht (

%)

MC

V (f

l)M

CH

C

(%)

Plat

elet

s (g

/dl)

Neu

troph

ils

(%)

Lym

phoc

ytes

(%

)Eo

sinop

hils

(%)

Mon

ocyt

es

(%)

Eryt

hroc

ytes

(×

106 /m

m3 )

Leuc

ocyt

es

(× 1

03 /mm

3 )

Fem

ales

5000

1011

.6 ±

0.4

39.1

± 1

.861

.5 ±

4.2

28.8

± 1

.66.

4 ±

0.5

26.8

± 9

.970

.2 ±

11.

82.

8 ±

1.7

1.6

± 2.

66.

4 ±

0.4

2.0

± 1.

310

,000

1011

.9 ±

0.5

40.2

± 1

.870

.2 ±

4.6

29.7

± 2

.06.

2 ±

0.2

19.6

± 7

.273

.4 ±

6.7

2.6

± 2.

74.

0 ±

2.8

5.7

± 0.

4*1.

4 ±

0.7

50,0

0010

12.5

± 0

.442

.3 ±

1.6

65.7

± 3

.129

.5 ±

0.5

6.3

± 0.

321

.8 ±

7.7

74.6

± 6

.81.

4 ±

1.7

2.2

± 2.

26.

4 ±

0.2

1.7

± 1.

1C

ontro

l08

11.4

± 4

.237

.1 ±

13.

459

.2 ±

21.

227

.4 ±

10.

45.

8 ±

2.1

21.5

± 1

2.8

66.1

± 2

5.2

1.3

± 2.

62.

0 ±

1.7

5.6

± 2.

01.

5 ±

0.6

Mal

es50

0010

13.6

± 0

.6*

44.1

0 ±

2.2

70.4

± 1

.331

.0 ±

0.8

6.1

± 0.

320

.0 ±

5.3

75.0

± 5

.82.

0 ±

2.5

3.0

± 2.

56.

3 ±

0.3

2.0

± 0.

710

,000

1013

.6 ±

0.4

*43

.0 ±

1.3

71.0

± 2

.031

.5 ±

0.4

*5.

9 ±

0.3*

24.4

± 7

.069

.6 ±

7.0

*2.

4 ±

2.1

3.4

± 1.

96.

1 ±

0.2

2.6

± 0.

650

,000

1012

.6 ±

0.4

42.6

± 1

.767

.2 ±

4.3

29.5

± 1

.26.

2 ±

0.2

22.0

± 5

.675

.2 ±

5.8

1.6

± 2.

31.

2 ±

1.4

6.4

± 0.

52.

0 ±

0.7

Con

trol

0812

.5 ±

0.2

42.8

± 0

.770

.8 ±

2.0

29.3

± 0

.66.

2 ±

0.2

17.5

± 6

.377

.9 ±

6.0

2.5

± 2.

12.

1 ±

3.1

6.0

± 0.

22.

3 ±

0.8

1 Valu

es a

re m

ean

± SD

; n

= n

umbe

r of

ani

mal

s; H

b, h

emog

lobi

n; H

t, he

mat

ocrit

; M

CV

, mea

n co

rpus

cula

r vo

lum

e; M

CH

C, m

ean

corp

uscu

lar

hem

oglo

bin

volu

me.

Aste

risk

indi

cate

s sig

nific

antly

diff

eren

t fro

m c

ontro

l, p

< .0

5.

Downloaded By: [Francisco Lozano Luvizutto, João] At: 22:17 17 June 2010

922 J. F. L. LUVIZUTTO ET AL.

induced changes (Michael et al., 2007), thisobservation brings further evidence of theabsence of toxicity after a 90-d period of expo-sure to the SS sample studied.

