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On-line version ISSN 1678-4790
J. Braz. Chem. Soc. vol.15 no.2 São Paulo Mar./Apr. 2004
Débora de A. Azevedo*; Elaine Gerchon; Ederson O. dos Reis
Instituto de Química, Universidade Federal do Rio de Janeiro, Ilha do Fundão, CT, Bl. A, s.603, 21949-900 Rio de Janeiro - RJ, Brazil
The Paraíba do Sul River, in the State of Rio de Janeiro, was studied for its water quality, by determining the levels of selected polycyclic aromatic hydrocarbons (PAHs) and pesticides from six sites in two cities, Resende and Campos dos Goytacazes, as they have industrial and agricultural activities. This study was carried out between July 2001 and March 2002. The method involved 200 mL samples taken by off-line, solid phase extraction by OASIS polymeric cartridges followed by gas chromatography-mass spectrometry (GC-MS). Recoveries and standard deviation of pesticides in non polluted real water sample spiked with a standard mixture were 82-119% and less then 20%. For PAH, recoveries and standard deviation were 56-78% and less then 18%, respectively, with exception to acenaphthylene, 23% and 2.7%. Atrazine was detected in the average concentration of 0.231 µg L-1 in two sites in Campos dos Goytacazes, near the sugar-cane power plants and plantations area, while no detection was observed in Resende. Irgarol was observed in Campos dos Goytacazes downtown at 0.138 µg L-1, an area of small boating activities. Benzo[a]pyrene was detected at 0.255 µg L-1 in Resende, near the Presidente Dutra highway. PAHs were not detected in the water samples from Campos dos Goytacazes.
Keywords: atrazine, water, PAHs, pesticides, GC-MS
Estudou-se a qualidade da água do Rio Paraíba do Sul através da determinação das concentrações de alguns poluentes orgânicos prioritários, a saber, hidrocarbonetos policíclicos aromáticos (HPAs) e pesticidas. Duas cidades foram selecionadas, Resende e Campos dos Goytacazes, três sítios em cada, por apresentarem atividades industriais e agrícolas. O estudo foi desenvolvido entre Julho de 2001 a Março de 2002. O método envolveu a extração de 200 mL de amostra por extração em fase sólida utilizando cartucho polimérico OASIS, seguido pela análise por cromatografia gasosa acoplada a espectrometria de massas (CG/EM). A recuperação e o desvio padrão para amostras de água real não poluída dopada com padrões de pesticidas foi de 82-119% e menos de 20%, respectivamente. Para os HPAs, a recuperação e o desvio padrão foram de 56-78% e menos de 18%, respectivamente, com exceção do acenaftileno, 23% e 2,7%. Atrazina foi detectada em uma concentração média de 0,231 µg L-1 em dois sítios em Campos dos Goytacazes, nas proximidades de usina e plantações de cana de açúcar, enquanto em Resende não foi detectada. Irgarol foi observado no centro de Campos de Goytacazes a uma concentração de 0,138 µg L-1, uma área com atividades de barcos de pequeno porte. Benzo[a]pireno foi detectado em Resende a uma concentração de 0,255 µg L-1, próximo a rodovia Presidente Dutra. HPAs não foram detectados nas amostras de água de Campos dos Goytacazes.
