Runoff of genotoxic compounds in river basin sediment under the influence of contaminated soils

Ecotoxicology and Environmental Safety 75 (2012) 63–72

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Runoff of genotoxic compounds in river basin sediment under the influence of contaminated soils Thatiana Cappi da Costa a,b, Kelly Cristina Tagliari de Brito a, Jocelita Aparecida Vaz Rocha a, Karen Alam Leal a,c, Maria Lucia Kolowski Rodrigues a,c, Jean Paolo Gomes Minella d, Silvia Tamie Matsumoto e, Vera Maria Ferra~ o Vargas a,b,n a

Programa de Pesquisas Ambientais, Fundac- a~ o Estadual de Protec- a~ o Ambiental Henrique Luı´s Roessler (FEPAM), Av. Salvador Franc- a, 1707, 90690-000, Porto Alegre, RS, Brazil ~ em Ecologia, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Bento Gonc- alves, 9500, 91501-970, Porto Alegre, RS, Brazil ´s-graduac- ao Programa de Po c ~ de Quı´mica, FEPAM, Rua Aure´lio Porto, 37, 90620-090, Porto Alegre, RS, Brazil Divisao d Universidade Federal de Santa Maria, Av. Roraima, 1000, 97105-900, Santa Maria, RS, Brazil e ´rio de Mutagˆenese Ambiental in vitro e in vivo, Universidade Federal do Espı´rito Santo (UFES), Av. Fernando Ferrari, 514, 29075-910, Vito ´ria, ES, Brazil Laborato b

a r t i c l e i n f o

abstract

Article history: Received 29 April 2011 Received in revised form 30 July 2011 Accepted 6 August 2011 Available online 3 September 2011

Contaminated sites must be analyzed as a source of hazardous compounds in the ecosystem. Contaminant mobility in the environment may affect sources of surface and groundwater, elevating potential risks. This study looked at the genotoxic potential of samples from a contaminated site on the banks of the Taquari River, RS, Brazil, where potential environmental problems had been identified (pentachlorophenol, creosote and hydrosalt CCA). Samplers were installed at the site to investigate the drainage material (water and particulate soil matter) collected after significant rainfall events. Organic extracts of this drained material, sediment river samples of the Taquari River (interstitial water and sediment organic extracts) were evaluated by the Salmonella/microsome assay to detect mutagenicity and by Allium cepa bioassays (interstitial water and whole sediment samples) to detect chromosomal alterations. Positive mutagenicity results in the Salmonella/microsome assay of the material exported from the area indicate that contaminant mixtures may have drained into the Taquari River. This was confirmed by the similarity of mutagenic responses (frameshift indirect mutagens) of organic extracts from soil and river sediment exported from the main area under the influence of the contaminated site. The Allium cepa test showed significant results of cytotoxicity, mutagenic index and chromosome aberration in the area under the same influence. However, it also showed the same similarity in positive results at an upstream site, which probably meant different contaminants. Chemical compounds such as PAHs, PCF and chromium, copper and arsenic were present in the runoff of pollutants characteristically found in the area. The strategy employed using the Salmonella/microsome assay to evaluate effects of complex contaminant mixtures, together with information about the main groups of compounds present, allowed the detection of pollutant dispersion routes from the contaminated site to the Taquari River sediment. & 2011 Elsevier Inc. All rights reserved.

Keywords: Salmonella/microsome assay Allium cepa test Wood preservation Contaminated soil site

1. Introduction Chemical substances intentionally introduced or toxic pollutants accidentally discharged onto the soil may cause contamination at the site. In some cases (e.g., wood preserving wastes, coal-tar, airborne combustion by-products), the contaminated soil creates a genotoxic hazard (White and Claxton, 2004). Inventories performed by national environmental agencies indicated that the number of contaminated sites per 1000 km2 in several European countries revealed the highest concentrations and values investigated, ranging from 6.5 in Norway to 868.7 in n Corresponding author at: Programa de Pesquisas Ambientais, Fundac- a~ o Estadual de Protec- a~ o Ambiental Henrique Luı´s Roessler (FEPAM), Avenida Salvador Franc-a, 1707, CEP: 90690-000, Porto Alegre, RS, Brazil. Fax: þ55 51 33346765. E-mail address: [email protected] (V.M.F. Vargas).

0147-6513/$ - see front matter & 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2011.08.007

Denmark, 847.7 in Switzerland and 568.4 in Germany. In the United States it is up to 4.7 and in Canada 1.5–4.0. In Brazil, Sa~ o Paulo, the state with the highest level of industrial development alone, over 2372 contaminated sites are listed (CETESB, 2008; White and Claxton, 2004). New laws have been enacted in Brazil to deal specifically with soil quality (Brazil, 2009). Contaminated soil sites may be harmful to the ecosystem, due to the runoff of organic compounds and heavy metals. Most of the chemicals released into the surface water are carcinogenic and become part of complex environmental mixtures (Ohe et al., 2004; Vargas et al., 1993; Vargas et al., 2008). The components of these mixtures can have adverse health effects on humans and indigenous biota (Dearfield et al., 2002; Vargas et al., 2008). There has been a growing interest in identifying chemicals associated with the use of biomarkers to monitor environmental quality (Cazenave et al., 2009). Bioassays, especially the Salmonella mutagenicity test, have

