Arch Environ Contam Toxicol DOI 10.1007/s00244-014-0072-0

Ecotoxicological and Genotoxic Assessment of Hospital Laundry Wastewaters Deivid Ismael Kern • Roˆmulo de Oliveira Schwaickhardt Carlos Alexandre Lutterbeck • Lourdes Teresinha Kist • Eduardo Alexis Lobo Alcayaga • Eˆnio Leandro Machado

•

Received: 31 March 2014 / Accepted: 8 July 2014 Ó Springer Science+Business Media New York 2014

Abstract The aim of the present study was to assess the ecotoxicity and genotoxicity of hospital laundry wastewaters generated from a regional hospital located in Rio Pardo Valley in the state of Rio Grande do Sul, Brazil. Physicochemical, microbiological, ecotoxicological, and genotoxic analyses were performed, and the results indicate that some parameters were not in accordance with the limit concentrations established by Brazilian and international guidelines for urban wastewaters. Daphnia magna (EC50 2.01 %) and Danio rerio (LC50 29.25 %) acute toxicity was detected, and sublethal effects were identified in Lactuca sativa (IC25 12.50 %) and Allium cepa (IC25 51.25 %). Cytotoxicity was observed at the five wastewater concentrations used yielding statistically significant differences (p \ 0.05) in the meristematic cells of A. cepa compared with the negative control. The results obtained here warn about the necessity to develop treatment

D. I. Kern  R. de Oliveira Schwaickhardt Graduate Program in Environmental Technology, Universidade de Santa Cruz do Sul, UNISC, Av. Independeˆncia, 2293, Bairro Universita´rio, CEP, Santa Cruz do Sul, RS 96815-900, Brazil C. A. Lutterbeck Institute of Sustainable and Environmental Chemistry, Leuphana University Lu¨neburg, 21335 Lu¨neburg, Germany L. T. Kist  Eˆ. L. Machado (&) Department of Chemistry and Physics, Universidade de Santa Cruz do Sul, UNISC, Av. Independeˆncia, 2293, Bairro Universita´rio, CEP, Santa Cruz do Sul, RS 96815-900, Brazil e-mail: [email protected] E. A. L. Alcayaga Department of Biology and Pharmacology, Universidade de Santa Cruz do Sul, UNISC, Av. Independeˆncia, 2293, Bairro Universita´rio, CEP, Santa Cruz do Sul, RS 96815-900, Brazil

methods that can mitigate the environmental impacts caused by the ecotoxicity and genotoxicity of hospital laundry wastewaters. Wastewaters generated from health facilities pose a potential threat to the environment and to public health due to the discharge of toxic chemical substances affecting several aquatic species (Paz et al. 2006). These wastewaters are classified as dangerous sources of pollution, especially because they consist of complex mixtures of numerous substances, such as organic matter detergents, surfactants, antibiotics, antiseptics, solvents, medical drugs, heavy metals, and even radioactive substances (Emmanuel et al. 2005; Nun˜ez and Moretton 2007; Gautam et al. 2007; Tsakona et al. 2007; Verlicchi et al. 2010), which are generated intermittently by different services of a hospital. When discharged directly into the sewage systems, water bodies and sources of water for agriculture and human consumption will become contaminated (Emmanuel et al. 2005; Ortolan and Ayub 2007). The scientific community has shown interest in the hazardous potential of hospital wastewaters. Publications can be found that provide general characterization on emerging pollutants (Ku¨mmerer et al. 2002; Drillia et al. 2005; Herberer and Feldmann 2005; Larsson et al. 2007; Verlicchi et al. 2010), on the assessment of toxicity (Emmanuel et al. 2004, 2005; Tsakona et al. 2007; Boillot and Perrodin 2008; Santos et al. 2010) and genotoxicity (Paz et al. 2006; Bagatini et al. 2009; Gupta et al. 2009), and on microbiological investigation (Nun˜ez and Moretton 2007; Fuentefria et al. 2008). In the same way the use of several treatment methods—such as physicochemical processes (Gautam et al. 2007; Suarez et al. 2009), membrane bioreactors (Wen et al. 2004; Kovalova et al. 2012) and advanced oxidative processes (Kajitvichyanukul and Suntronvipart 2006; Machado et al. 2007; Kist et al. 2008; Berto et al. 2009; Vasconcelos et al. 2009; Machado et al.

