the sewage produced in urban centers contains numerous pollutants from domestic and industrial sources , and treated sewage may contain complex mixtures of organic and inorganic compounds that may not be degraded during treatment ( rank and nielsen , 1998 ) . several studies have investigated the genotoxicity of industrial effluents ( houk , 1992 ; ruiz et al . , 1992 ; nielsen and rank , 1994 ; rodrigues et al . , 2010 ) and the water bodies that receive them ( ohe et al . , 2004 ; the sewage sludge from municipal treatment plants has also been tested for toxicity and genotoxicity ( hopke et al . , 1984 ; ottaviani et al . , 1993 however , liquid effluents from municipal sewage plants have been evaluated using chemical analyses primarily to meet legal obligations , and less attention has been paid to their genotoxic effects on organisms ( grisolia et al . , 2005 ) . in 2006 , the brazilian state of rio grande do sul issued a regulation to establish criteria for the release of domestic and industrial liquid effluents into the environment based on their toxicity , genotoxicity and type of sewage ( consema , 2006a ) . over the next 812 years , the state environmental control agencies will define the test organisms and protocols to be used in monitoring these effluents . the tradescantia micronucleus ( trad - mcn ) assay , a sensitive test for evaluating genotoxicity ( crebelli et al . , 2005 1997 ) , is based on the small chromatin masses derived from chromosomal breakages or aneuploidy during meiosis of the pollen mother cells ( ma et al . , 1984 ) . clone # 4430 , a hybrid of t. subacaulis bush and t. hirsutiflora bush , has been successfully used for bioassays ( ma et al . , 1994 ) . purpurea boom is an appropriate alternative for genotoxicity testing because it is well adapted to and extensively cultivated in tropical and subtropical regions ( batalha et al . , 1999 ) . in this work , we used the trad - mcn assay to evaluate and compare the genotoxicities of untreated and treated sewage from two municipal wastewater treatment plants ( wtps ) over a one - year period . the plants are located in the hydrographic basin of guaba lake , in the southern brazilian state of rio grande do sul , and receive wastewater from the municipality of porto alegre ; both are operated by the municipal department of water and sewage ( dmae ) . wtp sjn is located in a densely populated urban area with commercial and industrial activities . the sewage is treated by biological oxidation using an activated sludge process followed by aerated lagoon treatment at a mean flow rate of 246 l / s ( range : 146323 l / s ) . wtp bn is located in a less populated urban residential area and the sewage is treated in conventional , australian - type stabilization lagoons at a mean flow rate of 23 l / s ( range : 860 l / s ) . in february , april , july and october 2009 ( representing summer , autumn , winter and spring , respectively ) , untreated and treated sewage samples were collected from both sampling sites , transported to the laboratories where the experiments were done and preserved according to criteria established by the brazilian association of technical standards ( abnt  nbr 9898 ) . untreated and treated sewage samples from both wtps were collected for physicochemical analyses at the intervals indicated above . for each sewage sample collected the biochemical oxygen demand ( bod ) , chemical oxygen demand ( cod ) , total suspended solid ( tss ) , total nitrogen ( tn ) , total phosphorus ( tp ) and heavy metal content were measured . the parameters tested included some of those used in monitoring the efficiency of the wtps operated by the dmae , in accordance with the standard recommendations of the state foundation for environmental protection ( fepam ) and the quality criteria of the state environmental council ( consema , 2006a ) . metals were analyzed by using inductively - coupled plasma optical emission spectrometry ( icp - oes ) ( usepa , 2001 ) . purpurea were grown in pots ( 37 cm  20 cm  20 cm ) containing 4 kg of commercial soil from the same batch , in a non - polluted area of feevale university . once a week , 100 ml of 1/3-strength hoagland solution was applied to each pot and 100 ml of an n - p - k fertilizer solution ( 10 - 10 - 10 , w / w / w ) was applied once a month . plant exposure , inflorescence fixation and storage , slide preparation and data analysis were done using a modified version of the protocol described by ma et al . cuttings collected from the plant stocks bearing young inflorescences were maintained partially immersed in distilled water for 24 h in groups of 15 to 20 . after this period of adaptation , only turgid cuttings were exposed to the following samples for 8 h : ( a ) untreated sewage , ( b ) treated sewage , ( c ) 0.1% formaldehyde solution ( positive control ) and ( d ) distilled water ( negative control ) . the inflorescences were fixed in 3:1 ( v / v ) ethanol / acetic acid for 24 h and stored in 70% ethanol . one bud per inflorescence was dissected and the anthers were squashed in 1% acetocarmine stain on a slide . only preparations with early tetrads were included in the analysis . the slides were coded and the number of mcn in a random set of 300 tetrads per slide was scored under 400x magnification ( olympus cx4 microscope ) . the mcn frequencies were calculated by dividing the total number of micronuclei by the total number of tetrads scored , and the results were expressed as mcn/100 tetrads . all of the experiments were done in the laboratory of plant biotechnology at feevale university . the trad - mcn bioassay data were analyzed using the software package spss 17.0 after initial natural logarithm ( ln ) data transformation to obtain a normal distribution of variables and homogenous variances . the mcn frequencies of the samples in each of the four months were subsequently compared by one - way anova followed by the tukey test for multiple comparisons . the plants are located in the hydrographic basin of guaba lake , in the southern brazilian state of rio grande do sul , and receive wastewater from the municipality of porto alegre ; both are operated by the municipal department of water and sewage ( dmae ) . wtp sjn is located in a densely populated urban area with commercial and industrial activities . the sewage is treated by biological oxidation using an activated sludge process followed by aerated lagoon treatment at a mean flow rate of 246 l / s ( range : 146323 l / s ) . wtp bn is located in a less populated urban residential area and the sewage is treated in conventional , australian - type stabilization lagoons at a mean flow rate of 23 l / s ( range : 860 l / s ) . in february , april , july and october 2009 ( representing summer , autumn , winter and spring , respectively ) , untreated and treated sewage samples were collected from both sampling sites , transported to the laboratories where the experiments were done and preserved according to criteria established by the brazilian association of technical standards ( abnt  nbr 9898 ) . untreated and treated sewage samples from both wtps were collected for physicochemical analyses at the intervals indicated above . for each sewage sample collected the biochemical oxygen demand ( bod ) , chemical oxygen demand ( cod ) , total suspended solid ( tss ) , total nitrogen ( tn ) , total phosphorus ( tp ) and heavy metal content were measured . the parameters tested included some of those used in monitoring the efficiency of the wtps operated by the dmae , in accordance with the standard recommendations of the state foundation for environmental protection ( fepam ) and the quality criteria of the state environmental council ( consema , 2006a ) . metals were analyzed by using inductively - coupled plasma optical emission spectrometry ( icp - oes ) ( usepa , 2001 ) . purpurea were grown in pots ( 37 cm  20 cm  20 cm ) containing 4 kg of commercial soil from the same batch , in a non - polluted area of feevale university . once a week , 100 ml of 1/3-strength hoagland solution was applied to each pot and 100 ml of an n - p - k fertilizer solution ( 10 - 10 - 10 , w / w / w ) was applied once a month . plant exposure , inflorescence fixation and storage , slide preparation and data analysis were done using a modified version of the protocol described by ma et al . cuttings collected from the plant stocks bearing young inflorescences were maintained partially immersed in distilled water for 24 h in groups of 15 to 20 . after this period of adaptation , only turgid cuttings were exposed to the following samples for 8 h : ( a ) untreated sewage , ( b ) treated sewage , ( c ) 0.1% formaldehyde solution ( positive control ) and ( d ) distilled water ( negative control ) . the inflorescences were fixed in 3:1 ( v / v ) ethanol / acetic acid for 24 h and stored in 70% ethanol . one bud per inflorescence was dissected and the anthers were squashed in 1% acetocarmine stain on a slide . only preparations with early tetrads were included in the analysis . seven slides were prepared for each sample . the slides were coded and the number of mcn in a random set of 300 tetrads per slide was scored under 400x magnification ( olympus cx4 microscope ) . the mcn frequencies were calculated by dividing the total number of micronuclei by the total number of tetrads scored , and the results were expressed as mcn/100 tetrads . all of the experiments were done in the laboratory of plant biotechnology at feevale university . the trad - mcn bioassay data were analyzed using the software package spss 17.0 after initial natural logarithm ( ln ) data transformation to obtain a normal distribution of variables and homogenous variances . the mcn frequencies of the samples in each of the four months were subsequently compared by one - way anova followed by the tukey test for multiple comparisons . tables 1 and 2 show the physicochemical properties of the samples collected from wtp sjn and wtp bn for genotoxicity evaluation during the four seasons of 2009 . the biochemical oxygen demand ( bod ) met legal criteria , except at wtp sjn in the summer and winter . total suspended solid ( tss ) values were above the maximum permitted , except in the winter at wtp bn and wtp sjn and in the summer at wtp sjn . wtp bn satisfactorily removed total nitrogen ( tn ) in the summer and autumn , whereas the removal at wtp sjn was unsatisfactory in all seasons . the total phosphorus ( tp ) concentrations were higher than recommended at both wtps in all seasons except at wtp bn in the spring . chromium ( cr ) was detected in untreated and treated sewage from wtp sjn in all seasons except the winter , when it was undetectable in treated sewage . in contrast , at wtp bn , low concentrations of this metal were detected in untreated sewage samples only in the summer and autumn , and no chromium was found in treated sewage . lead ( pb ) was found in all untreated sewage samples from wtp sjn , but was only found at wtp bn in the summer and autumn . the mcn frequencies of untreated sewage samples from wtp bn were significantly higher than those of the negative control in the summer and autumn , whereas treated