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T.C. da Costa et al. / Ecotoxicology and Environmental Safety 75 (2012) 63–72

been used in most assessments of genotoxic hazards of surface water, sediments and soil. The Salmonella/microsome assay (Ames test) is a widely accepted short-term assay for identifying substances that can produce genetic damage (Mortelmans and Zeiger, 2000). It is used worldwide to detect the mutagenicity of biological mixtures such as water, sediment, soil, atmospheric environmental samples and pure chemicals (Chen and White, 2004; Ducatti and Vargas, 2003; Horn and Vargas, 2008; Ohe et al., 2004; Tagliari et al., 2004; Vargas et al., 2008; White and Claxton, 2004). Aspects ranging from gene mutations to chromosome damage and aneuploidies can be identified by analysis of eukaryotes. Higher plants present characteristics that make genetic models to assess environmental pollutants, and they are often used in monitoring studies (Leme and Marin-Morales, 2009). Tests were performed employing a variety of plant species such as Tradescantia, Vicia faba, Zea mays and Allium cepa (Chen and White, 2004; Mortelmans and Zeiger, 2000; Ohe et al., 2004; White and Claxton, 2004; Fernandes et al., 2007). The study intends to define the contribution of rainfall runoff to the dispersion routes of genotoxic contaminants from a soil contaminated site where chemicals were used by the wood preservation industry on Taquari River sediments. The most widely used method to prevent attacks by xylophagous insects and lignivorous fungi is the introduction of chemical substances that are toxic to these organisms (Appel et al., 2007). Currently, the most widely used wood preservative is chromated copper arsenate (CCA), which has replaced alternative organic preservatives such as pentachlorophenol (PCF) and creosote due to environmental and human health concerns (Hingston et al., 2001). There is a very high risk of transfer and impact of wood preservatives on the aquatic environment in such areas (Adam et al., 2009).

north–south direction, and is 140 km long (Fig. 1). It is characterized as a plain river, with a mild declivity, rare rapids, and wide outflow. The river is used for water supply, irrigation, recreation, fishing and various other purposes (FEPAM, 2010a,b). The specific study area is under the influence of a site whose soil is contaminated with chemicals used at a wood preservation plant located in the municipality of Triunfo, Rio Grande do Sul State, Brazil. This contaminated site is located between the Taquari riverbank and a residential area. The 18 ha area is flat with small streams flowing towards the main river drainage. Part of the area was landfilled using local soil and debris, with a 0.5–5 m thick layer. The local aquifer is phreatic, with water table depths between 1.6 and 6 m. Because of its physical characteristics, the site is highly susceptible to contamination of soil, groundwater and surface water (FEPAM, 2010a,b). During the time when the plant operated – from 1960 to 2005 – wood preservatives such as pentachlorophenol (PCF), creosote and hydrosalt CCA (chromated copper arsenate) were employed, treating large volumes of wood poles, consequently releasing large amounts of hazardous chemical wastes into the environment. The area is potentially a major source of pollutants in natural resources of this basin area (river water, sediment and soil). The region is next to a residential area and to agricultural crops. Early chemical analyses of the industrial area confirmed the contamination of groundwater and soil by polycyclic aromatic hydrocarbons (PAHs), Cr, Cu, As, PCF, dioxins and furans (FEPAM, 2010a,b). This basin area is potentially a major source of pollutants in natural resources. The relief of the contaminated site is flat with three distinct parts (Fig. 2): (i) the first area consists of a plateau located in the center of the terrain, where the wood treatment infrastructure was installed. This included treatment tanks and storage tanks for chemicals, autoclaves and drying sheds. This part of the terrain is more elevated than the others and drains towards the sides (industrial area A1); (ii) To the left of the central plateau, towards the river, there is an ill drained flood plain with a waste reservoir that is partially isolated. In this area there used to be storage places for treated woods and buried residues that could contaminate the base flows and groundwater flows, with possible surface runoff towards the river (drainage area A2); (iii) To the right of the plateau is another flat area with better drainage used as a storage place for treated wood. This area drains towards the river through a small stream that receives surface runoff from the plateau (drainage area A3).

2. Materials and methods

In order to evaluate the contaminated soils site, two types of sampler manufactured from neutral glass were placed inside the contaminated soil site to evaluate organic compound runoff from the soil in the plant area. One of them was placed on the surface of the soil (Sampler 1) in a flat area with better drainage (291520 17.8200 S 511430 7.8300 W) (A3). This sampler was made of dark glass with a conical, funnel-shaped structure, 30 cm long and 18 cm in diameter, connected to a glass reservoir with an approximately 2-liter capacity. The sampler was placed

2.1. Area of study The study was developed in the Taquari River, Taquari–Antas basin, in the northeast of Rio Grande do Sul State, Brazil. This river lies predominantly in the

2.2. Sampling sites

Fig. 1. The Taquari–Antas river basin, sampling areas in the Taquari River, Ta 032, Ta 010 and Ta 006. The numbers show the distance from the mouth in kilometers.

T.C. da Costa et al. / Ecotoxicology and Environmental Safety 75 (2012) 63–72 on the ground in an area used for the industrial processes (A1, next to the plant). It was intended (used) to characterize the eroded material from the plateau where the poles were treated (Figs. 2 and 3). According to hydrosedimentological studies (FEPAM, 2010a,b) the area drains into the Taquari River through the streams flowing across the site. Sampler 2 was placed in one of these streams under the influence of the drainage area A3 and secondarily of the industrial area (A1) (291520 14.1900 S 511430 06.7300 W; Figs. 2 and 3). This torpedo-type glass/stainless or plastic sampler has a small orifice on each side (both 4 mm in size), and is one meter long with a 100 mm diameter. When the flow enters the sampler the flow speed (velocity) diminishes, increasing the sediment deposition rate inside it (Figs. 2 and 3). Sampler 2 collected samples of suspended material, integrated over time for all rainfall events that occurred during the study period, especially the influence of Area A3.