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2012; Kist et al. 2013)—has also been the subject of various investigations concerning these wastewaters. Among the different types of hospital wastewaters, those generated from laundries are a major problem because they have a high chemical oxygen demand (COD), biochemical oxygen demand for five days (BOD5), microbial load, and toxicity depending on the washing stage, and they also have a high concentration of particulate matter, proteins, starch, fat, oils and grease, detergents, disinfectants, and pharmaceutical products (Kist et al. 2008; Machado et al. 2012). Many of the pollutants in these wastewaters are ‘‘emerging pollutants,’’ which are not yet considered by regulatory agencies, but which can be regulated in the future depending on the study of their effects on the environment and on human health (Verlicchi et al. 2010). The discharge of this type of wastewater within the environment can produce negative impacts in the aquatic ecosystems directly by causing imbalance at different trophic levels from ecotoxicological effects and indirectly by eutrophication (Ku¨mmerer et al. 2002; Berto et al. 2009). Because of its high sensitivity against different wastewaters and capacity to detect toxic effects at several end points, plant bioassays have been extensively used during the last years in the environmental monitoring (Leme and MarinMorales 2009; Sobrero and Ronco 2004). The assay involving Lactuca sativa is a simple and fast test with a low cost, and it allows comparison of bioassay results for many different compounds. Furthermore, this species is widely consumed, has economic and ecological relevance, and was proposed for use in toxicity studies by the United States Environmental Protection Agency (USEPA) and the Organisation for Economic Co-operation and Development (OECD) (Migliore et al. 2003; Sobrero and Ronco 2004). Among several assays using higher-trophic plants, the test with Allium cepa has been receiving special attention and has been frequently used in the biomonitoring of a wide range of compounds. In addition to its sensitivity, the A. cepa test is known for its good correlation to other test systems, thus allowing for accurate assessment of environmental risks and to the successful extrapolation of test results obtained in exposed organisms to other species (Leme et al. 2008b). The use of aquatic organisms for biomonitoring is an important tool in aquatic ecotoxicology that allows for assessment of the level of pollution and degree of toxicity of wastewaters into the environment. Daphnia magna is a microcrustacean with worldwide distribution in freshwater that has been widely used as a test organism in ecotoxicological assays involving several chemical compounds present in aquatic ecosystems. Due to its importance in the food chain, sensitivity to toxic agents, and easy handling in the laboratory, standard methods with D. magna have been recommended by international environmental agencies (OECD 202 2004; USEPA 2002). The use of the fish Danio rerio in

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ecotoxicological assays may be attributed mainly to its availability in many countries, easy growth in the laboratory, and satisfactory sensitivity to wide range of chemicals. Furthermore, it is internationally recognized as a species internationally standardized for use in toxicity test ( OECD 1992). Therefore, the aim of the present study was to assess the ecotoxicity and genotoxicity of hospital laundry wastewaters. Physicochemical and microbiological analyses were performed to verify critical load parameters and compare them with national and international regulations.

Materials and Methods Characterization of the Study Site Wastewater samples were collected from a regional hospital located in Santa Cruz do Sul, central region of the state of Rio Grande do Sul, Brazil. Approximately 150 m3 day-1 of wastewaters is generated by the hospital, which is discharged into the local sewage system without any previous treatment. The hospital laundry accounts for 48–50 m3 day-1 of wastewaters generated from the average washing of 970 kg day-1 of textile items, thus contributing 33 % to the generation of hospital wastewaters. Laundry wastewater has a distinct composition if compared with those generated by other hospital departments: It has a high concentration of organic and microbial load in the early washing stage. The initial washing stage of surgical pads accounts for 12.8 m3 day-1 of wastewaters discharged in the three initial rinses. Specifically, the high content of organic matter at this stage produces hazardous wastewaters with high concentrations of bodily fluids, such as blood, feces, vomit, and others, in addition to a high microbial load and the potential presence of viruses, drugs, detergents and other cleaning products, such as sanitizers and disinfectants, adhering to the pads. Methods for Physicochemical, Chromatographic, and Toxicological Characterization The characterization of untreated wastewaters followed the American Public Health Association/American Water Works Association Standard Methods for the Examination of Water and Wastewater (American Public Health Association/American Water Works Association 2008) for determination of COD, BOD5, total Kjeldahl nitrogen (TKN), total phosphorus (totalP), pH, turbidity, conductivity, total coliforms, and Escherichia coli. A method of solid phase extraction (SPE) was used for the clean-up of samples on a Strata C18-E column (500 mg/3 ml cartridges; Phenomenex, Torrance, California, USA). For preparation of the column, 4 ml of methanol 2 ml H2Od and