sewage did not differ significantly from the negative control throughout the monitoring period ( figure 1 ) . in contrast , the mcn frequency in plants exposed to untreated sewage from wtp sjn was significantly higher than in the negative control in all seasons , except spring . at this wtp , treated sewage samples had high mcn frequencies that were equal to or greater than those in positive control samples , except in the spring . the mcn frequencies in untreated sewage from both wtps were lower in the spring than in other seasons and did not differ from the negative control . except in the spring , when all samples had low mcn frequencies , treated sewage from wtp sjn was significantly more genotoxic than treated sewage from wtp bn . effluents from municipal sewage treatment plants have been tested for mutagenicity using the salmonella / microsome test ( meier et al . , 1987 ) , the polychaete platynesis dumerilli ( jha et al . , 1997 ) , allium cepa ( nielsen and rank , 1994 ; grisolia et al . , 2005 ) and fish ( grisolia et al . , 2005 ) . however , tradescantia is apparently more sensitive to genotoxic substances than allium cepa , a widely used bioindicator of water quality ( crebelli et al . , 2005 ) . in contrast , there have been no reports on the use of t. pallida var . purpurea has been successfully used to evaluate genotoxicity by in situ exposure in a brazilian river ( umbuzeiro et al . , 2007 ) and by long - term exposure to treated sludge samples from wtps in the state of so paulo ( mielli et al . , 2009 ) , with the latter study showing that this organism is as sensitive as clone # 4430 . for samples from wtp bn , the high genotoxicity of untreated sewage was not observed in treated sewage . in contrast , both untreated and treated sewage samples from wtp sjn had high mcn frequencies . in general , the values of the physicochemical parameters for treated sewage from wtp bn were below the legal emission limits , whereas the corresponding values for wtp sjn were higher . the total nitrogen and total phosphorus concentrations of samples from wtp sjn were above the legal limits . of all the metals analyzed , chromium was detected in more seasons and at higher concentrations in samples from wtp sjn than in those from wtp bn . these differences in the physicochemical characteristics between the two wtps may have influenced the mcn frequencies , which were higher for wtp sjn . industrial effluents containing chemicals other than those included in our tests may have been released unexpectedly into the sewer system , which was built to treat only domestic sewage . in large metropolitan areas , sewage from different sources may form complex mixtures that have marked effects on organisms ( white and rasmussen , 1998 ) , and sludge from domestic sewage is less genotoxic than that from other sources ( hopke et al . , 1984 ) . heavy metals in different substrates can induce micronuclei in tradescantia ( majer et al . , 2002 ) . however , in the present study , we did not identify which chemicals were responsible for genotoxicity . further studies should investigate how the complex mixtures of pollutants influence micronucleus formation . in the spring , the mcn frequencies of untreated and treated sewage from both wtps were not significantly different from the negative control . these findings may be explained by the cumulative volume of rainfall during the four days before sampling . in the first three seasons of the year , rainfall before sampling was zero or very close to zero , but was much higher ( 37.6 mm ) in the spring and may have diluted the sewage . these findings indicate that monitoring should be done at various time points throughout the year . furthermore , tests should include both untreated and treated sewage to determine whether the test organism is a suitable bioindicator for the samples under evaluation . otherwise , it may be unclear whether negative results are indicative of a lack of genotoxicity or simply reflect the low sensitivity of the bioindicator used to test the samples . our results indicate that this short - term assay is a useful tool for assessing the genotoxicity of sewage .
the genotoxicity of untreated and treated sewage from two municipal wastewater treatment plants ( wtp bn and wtp sjn ) in the municipality of porto alegre , in the southern brazilian state of rio grande do sul , was evaluated over a one - year period using the tradescantia pallida var . 
 purpurea ( trad - mcn ) bioassay . 
 inflorescences of t. pallida var . 
 purpurea were exposed to sewage samples in february ( summer ) , april ( autumn ) , july ( winter ) and october ( spring ) 2009 , and the micronuclei ( mcn ) frequencies were estimated in each period . 
 the high genotoxicity of untreated sewage from wtp bn in february and april was not observed in treated sewage , indicating the efficiency of treatment at this wtp . 
 however , untreated and treated sewage samples from wtp sjn had high mcn frequencies , except in october , when rainfall may have been responsible for reducing these frequencies at both wtps . 
 physicochemical analyses of sewage from both wtps indicated elevated concentrations of organic matter that were higher at wtp sjn than at wtp bn . 
 chromium was detected in untreated and treated sewage from wtp sjn , but not in treated sewage from wtp bn . 
 lead was found in all untreated sewage samples from wtp sjn , but only in the summer and autumn at wtp bn . 
 these results indicate that the short - term trad - mcn genotoxicity assay may be useful for regular monitoring of municipal wtps .