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Three sampling points in the Taquari River (Fig. 1) were chosen for the study. One in the city of Taquari as reference area, Ta 032 (291480 19.200 S 511520 50.200 W), another in the city of Triunfo at the site contaminated by wood preservatives, Ta 010 (291520 23.900 S 511430 21.9900 W; Fig. 2), and lastly in the city of General Cˆamara, Ta 006 (291540 09.700 S 511450 05.100 W). Site Ta 010 is potentially influenced by the contaminated site through two streams draining into the Taquari River. The numbers show the distance from the mouth in kilometers. The areas are located in the south region of the Taquari–Antas basin, at an altitude of 6–100 m above sea level, with a 0 to three percent slope. These are plain areas and favor the development of annual crops, such as maize, soy, wheat and rice. They also are used for cattle-raising and to cultivate species, such as Acacia, Eucalyptus and Pinus. Native species are found in the vicinity of watercourses, constituting riparian formations (De Lima et al., 2007). The Ta 032 area, in particular, is used for recreation activities. The Ta 010 site suffers the influence of an area contaminated by chemicals used for wood preservation, with a possible contribution of organic pollutants and heavy metals to the Taquari River and TA 006, which is under the influence of agricultural activities (rice). 2.3. Sample collection At the contaminated soil site both samplers were installed on 12/19/2007 and their material was collected in two periods, first on 08/19/2008 (winter) and the second on 10/26/2008 (spring), after a major rainfall event. The second sampling presented high mean relative humidity, temperature and mean precipitation compared to the first sampling (Fig. 4). The samples of water and sediment collected were placed at 4 1C in dark glass flasks protected from light until they were used for the tests (glass/stainless Sampler 2 for organic compounds and mutagenic analyses; plastic Sampler 2 for metals analyses). The first sediment sampling was performed in Taquari River in 12/2007 (summer) and the second in 09/2008 (winter) to study mutagenicity. At both samplings the mean relative humidity was similar and the mean temperature in summer was 24 1C and in winter 15 1C. Mean precipitation data were also looked at, and 12/2007 was dry compared to 09/2008 (Fig. 3). The sediment samples were collectedat at about 20 cm from the riverbed in a compound sample, placed at 4 1C in dark glass flasks protected from light until they were used for the tests (Tagliari et al., 2004; Vargas et al., 2001). The granulometric characterization of sediment samplings had a simple classification, where in the first sampling Ta 032 was classified as sand with mud, Ta 010 as mud and Ta 006 as mud with sand. In the second sampling Ta 032 was classified as gravel with sand, Ta 010 as mud with sand and Ta 006 as mud with sand (Fig. 4). 2.4. Sample preparation

Fig. 2. Internal area of the wood plant. Sample localization: Ta 010 in the river, Sampler 1 in the soil and Sampler 2 in the water body. A1—industrial area; A2—drainage area; A3—drainage area.

2.4.1. Organic liquid–liquid extraction The liquid part of superficial soil draining samples (Sampler 1 and Sampler 2) was submitted an organic extraction procedure, as described in Vargas (1992)

Fig. 3. Sampler 1 (A) and Sampler 2: glass/stainless and plastic (B).

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T.C. da Costa et al. / Ecotoxicology and Environmental Safety 75 (2012) 63–72

Fig. 4. Meteorological data: relative humidity, temperature and precipitation during the study period (December/2007 to October/2008) and grain size classification of the Taquari River sediment samples (Ta 032, Ta 010 and Ta 006) in two samplings. 1 First sediment sampling from Taquari River. 2 First sampling of drained soil after a significant rainfall event. 3 Second sediment sampling from Taquari River. 4 Second sampling of drained soil after a significant rainfall event. with modifications (water extracts). Two extraction steps were performed with a sample of 800 ml of the superficial soil drainage, using the pesticide-grade dichloromethane solvent (CASRN 75-09-2), resulting in sample fractionation into two extracts of compounds having affinity with neutral and acid pH. The first step was to add dichloromethane and manually shake for 2 min. Then the neutral fraction was extracted and filtered in glass wool with sodium sulfate. This procedure was repeated twice more. In the second step, the same sample was acidified (pH 2) with sulfuric acid (1:1) and the procedure to extract the acid fraction was similar, thus obtaining the second extract. The EOM (extractable organic matter) was measured in a 10  4 analytic precision scale.

2.4.2. Sediment organic extracts Organic sediment extracts were obtained from the Taquari River and from the particulate soil matter, forming sedimentary material (Sampler 2) to analyze mutagenicity as described in previous papers (Vargas et al., 2001; Tagliari et al., 2004). The sediment (50 g) or particulate soil material (25 g) samples were filtered in glass wool with sodium sulfate. The solvent dichloromethane (CASRN 75-09-2) was added, mixed and sonicated for 5 min at 100 W to extract moderately polar fractions. This operation was repeated five times and the filtrate obtained passed through a chromatographic column containing sodium sulfate and celite. The extract was concentrated in a rotary evaporator (40 1C), stored at  20 1C for up to 14 days, and the density was determined. At the time of analysis, the extracts were dried by evaporation under a nitrogen gas flow and resuspended in dimethylsulf¨ CASRN 67-68-5), spectrophotometric grade. oxide (DMSO—Riedel-de Haen,

2.4.3. Interstitial water and gross sediment The sediment samples from the Taquari River were centrifuged at 10,000  g, for 10 min at 4 1C to extract interstitial water. The water extracted was filtered using a 0.45 mm Milipore filter, divided into aliquots and frozen at  20 1C to evaluate the mutagenicity. For the Ames test, the samples were sterilized in 0.22 mm Milipore filter (APHA, 1992). These filters were submitted to extractions procedures (after the samples were filtered, the filters were used to extract the compounds adhered to them with water and DMSO) for analysis of the presence of possible mutagens retained, during the filtration process. The river sediment samples were frozen to evaluate the mutagenicity using the Allium cepa test.