Arch Environ Contam Toxicol

0.5 ml of a buffer solution (pH 6.8) were used. One hundred milliliters of the sample were eluted on the cartridges; afterwards 4 ml of the solution acetonitrile and methanol (1:1) were collected (Donato et al. 2012). A gas chromatograph GC-2010 (Shimadzu, Kyoto, Japan) coupled to a mass spectrometer GCMS-QP2010 (Shimadzu) was used for the separation and identification of the organic compounds present in the samples. The separation was performed on a Zebron CP 7420 column (60 m 9 0.25 mm 9 0.25 mM; Phenomenex). A solution of acetonitrile and methanol (1:1) was used as mobile phase, and helium was used as carrier gas. The column temperature started at 80 °C during the analysis and increased B280 °C. Analyses were performed during the course of 70 min. Acute toxicity in D. magna Straus 1820 was assessed by immobility assays according to the recommendations of the ABNT-NBR 12713-04 (2004) standard. Neonates of D. magna were obtained from cultures kept at the Laboratory of Ecotoxicology of Universidade de Santa Cruz do Sul (UNISC). Organisms were fed daily with Scenedesmus subspicatus and maintained at 20 ± 1 °C under a controlled photoperiod (18:8 h of light to dark). The sensitivity of the Daphnia cultures was evaluated monthly using potassium dichromate (K2Cr2O7). The method consists of exposure of individuals at 2–26 h of life to different sample concentrations for 48 h in the dark at a controlled temperature (20 ± 1 °C). Samples were prepared with volumetric accuracy and geometric progression at a ratio of one half. Eight concentrations ranging between 0.4 and 50 % were tested in 50-mL beakers containing 25 mL of the samples. The different concentrations were tested in duplicate, so 10 individuals were placed in each beaker for a total of 20 individuals/concentration. Three independent experiments were performed, and the EC50 values with 95 % confidence intervals were estimated by the trimmed Spearman–Karber method (Hamilton et al. 1979). In fish toxicity assays, D. rerio was used according to the recommendations of the ABNT-NBR 15088-04 (2004) standard. Individuals of this species were obtained from the local market, and before their use they were acclimated under laboratory conditions to each assay for 1 week. During this period, the quality of the test organisms was controlled by removing diseased individuals or those that showed a different behavior (immobility) from the group of tested organisms. Before the start of the test water was aerated for at least 12 h for oxygen saturation and stabilization of pH ranging between 7.0 and 7. 4, and the fish were fed up until 24 h before the tests. Five wastewater concentrations ranging between 3 and 50 % as well as the negative control were tested in 2-L beakers containing 1.5 L of solution. Ten organisms per concentration were tested and kept at 25 ± 2 °C with a controlled 12-hour photoperiod for 48 h. Three independent experiments were