No alterations were observed in the kid-neys, but in liver a mild centrilobular hepato-cyte hypertrophy occurred in the majority ofthe 50,000 ppm- and in a few 10,000 ppm-fed

TABLE 3. Blood Clinical Chemistry Values in the Different Experimental Wistar Rat Groups After a 90-d Period ofDietary Exposure to Sewage Sludge Samples From the PCJ1 Treatment Plant

SS in the diet (ppm) n Urea (mg/dl) Creatinine (mg/dl) AST (UI/L) ALT (UI/L)

Females5,000 10 60.3 ± 4.7 0.7 ± 1.0 144.7 ± 26.0* 40.8 ± 15.210,000 10 57.8 ± 7.4 0.7 ± 0.1 136.3 ± 17.1* 34.0 ± 9.950,000 10 64.1 ± 5.5 0.7 ± 0.1 135.7 ± 16.0* 33.0 ± 4.1Control 8 64.6 ± 8.0 0.7 ± 0.1 115.3 ± 11.0 38.3 ± 6.8Males5,000 10 71.4 ± 6.8 0.6 ± 0.1 137.5 ± 20.3 37.6 ± 9.210,000 10 68.7 ± 3.4 0.6 ± 0.1 127.2 ± 15.0 37.1 ± 6.050,000 10 75.4 ± 5.3 0.6 ± 0.1 135.1 ± 24.4 31.2 ± 4.6Control 8 73.0 ± 4.4 0.6 ± 0 117.6 ± 16.7 34.1 ± 3.8

Note. Values are mean ± SD; n = number of animals; ALT, alanine aminotransferase; AST, aspartate aminotransferase.Asterisk indicates significant at p < .05 when compared to control.

FIGURE 1.

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SUBCHRONIC TOXICITY OF TREATED SEWAGE SLUDGE 923

male animals. This alteration usually indicatedhyperplasia of the smooth endoplasmic reticu-lum due to increased activity of the cyto-chrome P-450 enzymatic system, which isinvolved in the biotransformation of xenobi-otic. It is an adaptative process, reversibleupon withdrawal of the stressing stimulus(Schoket & Vincze, 1985, 1990; Grasso et al.,1991). When the induction of the metaboliz-ing system surpasses a threshold, centrilobularhypertrophy is generally accompanied byincreased liver weight (Grasso et al., 1991),which did not occur in the present study.Female rats did not develop this centrilobular-cell hypertrophy, indicating a gender differ-ence in susceptibility to the influence of SS.However, female animals did presentincreased AST activity levels in all SS-fedgroups. Overall these findings suggest thatdietary SS may exert a mild adaptative influ-ence on male and female rat liver. This obser-vation may be attributed to the influence ofcertain of the SS components, not identifiedconstituents of the tested SS.

The maintenance of the liver enzymaticinduction by xenobiotics may be associatedwith centrilobular hypertrophy followed bysustained cell proliferation and eventualappearance of altered foci of hepatocytes(AFH), a putative preneoplastic alteration thatwas found in different models of hepatocar-cinogenesis (Bannasch et al., 1989; Bannasch& Zerban, 1992). Under routine H&E stainingthese foci may present different phenotypesdepending upon the appearance of the pre-dominant constituent cells, as clear, eosino-philic, or basophilic cells. Most of these focialso express immunohistochemically the enzymeGST-P, which has been regarded as a reliablemarker of putative preneoplastic alteration inrodent liver (Satoh et al., 1985; Ito et al., 1998,2003). The development of AFH as observedunder H&E staining was rare in this study: onlya single male rat and none of the femalesexposed to 50,000 ppm SS developed onesmall clear-cell foci in liver. Under immunohis-tochemistry, GST-P-positive foci were small andrare, which was the basis for estimating only thenumber of cells expressing the placental form of

GST-P. These positive cells were counted atthe same magnitude in both male and femalecontrol and 50,000 ppm SS-fed rats, indicatingthat SS did not influence the development ofthese altered cells at 90 d. Therefore, theobservation of a single male rat with a rareGST-P positive focus may be due to spontane-ity and not a result of xenobiotics challenge, asreported in aging and control rats in otherstudies (Popp et al., 1985; Michel et al., 2007).

Apart from the economic and sanitary con-siderations, the reutilization of this type of SSin agriculture soil can be an interesting alterna-tive, considering the progressive mineralizationof some soils due to the excessive use of inor-ganic chemical fertilizers and extensive cultiva-tion of only one kind of crop (Cocker & Cassells,1983). Data reported in this study may repre-sent important information to assess the risks topublic health and the environment related toexposure to SS generated in a local wastewatertreatment plant in Brazil.

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