2.5. Mutagenicity assays 2.5.1. Salmonella microsuspension bioassay The mutagenicity of interstitial water, sediment organic extracts and liquid– liquid organic extract (superficial soil draining) was evaluated using the Salmonella/ microsome assay by pre-incubation method. In this study three types Salmonella typhimurium strains were used, TA98 (hisD3052, frameshift GC, rfa, Duvrb, pKM101), TA97a (hisD6610, frameshift GC, rfa, Duvrb, pKM101) and TA100 (hisG46, base-pair substitution GC, rfa, Duvrb, pKM101). TA98 and TA100 are generally used as standard strains and give a screening answer. The strain most often indicated to evaluate the mutagenicity of heavy metals is TA97a (Levin et al., 1982). The interstitial water volumes evaluated were 50, 100, 150, 250 and 400 mL per plate, expressed as 0.05, 0.1, 0.15, 0.25 and 0.4 ml per plate, respectively, the liquid– liquid organic extract was analyzed at volumes of 0.62, 1.25, 2.5, 5 and 10 mL expressed in 0.00125, 0.0025, 0.005, 0.01 and 0.02 L equivalent per plate, respectively, and the organic sediment extract assessment was performed at 2.5, 10, 40 and 80 ug concentrations per plate of organic extract obtained, expressed in g of dry sediment

equivalents. To analyze the liquid–liquid extract the two extracts were joined (50 percent acid and 50 percent neutral compounds) according to the volumes. The Salmonella/microsome assay was performed with modifications (Kado et al., 1983) in the presence and absence of a microsomal fraction (prepared from Sprague Dawley rat liver, Aroclor activator 1254, Moltox S.A., USA) (S9 mix with four percent of rat liver S9). The samples were incubated in a water bath without shaking, with 100 mL of the tester bacterial culture (1  1010 cells/ml) in the absence and presence of metabolic activation (100 mL) solution, for 90 min, in the dark, at 37 1C. Plating was performed as described in Ames et al. (1973). Cytotoxic activity was also assessed by a survival curve of the test organism compared with different concentrations of the samples (Vargas et al., 1993) or analyzing the background lawn when the sample was not enough for all the assays (Ames et al., 1973; Mortelmans and Zeiger, 2000). The mutagenicity and cytotoxicity were evaluated after 72 h of incubation (37 1C) for all strains. The negative and positive control was done concomitantly. DMSO (spectrophotometric grade) or sterile deionized water were used as a negative control and sodium azide (SAZ) and 4-nitroquinoline oxide (4NQO) were used as a positive control according to strain in assays without S9 mix, and 2-aminofluorene with S9 mix. As described in Da Silva Jr. and Vargas (2009), the samples were considered positive for mutagenicity when the linear portion of the dose–response curve was significant using Salanal software (Salmonella Assay Analysis) version 1.0 of Research Triangle Institute, RTP, NC, USA. The results of interstitial water, sediment organic extracts and liquid–liquid organic extract (superficial soil draining) were expressed as: (a) revertants per milliliter of interstitial water, (b) revertants per gram of dry sediment equivalent and (c) revertants per liter of water equivalent. The cytotoxic response was considered positive when the percentage of surviving cells was less than 60 percent of the colonies compared with the negative control in at least one dose or when thinning or absence of background lawn was observed (Mortelmans and Zeiger, 2000; Vargas et al., 1993; Vargas et al., 2001).

2.5.2. Allium cepa assay The assay was carried out with Allium cepa seeds (Baia Periforme variety), that are genetically and physiologically homogeneous. The test consisted of evaluating different parameters of meristematic cells, such as genotoxicity, mutagenicity, cytotoxicity and toxicity. The assays were performed according to a modified version of Grants protocol (Matsumoto et al., 2006). In this study two treatments were carried out, one with interstitial water and the second with gross sediment. To evaluate interstitial water, several petri dishes were covered with filter paper, then one hundred (100) onion seeds were germinated in each one, at room temperature (20 75 1C), with 5 ml of the interstitial water sample. Deionized water (5 ml) was used as a negative control and 5 ml of 4  10  4 M of methylmethanesulfonate (MMS, Sigma–Aldrich, CAS 66-27-3) as a positive control. In order to analyze in direct sediment, onion seeds were germinated (100 seeds in each petri plate), at room temperature (207 5 1C), in several petri plates containing 15 g of gross sediment from different sites to which 5 ml of deionized water were added. The negative and positive controls were done in parallel. In the negative control the seeds were germinated in the reference area with 5 ml of deionized water, and the positive control was carried out in the reference area sediment adding 5 ml of 4  10  4 M of methylmethanesulfonate (MMS, Sigma– Aldrich, CAS 66-27-3). After germination, the roots were fixed in alcohol–acetic acid (3:1) for 24 h. Slides were prepared with meristematic and F1 cells by Feulgen methodology (Fernandes et al., 2007). In the meristematic cells the mitotic index (MI) is the first category analyzed for the number of cells in the division. This index evaluated the cytotoxicity. The mutagenic index (Mut I) is the second category analyzed by quantification of

T.C. da Costa et al. / Ecotoxicology and Environmental Safety 75 (2012) 63–72 micronucleus and chromosomes breaks. The third category analyzed is the chromosome aberration index (CA) that identifies genotoxic activity, evaluated from chromosome aberration analysis, considering several types of aberration within different cell division stages (metaphase, anaphase and telophase), for instance, chromosome losses and bridges, laggard or vagrant chromosomes and other aberrations. And the germination index (GI) is the fourth category analyzed from the number of roots growing, assessing toxicity. All the categories were analyzed by counting 5000 meristematic cells per site (500 cells per slide), comprising a total of ten slides (Carita´ and Marin-Morales, 2008). Micronuclei in F1 cells were also evaluated counting 5000 cells per site. The results obtained in the different assays were analyzed using a non-parametric statistical analysis—the Kruskal–Wallis test. The statistical method was carried out with Bioestat Software version 5.0.