performed (one per sample). After the test, the number of dead individuals was determined, and the LC50 values with 95 % confidence intervals were estimated by the trimmed Spearman–Karber method (Hamilton et al. 1979). Phytotoxicity assays were performed by adapting the method of Sobrero and Ronco (2004), using L. sativa L. and A. cepa L. seeds. The assay consists of exposure of the seeds plated on Petri dishes with a diameter of 100 mm and lined with 90-mm Whatman filter paper. Before exposure of the seeds, the filter paper was soaked in 4 mL of samples at five different concentrations at a ratio of one half, ranging between 6 and 100 %, in duplicate. Distilled deionized water was used as negative control. Twenty seeds per dish were exposed for a total of 40 individuals/ concentration in each test. Three independent assays were performed amounting to 120 seeds/concentration at the end of the experiments. After exposure, the seeds were incubated in the dark at a controlled temperature of 20 ± 1 °C. Lactuca sativa seeds were incubated for 120 h, whereas A. cepa seeds were exposed for 168 h. Thereafter, root length per concentration was recorded, and the inhibition coefficient (IC) was estimated using the statistical software proposed by Norberg King (1993). The assay results were expressed the IC25, i.e., initial nominal concentration that decreases the growth of roots exposed to different concentrations by 25 % compared with the negative control. Cytogenetic Assessment Using Meristematic A. cepa Cells The cytogenetic assessment was performed in seeds by adapting the methods used in Grant 1982; (Fiskesjo 1985, 1993), Rank and Nielsen 1993; Grover and Kaur 1999; Yi et al. 2007, and (Leme and Marin-Morales 2008a, 2009). The method consisted of exposure of seeds in Petri dishes, lined with paper filter and saturated with 4-mL of samples, which, in this case, corresponded to the wastewater samples at five concentrations, 6.25–100 %, in triplicate, totaling 60 individuals exposed per concentration. Distilled deionized water was used as negative control, whereas K2Cr2O7 at two different concentrations, 1.4 and 10 mg L-1, was used as positive control. After the exposure, the seeds were incubated at 20 ± 1 °C, in the dark, for 168 h and fixed thereafter in Carnoy’s solution (absolute ethanol and glacial acetic acid at a 3:1 ratio) for 24 h at 25 ± 2 °C. For slide preparation, the material fixed in Carnoy’s solution was submitted to acid cell lysis with hydrochloric acid (HCl) 1 mol L-1 at 60 °C for 11 min and later exposed to Schiff reactive staining for 20 min in the dark. Thereafter, the meristematic region was removed and softly crushed in two droplets of 45 % acetic acid on a glass slide and covered by a cover slip. Five treatment slides were analyzed, 1

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Arch Environ Contam Toxicol Table 1 Physicochemical and microbiological characterization of hospital laundry wastewaters

UWTD urban wastewater treatment directive

Parameter

Unit

Wastewater

CONSEMA 128/2006a

UWTD 91/271/EECb

COD

mg L-1

3,385 ± 315.35

360

125

BOD5

mg L-1

2,042 ± 201.39

150

25

COD/BOD5

–

1.65

–

–

Total P

mg L-1

0.73

4

1

TKN

mg L-1

76.6

20

10

pH

–

8.33 ± 0.23

6.0–9.0

6.0–9.0

Turbidity

NTU

178.7 ± 42.93

–

–

Conductivity

lS cm-1

411.53 ± 53.47

–

–

Total coliforms

CFU/100 mL

2.8 9 107

–

–

E. coli

CFU/100 mL

2.1 9 107

105

–

meristem/slide, each representing by one individual. The observations were made under a light microscope, using the count of 5,000–8,000 cells/group of five slides/ concentration. The cytogenetic analysis consisted of the assessment of cytotoxicity, inherent to the record of changes in the mitotic index (MI); genotoxicity, which is characterized by chromosome aberrations (CA)—such as losses, fragments, delays, bridges, adhesions, and viscosity, among others— observed at metaphase, anaphase and telophase; and mutagenicity, which is determined by the frequency of MCN. MI was determined by the cell division rate using the following equation: MI ¼ number of cells in mitosis ðfrom prophase to anaphaseÞ total number of cells x 100 CA and MCN were determined by their frequency in the total number of cells counted per concentration. The data were analyzed by each indicator, and differences were examined by Chi square test (V2) in a 2V2 contingency table at a significance level of p \ 0.05. The Instat Graphpad program version 5.00 (Spiegel 2004) was used for statistical analysis. Results and Discussion General Analytical Characterization of the Wastewater The physicochemical characterization of the initial rinsing stage showed the presence of high pollutant load, with COD, BOD5, and TKN of 89.36, 92.65 and 74.65 %, respectively, for flows \100 m3 d-1 (Table 1), which are above the limits allowed by Resolution 128/06 established by Rio Grande do Sul State Council on the Environment (CONSEMA), the state council for wastewater disposal in

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Rio Grande do Sul. When considering the European guideline (Directive 91/271/EEC) for urban wastewater treatment, the high pollutant load of this effluent become still more evident. The COD is 96.31 % greater the maximal concentration limit considered in this directive, whereas the measured values of for BOD5 and TKN are 98.78 and 86.95 % greater, respectively, than the allowed concentrations. COD and BOD5 values were high and way greater than those described in some studies addressing hospital wastewaters where COD and BOD5 ranged from 1,388 to 632 mg L-1(Beyene and Redaie 2011), from 1,350 to 410 mg L-1 (Kajitvichyanukul and Suntronvipart 2006), from 880 to 550 mg L-1 (Tsakona et al. 2007), from 855 to 603 (Emmanuel et al. 2002). The presence of low COD and BOD values led some investigators to include hospital wastewaters in the same category as domestic wastewaters. However, in some cases, these values are much greater and exceed the limits established by regulatory agencies (Emmanuel et al. 2005). The values found in this article are close to those of Berto et al. (2009) with maximum COD and BOD5 values, respectively, of 2,480 and 1,268 mg L-1 and those of Emmanuel et al. (2005) with 2,516 and 1,559 mg L-1. The nitrogen load was close to that observed by Berto et al. (2009), i.e., 85.5 mg L-1 and shows the need to treat the effluents to adjust these parameters and avoid environmental hazards. The analytical characterization of the studied wastewaters shows the presence of low N-NH3 values (2.3 mg L-1) because the values determined for TKN were in the range of 70 mg L-1. (Du¨pont and Lobo 2012) associated values of N-NH3 in the range of 70 mg L-1 to the acute toxicity effects to D. magna in a study performed with urban wastewaters. Therefore, the low values of N-NH3 obtained in our results are unexpected to confer acute toxicity. The results of gas chromatography analysis indicated the occurrence of some important classes of compounds in the laundry wastewater, such as surfactants, organic acids, and

Arch Environ Contam Toxicol

products containing aromatic rings. Among them we can mention the presence of lauryl ethoxylate, benzoic acid, n-(4-aminophenyl)-n-methylacetamide 2-(diethylamino)-n(2,6-dimethylphenyl)-acetamide),and methyl-m-(1-methylbutyl) phenyl ester. Nonetheless, the compounds identified in the analyses presented low peak intensity (low peak area). Quantitative analysis may indicate future studies for the quantification of the compounds as well as establish a correlation with the ecotoxicity. pH varied slightly, less than 1 U, and showed the existence of alkaline conditions. The conductivity values indicate the existence of different concentrations of mineral substances (Emmanuel et al. 2009), whereas turbidity is due to the presence of particulate matter such as blood and cotton fibers (Table 1). In addition to COD and BOD5, the pathogen load was also high, exceeding by [100 % the limits established by the national law (CONSEMA 128/06) (Table 1). The high incidence of E. coli indicates contamination of the wastewater by human fecal matter; this bacterium is one of the major organisms in the group of thermo-tolerant coliforms and may cause infection in humans by different modes of transmission (Emmanuel et al. 2009). Several works reported on the high incidence of coliforms and E. coli in hospital wastewaters. The incidence rates range from 1.6 9 102 (Wen et al. 2004) 1.1 9 105 (Nun˜ez and Moretton 2007) 2.9 9 105 (Machado et al. 2012) 1 9 106 (Emmanuel et al. 2005) 1.23 9 106 (Beyene and Redaie 2011), and 3.0 9 106 (Kist et al. 2008) to 1.6 9 108 (Berto et al. 2009). These high pollutant and microbial loads indicate the need to treat these wastewaters before their disposal into the local sewage system. The low COD-to-BOD5 ratio shows potential biodegradability, which somehow is favorable to the implementation of biological treatment systems. Ecotoxicological Characterization of Wastewater Acute toxicity was investigated by assays with aquatic species from two trophic levels: D. magna (primary consumer) and D. rerio (secondary consumer). The data showed extremely toxic responses of D. magna with a mean EC50 of 2.01 % and EC50 [ 1.0 for sample 2 (Table 2). Acute toxicity was less pronounced for D. rerio with a mean LC50 of 29.25 %. The highest toxicity, as with D. magna, was also observed for sample 2 (Table 2). In the literature, high acute toxicity of hospital wastewaters was detected in D. magna with an EC50 [ 2.0 % (Emmanuel et al. 2005) and of hospital laundry wastewaters as well, especially surgical pads, with an EC50 of 28.8 % (Machado et al. 2012). Detergents, surfactants, and disinfectants are the main substances that cause toxicity in aquatic species (Boillot and Perrodin 2008). Some