2.6. Chemical analyses The chemical analyses prioritized organic compounds such as PHAs, considered most important by the Environmental Protection Agency of United States—USEPA (ATSDR, 2008): acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, indeno(1,2,3-cd)pyrene, dibenz(a,h)anthracene and benzo(ghi)perylene. The amount of sample used in the extraction was determined based on the 3550C method of the Environmental Protection Agency of United States (USEPA, 2008), which refer distinct methodologies for analysis of PAHs in soil, considering the presence of low and high concentrations. The description of the activities previously developed in the region indicated that the approximate amount of PAHs in the samples would be high. This information was essential to assist in choosing the methodology, using 2 g of dry sample in sodium sulfate. Then dichloromethane was added and placed in ultrasound for 3 min. Next the sample was filtered and the volume reduced with nitrogen gas. Later, the extracts were cleaned using a silica gel column, elution was performed with hexane/dichloromethane and the volume was reduced to 1 ml. The extract was analyzed by gas chromatography connected to a mass spectrometer (USEPA TO 13-A), using external standardization. The PAHs dosage was measured in the Taquari River sediment samples (September/2008) and in the sediment fraction obtained in Sampler 2 (August and October/2008). The total metal content of sediment samples was analyzed in the silt-clay fraction by X-Ray fluorescence spectrometry (XRF), (Philips, model PW 2404, Netherlands) at the Geochemistry Laboratory at UNICAMP, Campinas, Sa~ o Paulo, Brazil. Organic extraction with dichloromethane was used to look for pentachlorophenol (PCP) using GC/MS at Bioagri Ambiental Laboratory, in Piracicaba, Sa~ o Paulo, Brazil.

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3. Results and discussion 3.1. Assessment of drained superficial soil from the contaminated site According to Vargas et al. (2001), Ohe et al. (2004) and Vargas et al. (2008), several studies have reported the presence of xenobiotics of organic origin and genotoxic activity in the aquatic environment. Studies identifying the chemical fractions that are mutagenic to Salmonella have shown that much of the mutagenic activity of several complex mixtures is caused by compounds within one or a few classes of chemicals present in the mixture such as PAHs, pesticides, heterocyclic amines and heavy metals (Vargas et al., 2008). Early studies confirm the presence of heavy metals, PAHs, dioxins and furans at values above the level recommended for intervention (DRF, 2002) in soil and groundwater at the site studied (FEPAM, 2010a,b). These soils are a potential source of diffusion of contaminants into the Taquari River sediment. As a measurement to define the pollutant dispersion route, the mutagenic activity of the eroded material after rainfall events was evaluated by Salmonella microsuspension bioassay using the TA98, TA97a and TA100 strains, in the presence/absence of metabolic activation. It was decided to analyze organic extracts at this stage in the study due to the small amount of sample obtained in these samplers and the known sensitivity of the Ames test to organic compounds such as PAHs, heterocyclic compounds and aromatic amines (White and Claxton, 2004). Results of mutagenic activity in the liquid–liquid organic extracts (water extracts) and organic extracts of sediment from superficial soil drainage samples are summarized in Table 1. In the assessment of liquid–liquid Sampler 1 organic extracts, a positive response for frameshift (TA98 and TA97a) and base-pair substitution (TA100) mutation in the absence of metabolic activation was detected in the 08/2008 samplings. During the second sampling period (10/2010) a positive result was only detected by TA98–S9 mix. For Sampler 2 a negative mutagenic response was

Table 1 Mutagenic responses of organic extract liquid–liquid (revertants/L water equivalent) and organic extract of sediment (revertants/g dry sediment equivalent) and cytotoxicity (L or g dry sediment equivalent) in presence and absence of S9 mix, from superficial soil drainage in Winter (W) and Spring (S) of 2008. Area/Sampling

08/2008 W

10/2008 S

08/2008 W 10/2008 S

TA98

TA97a

Cytotoxicitya

TA100

 S9 mix

S9 mix

 S9 mix

S9 mix

 S9 mix

S9 mix

–S9 mix

S9 mix

717 7 315.98b

nsc

2,184 7899.60

ns

1,149 7 594.53

ns

–

–

ns

ns

ns

ns

ns

ns

–

0.01

439 7 209.36

ns

ns

ns

ns

ns

–

0.02

ns

ns

ns

ns

ns

ns

–

–

Sampler 2 Organic extract

ns

ns

ns

ns

ns

ns

–

0.04

Sampler 2 Organic extract

ns

21 74.00d

ns

102 7 26.00

ns

ns

0.61

0.61

Sampler 1 liq–liq extract Sampler 2 liq–liq extract Sampler 1 liq–liq extract Sampler 2 liq–liq extract

Negative control (rev/plate7 standard deviation) (5 mL DMSO/plate)  S9 mix: 31.5 7 4.95 (TA98), 210.3 7 14.22 (TA97a), 257.0 74.36 (TA100); þS9 mix: 37.0 72.65 (TA98), 229.3 7 8.08 (TA97a), 235.7 7 13.58 (TA100); Positive control  S9 mix: 4NQO (0.5 mg/plate) 357.0 7 52.32 (TA98), 700.57 10.6 (TA97a) and AZS (5 mg/plate) 1022.5 7 133.60 (TA100); þS9 mix: 2AF (10 mg/plate) 338.0 7 50.91 (TA98), 511.0 78.48 (TA97a) and 1051.0 7 76.3 (TA100). a First dosage Cytotoxic, non-cytotoxic samples in the presence of at least one dosage, cell survival is less than 60 percent of negative control. Least one dosage, cell survival is less than 60 percent of negative control. b Number of revertants/L superficial soil drainage (liquid part). c ns, non-significant response. d Number of revertants/g dry sediment equivalent.

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found at both samplings. Cytotoxicity was detected for Sampler 1 (0.02 L water equivalent dosage) in the October sampling, and for Sampler 2 in the August sampling based on lower dosages (0.01 L water equivalent dosage), both with S9 mix. The sediment organic extract was analyzed only in Sampler 2, due to the absence of sufficient sediment in Sampler 1. The results in Sampler 2 were negative in the first sampling (08/2008). Positive results were found in the presence of S9 mix for two frameshift mutation strains (TA98 and TA97a) in the October sampling, indicating that organic pollutants accumulate in the particulate material in soil, and are potentially carried into the stream sediment. Cytotoxic activity was found only at the highest dosage (0.61 dry sediment) in the second sampling, and at all dosages tested in the first sampling, and the lowest cytotoxic dosage is 0.046 g of dry sediment. This cytotoxicity may have prevented the detection of mutagenicity, because of death in this first sampling. The mutagenicity and cytotoxicity responses observed in Sampler 2 characterize a possible contaminant dispersion route from soil into the stream sediment. According to Da Silva Jr. and Vargas (2009) the mutagenic potency was expressed adding up the results obtained for each strain used in the Salmonella assay in the absence and presence of S9 mix for a graphic and comparative representation of events observed in the different strains and assay conditions. Results of water and sediment organic extracts from drained soil material were organized and shown in Fig. 5 summarizing the frameshift (TA98 and TA97) and base-pair substitution mutations (TA100) per site and sampling. Observing these results and the precipitations in rainfall events (Fig. 4), it is possible to consider that: (1) for Sampler 1 (water extracts) a higher and more diversified potency of direct mutagenicity was observed in the first rainfall event than in the second; (2) for the water extracts, probably due to the dry period, the accumulated pollutants in the superficial soil layers were released in the first significant rainfall event, resulting in a more concentrated sample than the second one; (3) the differences observed in the mutagenicity of water extracts for the two samplers may reflect their location. Sampler 1, beyond treated wood storage area also was under the influence of the old industrial area; (4) compounds that are not highly soluble can be absorbed in the soil particles, where they undergo interaction and degradation processes resulting in a range of different responses, as seen in Sampler 2, which accumulated sedimentary material. The PAHs organic chemical analyses (Fig. 6) showed the presence of PAHs during the sediment sampling events for Sampler 2, with high concentrations in the first. The cytotoxic responses (first event) and mutagenic responses (second event), predominantly for assays after metabolization, may reflect the presence of these compounds. Hence the high cytotoxic response already observed at low concentrations of extract analyzed (0.04 g dry sediment equivalent), prevented the detection of expected mutagenicity in the first event.

The concentration of pentachlorophenol as a specific marker of the area was investigated in the sediment of the stream where Sampler 2 had been placed. A concentration of 0.664 mg/kg was observed, confirming the evidence of contaminant runoff from the area to the Taquari River. These values were higher than the prevention value established for soils in Brazil (0.16 mg/kg dry soil; Brazil, 2009). As to the study of metals of interest due to the presence of CCA, the concentration of Cr(VI), arsenic and copper was evaluated in the stream water where Sampler 2 was located. The results showed that the Cr(VI) content remained below the limit of detection of the analytical method (o3 mg/L). For total arsenic, 136 mg/L was found in the stream water, near the source (Industrial area) ranging to 8.7 mg/L near the Taquari River, suggesting a major incorporation of the metalloid into the stream sediments. As to total copper, reached 18.1 mg/L in stream waters, much lower than at the source (126.000 mg/L). The presence of characteristic chemical compounds in the area of the stream that receives the main drainage from the industrial area supports the potential dispersion of contaminants into the Taquari River.

3.2. Taquari River sediment The study of mutagenic activity in the Taquari River sediment, together with the investigation of contaminant runoff from soil, made it possible to show this pollutant dispersion route, helping define the sources of compounds that contribute to the complexity of hazardous mixtures at these sources. The evaluation was performed in organic extracts, interstitial water and whole sediment, from different sites in the Taquari River basin during two seasons. Organic extract was investigated only with the Salmonella microsuspension test using the TA98, TA97a and TA100 strains, in the presence/absence of metabolic activation. Interstitial water was tested using the same samples and conditions by the Allium cepa bioassay. The gross sediment was available only by Allium test. Table 2 shows the results observed for the Salmonella test in sediment organic extracts (number of revertants/g sediment equivalent) and interstitial water (number of revertants/ml interstitial water equivalent). In organic extracts the sample Ta 032 presented mutagenic activity with frameshift mutation, TA98–S9 mix in the September sampling. In Ta 010 extracts, the mutagenicity was significant in both samplings with S9 mix. The category of mutation detected in the December sampling was frameshift (TA98 and TA97a) and in the September sampling was frameshift (TA98 and TA97) and base- pair substitution (TA100). Ta 006 site proves to be mutagenic in presence of metabolic activation in both samplings, where frameshift mutation (TA98) was identified. Cytotoxicity was found in Ta 010 and Ta 006 in the largest dosages in organic extracts in Summer, and in the winter dosages values were observed at the lowest concentrations.