Table 2 Acute and chronic toxic effects of hospital laundry wastewaters Toxicity

EC50 D. magna

LC50 D. rerio

IC25 L. sativa

IC25 A. cepa

Sample 1

2.29 (2.14–2.45)a

34.72 (25.69–45.93)

10.8

58.09

Sample 2

0.94 (0.81–1.06)

17.68 (13.8–22.65)

Sample 3

2.81(2.43–3.26)

35.36 (26.61–46.97)

17.9

55.12

Mean

2.01

29,25

12.50

51.25

SD

0.97

10.03

4.79

9.39

CV (%)

47.94

34.28

38.30

18.32

8.79

40.55

Index values obtained for L. sativa and A. cepa were estimated by linear interpolation and therefore lack confidence interval values EC50 median effective concentration capable of causing an effect on 50 % of exposed organisms, IC25 concentration capable of inhibiting 25 % of the growth of exposed individuals, CV (%) coefficient of variation a

95 % confidence intervals are shown in parentheses

chemical substances with high toxicity, such as glutaraldehydes and surfactants, have been widely used in hospitals and detected in sewage systems (Boillot and Perrodin 2008). The high toxicity caused by the mix of glutaraldehyde and surfactant was also referenced for D. magna with an EC50 of 10–0.02 % (Emmanuel et al. 2004). Mixtures of glutaraldehyde and different surfactants were also investigated as to type of surfactant and were toxic to D. magna in the following order: cationic [ glutaraldehyde [ anionic [ nonionic (Boillot and Perrodin 2008; Kist et al. 2013). In addition to glutaraldehyde and surfactants, active ingredients of drugs cause high toxicity in D. magna with an EC50 of 6.7–7.2 % as a result of the exposure to pharmaceutical wastewaters consisting mostly of ciprofloxacin and losartan (Larsson et al. 2007). The toxicity of hospital wastewaters to aquatic species can also be attributed to high nitrogen loads, especially ammonium and ammonia (Emmanuel et al. 2005). In our study, ammoniacal nitrogen was not estimated, but it may be present at high concentrations in the TKN rate, detected at the average concentration of 76.6 mg L-1, which corresponds to all forms of nitrogen found in the wastewater (Table 1). The toxic effects of wastewaters on A. cepa and L. sativa were observed by the root growth-inhibition rate. The IC25 allows determination of the initial wastewater concentration that inhibits root growth by 25 % in both analyzed species. The toxic effect of the wastewater was more pronounced on L. sativa than on A. cepa. The mean IC25 was 12.50 % for L. sativa and 51.25 % for A. cepa. The maximum toxicity for both species was detected for sample

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2, in which 8.79 % was enough to cause a toxic effect on L. sativa, compared 40.55 % for A. cepa (Table 2). Lactuca sativa roots were sensitive to the analyzed wastewater. This species also showed sensitivity in the detection of toxicity of active drugs in pharmaceutical wastewaters, which predominantly consist of antibiotics that are not easily biodegradable, such as ciprofloxacin (Larsson et al. 2007) and enrofloxacin (Migliore et al. 2003), thus inhibiting the development of cotyledons with an EC50 of 1.2–3.2 % at 28,000–31,000 lg L-1 (Larsson et al. 2007) and of roots at concentrations of 5,000 lg L-1 (Migliore et al. 2003). In a study performed with 10 antibiotics, Hillis et al. (2011) obtained EC25 values for root growth of L. sativa, Medicago sativa, and Daucus carota ranging from 3.9 to 10,000 lg L-1. According to the investigators, chlortetracycline, levofloxacin, and sulfamethoxazole were the most phytotoxic compounds, whereas M. sativa was the most sensitive plant species followed by L. sativa and D. carota. The effects on growth proved to be the most sensitive aspect in the assessment of toxic responses of the species to the drug (Hillis et al. 2011). The uptake of pollutants affects plant growth. However, even when pollutants are absorbed, they can be converted by the metabolism giving rise to different toxic responses, which in some cases are undetectable (Migliore et al. 2003). In our study, the hospital laundry wastewater showed different toxic responses in the two plant species used. The potential risks of the wastewater for terrestrial species were then determined. Cytogenetic Assessment of Wastewater by A. cepa Test Compared with the negative control (18.9 % of mitotic cells), statistically significant differences were observed at all five concentrations of the raw wastewater as well as for the positive controls at the two concentrations of K2Cr2O7. These results show the cytotoxic potential of the effluent even for more diluted samples. Hospital wastewater had the greatest response to toxicity at a concentration of 50 %, at which growth-inhibition rates, compared with that of the negative control, were greater than those detected for the positive control (Table 3). The number of mitotic cells increased after the dilution of the untreated wastewater to 6.25 %; however, significant differences were still observed. No genotoxic effects were observed in cells regarding chromosome aberrations; nonetheless, there were significant differences from the positive control at the concentration of 10 mg L-1 of K2Cr2O7. The incidence of cells with chromosome aberrations in raw wastewater increased by 28.4 % compared with the negative control, but this was still not statistically significant (Table 3).