Fig. 5. Mutagenic potency of water (revertants/L of superficial soil drainage) and sediment (revertants/g of dry sediment equivalent) from superficial soil drainage to total sum of the strains that detect frameshift (TA98 and TA97A) and base-pair substitution (TA100) mutagens in the absence and presence of metabolic activation in site and sampling carried out.

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69

Fig. 6. Organic chemical analyses for PAHs in mg/kg of sediment runoff from the contaminated soil site in 08/2008 and 10/2008 sampling (A) and a characterization of Taquari sediment (B).

Table 2 Mutagenic response of sediment organic extract (revertants/g dry sediment equivalent) and interstitial water (revertants/ml) and cytotoxicity (ml or g dry sediment equivalent) in presence and absence of S9 mix, from Taquari river basin in two sampling in Summer (S) and winter (W). Area/Sampling

12/2007 S

09/2008 W

12/2007 S

09/2008 W

TA98

TA97a

Cytotoxicitya

TA100

 S9 mix

S9 mix

 S9 mix

S9 mix

 S9 mix

S9 mix

 S9 mix

S9 mix

Org. Extract Ta 032 Org. Extract Ta 010 Org. Extract Ta 006

nsb ns ns

ns 87 3.54 247 745.28

ns ns ns

ns 46 7 7.79 ns

ns ns ns

ns ns ns

– 1.22 0.72

– 0.61 0.36

Org. Extract Ta 032 Org. Extract Ta 010 Org. Extract Ta 006

64 725.60 ns ns

ns 147 5.82 427 9.26

ns ns ns

ns 92 7 7.62 ns

ns ns ns

ns 73 7 24.47 ns

– – 0.52

– – 0.13

Int. water Ta 032 Int. water Ta 010 Int. water Ta 006

ns ns 34 714.25

ns ns ns

ns ns ns

ns ns ns

ns ns ns

ns ns ns

– 0.1 –

– – –

Int. water Ta 032 Int. water Ta 010 Int. water Ta 006

ns ns ns

ns ns ns

ns ns ns

ns ns ns

ns ns ns

ns ns ns

– – –

– – –

Negative control (rev/plate7standard deviation) (interstitial water, 100 mL deionized water/ plate)  S9 mix: 38.779.02 (TA98), 225.0723.81 (TA97a), 214.3711.50 (TA100); þ S9 mix: 3378.72 (TA98), 180.7727.10 (TA100), 194.0733.94 (TA97a); (organic extract, 5 mL DMSO/plate)  S9 mix: 28.374.04 (TA98), 187.7710.12 (TA97a), 169.3719.76 (TA100); þ S9 mix: 41.779.29 (TA98), 231.3713.43 (TA97a), 235.7717.39 (TA100); Positive control  S9 mix: 4NQO (0.5 mg/plate) 410.5799.70 (TA98), 933.07169.7 (TA97a) and AZS (5 mg/plate) 984.5717.67(TA100); þ S9 mix: 2AF (10 mg/plate) 575.0719.79 (TA98), 940.5734.64 (TA97a) and 1015.5735.30 (TA100). a b

First dosage Cytotoxic, non-cytotoxic samples in the presence of at least one dosage cell survival is less than 60 percent of negative control. ns, non-significant.

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Fig. 7. Mutagenic potency in revertants per gram of dry sediment equivalent to total sum of the strains that detect frameshift (TA98 and TA97A) and base-pair substitution (TA100) mutagens in the absence and presence of metabolic activation in site and sampling carried out.

Only site Ta 010 presented a direct cytotoxic response at lower concentrations in summer for analyses of interstitial water. Fig. 7 summarizes the analysis of mutagenic potency by the Ames test for sediment organic extracts by the sum of the three strains tested. Different strains were used in the Ames test to assess different groups of compounds. According to the data, at the Ta 010 site different classes of mutagens are found, indicating contamination by a mixture of different organic compounds, probably due to the chemicals employed at the nearby wood plant. An observation is necessary concerning the TA98 strain that appears to indicate some mutagenicity in all samples associated with the presence of this contaminant in chemical analyses of PAHs (Fig. 6). Site Ta 006 presents higher mutagenic activity values for frameshift mutagens in the presence of S9 mix than for Ta 010, specifically detected by strain TA98 in both samplings. The TA98þS9 is a possible strain that identifies these PAHs, and is very sensitive to PAHs (White and Claxton, 2004; Coronas et al., 2008). It should also be considered that the granulometry of Ta 010 and Ta 006 is characterized by the presence of fine particles (Fig. 4). Many aquatic pollutants are predominantly associated with fine deposits that are rich in organic matter. The environmental destination, the bioavailability and toxicity of these pollutants, particularly semi volatile organic substances, is determined by the interaction of these deposits (Chen and White, 2004). Thus, the deposition of fine particles can occur from the Ta 010 site to Ta 006. Another point concerning the Ta 006 site is the possible occurrence of contamination from a different source than in this study, raising the PAH levels. Another investigation is being performed in the final third of this basin, in order to account for additional sources downstream from the contaminated site. Although it is considered the main source in this area of the basin, other influences, mostly agriculture and cattle-raising, should be considered. Hence, even the upstream site (Ta 032) presented a mutagenic response in organic sediment extracts, although of a different nature from that observed at sites close to the source investigated, characterizing the presence of frameshift direct mutagens (TA98–S9). The pentachlorophenol concentration in the river sediment samples found was below the method limit of detection. It should be mentioned that the presence of CCA residues in the contaminated site can generate a release of heavy metals such as chromium, copper and arsenic towards the main river. According to Tovar-Sanchez et al. (2006) the metals tend to concentrate in silt-clay size particles and this fraction is transported almost entirely by suspension. The transfer of toxic metals from contaminated sediments to the water column occurs via interstitial waters. This flux of metals out of the sediment is

responsible for the high metal levels measured in other environments even after the anthropogenic sources have been eliminated (Tovar-Sanchez et al., 2006). Heavy metal contamination of interest to the study, Cr, As and Cu, was evaluated in the surface sediments of the Taquari River in the reach under the potential influence of the plant (Ta 010, Ta 006), besides Ta 032. No major spatial variation was observed in the heavy metal contents between kilometer 32 (Ta 032) and the area close to the mouth (TA 006). The results show that the river sediments have no recorded contamination, which could be ascribed to the contribution from the contaminated area studied, probably due to the high erosive potential of the basin and the dilution of contaminants in the solid material transported by the river. In order to diagnose the presence of mutagenic activity by inorganic compounds in the Taquari River, the interstitial water of sediment was first evaluated using the Ames assay. In the Ames test a negative response predominated in the December and September samplings. Only sample Ta 006 from the December sampling showed a positive response for frameshift mutation (TA98) without metabolic activation (S9 mix). Cytotoxicity was found in samples Ta 010 in the December sampling without metabolic activation at the lower dosage. Analyses were also performed with the Allium cepa bioassay. Fig. 8 shows the data of indexes for interstitial water samples in 12/2007 and 10/2008. Samples Ta 032 and Ta 010 in the September sampling were statistically significant for mitotic index (Fig. 8A), indicating cytotoxicity. Fig. 8B and C illustrates the chromosome aberration index and mutagenic, respectively, where samples Ta 032 and Ta 010 in the December sampling obtained a positive response for mutagenic and genotoxic activities by signifying a lack of mutagenicity in these cells. The data show that the interstitial water investigation obtained different responses for Salmonella/microsuspension and Allium cepa tests. This leads to several conclusions: (1) possible identification of different compounds in the different assays, since the Ames test indicated positive results only in a sample without metabolic activation, and the Allium test, a system with proper metabolization, showed positive results in different samples. (2) Another assumption is the lower sensitivity of the Ames test to detect genotoxic effects caused by heavy metals (Lah et al., 2008; Monarca et al., 2002). According to Lah et al. (2008), some reasons for the lower sensitivity could be the filtration step (0.2 mm) used to eliminate the contaminant fraction potentially present in the sample. Some of the components could attach to the filters and are consequently lost to further testing. However the filters used in sample filtration for the Ames test were

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71

Fig. 8. Results of mitotic index (MI), chromosome aberration index (CA), mutagenic index (Mut I) and germination index (GI) of interstitial water from the Taquari River basin in December/2007 and September/2008.

submitted to a watery extract and also to DMSO solvent, but no mutagenicity was detected in composites adhered to the filter. (3) The Ta 032 site located upstream from the source analyzed can receive other contaminants. This response was also seen by Rank and Nielsen (1998). Since soil and sediment are the growth media for most plants, it seemed logical to perform an Allium cepa assay in direct sediment exposure. According to Ohe et al. (2004), although mutagenic potency can be detected in non-concentrated samples in many cases, each contaminant is usually present at such low levels that it is difficult to detect, which is a possible explanation for the absence of a positive response in the assessment of direct sediment exposure.

4. Conclusions It can be concluded that the contaminated soils sites can export aggressive genotoxic composites to the river by runoff. These composites are entrained from the source (soil) by pluvial erosion, changing and probably accumulating in fine sediment particles. The environmental quality in the area under the influence of the contaminated site was diagnosed using bioassays as early parameters for genotoxic assessment. The presence of a range of effects could be defined using especially the Salmonella/ microsome assay, together with the analysis of the main groups of compounds present in the area investigated. Comparing the data to composites used in wood conservation, besides the PAHs analyzed chemically that probably originate in the creosote, the presence of other organics, such as dioxins and furans, is expected

in the investigated sediment as a result of the pentachlorophenol, besides heavy metals derived from CCA. Although contamination with pentachlorophenol and Cr, AS and Cu is limited to the contaminated area, there is a clear risk of runoff into the river. The sampling points could be classified according to relative degree of contamination, using the results, and pathways from the source to potential receptors by runoff into the river after heavy rainfall events were identified.

Acknowledgments The authors thank Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico for the Masters scholarship (T.C. Costa). The research is a part of the project on Ecotoxicology strategies to characterize contaminated areas as measures of risk to the population health, supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico, CNPq (555187/2006-3). References Adam, O., Badot, P.M., Degiorgi, F., Crini, G., 2009. Mixture toxicity assessment of wood preservative pesticides in the freshwater amphipod Gammaruspulex (L.). Ecotoxicol. Environ. Saf. 72, 441–449. Ames, B.N., Durston, W.E., Yamasaki, E., Lee, F.D., 1973. Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc. Nat. Acad. Sci. 70, 2281–2285. APHA, 1992. Standard Methods for the Examination of Water and Wastewater, American Water Works Association and Water Environment Federation, 18th ed. Franson, M.A.H. (Ed.), Washington, DC. Appel, J.S.L., Terescova, V., Rodrigues, V.C.B., Vargas, V.M.F., 2007. Aspectos toxicolo´gicos do preservativo de madeira CCA (arseniato de cobre cromato):

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