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Regarding the presence of micronucleated cells, it was possible to notice a more linear behavior of the data, with a direct influence of wastewater concentration, on the presence of MCN. Statistically significant differences were observed compared with the negative control at concentrations ranging from 25 to 100 %, whereas no differences were detected at wastewater dilutions of 12.5 and 6.25 % (Table 3). The presence of MCN in the 100 % untreated wastewater was 84.12 % greater than that of NC, thus exceeding the frequency found in PC 2 (83.33 %). These findings indicate the high potential of the wastewater to induce mutations (Table 3) in exposed organisms. K2Cr2O7 has been commonly used as a positive control in cytotoxic, genotoxic, and mutagenic assays. The results obtained in our study present induction changes of MI at both concentrations (1.4 and 10 mg L-1) and the presence of CA and MCN at the highest concentration (10 mg L-1). Micronuclei are traditional and reliable indicators in mutagenicity assays. Figure 1 illustrates some micronucleated cells found in the microscopic examination of the test organism. The origin of micronucleated cells in different organisms is related to unrepaired DNA damage and even to the presence of some chromosome aberrations such as losses. These cells may derive from polyploidization processes, in which they arise from the elimination of DNA from the main nucleus in an attempt to restore normal polyploidy (Fernandes et al. 2007). Genotoxicity in hospital wastewaters has been proven by the Ames (Salmonella mutagenicity [Gupta et al. 2009] ) and A. cepa tests (Paz et al. 2006; Bagatini et al. 2009), and it is believed that the combination of genotoxic substances of these wastewaters may be one of the possible causes of cancer in the past few decades (Gupta et al. 2009). The genotoxicity can be attributed to the presence of some drug classes such as anticancer drugs (ifosfamide, cisplatin, doxorubicin) and antimicrobial agents (ciprofloxacin); however, the correlation of dose-genotoxic effect data on a given compound in wastewaters is not an easy task due mainly to the presence of few available data in the literature and the variable characteristics of wastewaters, which depend on the type of hospital activity (Jolibois et al. 2003). The results of a study performed by Jolibois et al. (2003) showed variations in the genotoxic effects of a hospital wastewater by the use of a chromotest in E. coli PQ37 and by the Ames test in S. typhimurium strains TA 98 and TA 100 without metabolic activation. According to the investigators, genotoxicity was directly affected by low pluviometry, i.e., months in which rainwater collection by the hospital collection system (which mixes rainwater and the wastewater) was low, thus causing increased genotoxic activity frequency (Jolibois et al. 2003). In the literature, hospital wastewater dilutions to 50 % caused a remarkable increase of chromosome aberrations

0.63* ± 0.27 21.05

84.12

DfNC (%)

MCN V2

DfNC (%)

80.39

0.51* ± 0.23 15.13

8.7

0.07

0.69 ± 0.41

36.54

119.7

11.98* ± 2.68

871

7266

273

31

62

505

6395

50 %

66.67

0.30* ± 0.14 4.76

1.56

0.01

0.64 ± 0.32

30.19

75.91

13.18* ± 6.00

892

6766

162

43

52

635

5874

25 %

41.17

0.17 ± 0.09 0.47

13.7

0.27

0.73 ± 0.29

29.08

68.37

13.29* ± 5.14

801

6027

242

26

69

464

5226

12.5 %

0

0.10 ± 0.03 0.06

0

0.02

0.60 ± 0.27

24.78

49.54

14.20* ± 3.77

954

6717

307

34

79

534

5763

6.25 %

0

0.10 ± 0.08 0

0

0

0.63 ± 0.26

0

0

18.88 ± 3.33

1,102

5837

169

20

63

850

4735

NC

-1

56.52

0.23 ± 0.17 2.17

0

0.29

0.54 ± 0.21

12.92

12.15

16.44* ± 8.08

1,001

6090

120

23

38

820

5089

PC1

83.33

0.60* ± 0.21 19.49

39.42

5.46

1.04* ± 0.62

31.46

137.7

12.94* ± 3.93

795

6146

289

33

68

405

5,351

PC2

* Indicates differences according to V2 test relative to the NC with p \ 0.05

Division total total number of mitotic cells, NC negative control with distilled deionized water, PC1 positive control with K2Cr2O7 at 1.4 mg L , PC positive control with K2Cr2O7 at 10 mg L-1, DfNC (%) percentage difference of the indices relative to the negative control

2,155

28.4

V2

0.88 ± 0.17

907

Division total

28.44

6714

Total

CA

168

Telophase

DfNC (%)

29

Anaphase

13.51* ± 5.56

55

Metaphase

66.59

655

Prophase

MI

5807

Interphase

V2

100 %

Parameters

Table 3 Cytogenetic assessment of hospital laundry wastewater at five concentrations using the A. cepa test

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Arch Environ Contam Toxicol

Fig. 1 Presence of different-sized micronuclei at the interphase of A. cepa meristematic cells exposed to different hospital laundry wastewater concentrations

in A. cepa, thus confirming that chemical substances may promote genotoxicity to the biomonitor (Paz et al. 2006). In another study, hospital wastewaters also showed high capacity to cause genotoxicity with a lower MI (62.5 %) than that of the NC for the untreated wastewater (Bagatini et al. 2009). The genotoxic effect of drugs on pharmaceutical wastewaters was also investigated by Akintonwa et al. (2009), who used different indicators, such as the modified Ames test for E. coli (0157:H7) plus the S9 mix metabolic activator, and also the A. cepa test. Of six types of wastewaters analyzed, three proved to be cytotoxic and genotoxic for A. cepa at concentrations of 5–15 %. The presence of mutagenic chemical agents is a significant problem in hospital wastewaters that are discharged directly into sewage systems, thus rendering waters inappropriate for irrigating crops and drinking. Results obtained for the genotoxicity of hospital wastewaters for three large hospitals in New Delhi, India, indicated that genotoxicity is remarkably decreased after the use of treatment systems; however, owing to the peculiar composition of wastewaters, they must be pretreated before a biological system is used (Gupta et al. 2009). In this study, cytotoxicity and mutagenicity were detected, also at lower concentrations, indicating the persistence of genotoxic chemical substances. The results warn about the necessity to adopt measures that allow developing treatment methods capable to mitigate the environmental impacts caused by the toxic and genotoxic effects of hospital laundry wastewaters before they are discharged into sewage systems and water bodies.

Conclusion This study showed that hospital laundry wastewaters, when not treated properly, can cause large environmental impacts and be hazardous to human health by contaminating water bodies with toxic and genotoxic pollutants. Toxicity was detected for two trophic levels within aquatic ecosystems and one trophic level in soil ecosystems.

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Cytogenetic changes led to the classification of hospital laundry wastewaters as cytotoxic at the five concentrations analyzed and as mutagenic for three concentrations in A. cepa. The wastewaters had a greater mutagenic effect than those caused by K2Cr2O7 at its highest concentration. These results show the need to conduct a more in-depth investigation into the different types of hospital wastewaters to know their toxic and genotoxic effects and to propose specific treatments before the use of any general biological treatment and their discharge into local sewage systems. Acknowledgments The authors acknowledge the support provided by the Research Incentive Fund of UNISC (Graduate Program in Environmental Technology) and financial support from Research Foundation of the State of Rio Grande do Sul (FAPERGS) and Commission for the Improvement of Higher Education Personnel (CAPES).

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