National and international pressure is growing for water quality improvement and better management. The need for monitoring some important groups of dangerous organic pollutants, such as polyaromatic hydrocarbons (PAHs), pesticides, phenolic compounds, alkyl and aromatic sulfonates in surface waters is essential for achieving good water-quality objectives. The European list of the priority organic compounds to be monitored from discharges is the Directive EC 76/4641 while Brazilian regulations are CONAMA no. 20 from the Environmental Ministry and regulation no. 1469 from the Health Ministry. Regulations for drinking water are well defined in North America with given analytical procedures, whereas in Europe the regulation for each pesticide is set at 0.1 µg L-1 and for polyaromatic hydrocarbons, total of 6 compounds is set at 0.2 µg L-1.2,3
Atrazine was introduced in the 1950s, and since that has become a commonplace in agricultural and forestry applications. For instance, atrazine is used to control annual grasses and broad-leaved weeds in selected vegetable and cereal crops, vines, sugar cane, grassland, fruit orchards, citrus groves, and forestry. It is the most widely used herbicide in the world, with between 70,000 and 90,000 tons applied per year. It is one of the most effective and inexpensive herbicides in the world and is consequently used more frequently than any other herbicide. In recent years, concerns have been raised regarding its persistence within the environment and entry into groundwater and the aquatic environment. Atrazine is most effective in wet soils and is therefore usually applied after significant winter rain when soils are at field capacity.4 Banning atrazine completely would cost $240 million, with no improvement in water quality. Banning all triazines would improve water quality, but it would cost nearly $740 million. The most effective policies appear to be atrazine restrictions based on federal health limits of 3 µg L-1 or 100 µg L-1, water quality was greatly improved at a cost of between $160-$230 million.5
Polycyclic aromatic hydrocarbons are widespread contaminants throughout the environment arising mainly from anthropogenic sources such as the combustion from fossil fuels and the direct release of oil and oil products. These widespread environmental contaminants have been intensively studied owing to their mutagenic and carcinogenic properties. Some of the PAHs are classified as priority pollutants by both the US EPA and the European Community.6
This study aims to carry out a survey of selected organic micropollutants in the water samples from Paraíba do Sul River, focusing on three sites each in two important cities in the State of Rio de Janeiro, Resende and Campos dos Goytacazes, that suffers industrial and agricultural impact, and also to carry out a partial validation of the used methodology.
The city of Campos dos Goytacazes (latitude 21o45'15"S and longitude 41o19'28"W) with about 398,547 inhabitants, is the most important city in the northern part of the state of Rio de Janeiro. It is basically an agricultural area, in which the sugar cane plantation is the main economic activity with sugar/alcohol power plants.
The city of Resende (around latitude 22o28'S and longitude 44o27'W) with about 120,000 inhabitants and an area of 407 km2, is an important city in the southern part of the state of Rio de Janeiro. It is an industrial area with agricultural (tomato, corn, rice, bean) and ecological activities. Some industries in this area are Clariant, Volkswagen Brazil, Norvartis and Cyanamid Brazil.
Three sites were chosen in each city for sampling: sites 1 and 4, just before downtown; 2 and 5, downtown the cities; 3 and 6, after passing through downtown. The unpolluted river water sample was sampled from an ecological site, Lumiar, Nova Friburgo (RJ; latitude 22o16'55"S and longitude 42o31'52"W). Figure 1 shows the Paraíba do Sul River map, with the two cities in study.
The standards used were 98-99% pure. It was studied 10 compounds (Table 1). Pesticides standards were obtained from Promochem (Wesel, Germany) and Riedel-de Haën (Seelze, Germany). Irgarol was from Ciba-Geigy (Barcelona, Spain). Polycyclic aromatic hydrocarbons standard as Phenanthrene, Fluoranthene, Pyrene, Chrysene and Benzo[a]pyrene are from Sigma/Aldrich (USA). Stock solutions of 1000 µg mL-1 were prepared by weighing 10 mg of each of the solutes and dissolving them in 10 mL of chromatographic-grade dichloromethane or acetonitrile (TediaBrazil, Rio de Janeiro). They were then stored at 20 oC. Dichloromethane and acetonitrile ABSOLV (chromatographic grade) were from TediaBrazil, acetic acid was purchased from Grupo Química (Rio de Janeiro, Brazil).
The samples were collected at July and October/2001 and March/2002. All the water samples were sampled at 20 cm depth using 1 L precleaned amber glass bottles (avoiding the surface micro-layer). Samples were acidified with acetic acid to pH 4.0, filtered through 1.2 and 0.45 µm glass fiber filters in order to remove suspended particles and kept at 4 oC in the dark until analysis.
The OASIS cartridges (60 mg, Waters, USA) were washed sequentially with 6 mL of dichloromethane, 6 mL of acetonitrile and 6 mL of water. A 200-mL aliquot of sample was passed though the cartridge and then washed with 1 mL of water. Water residues from cartridges were eliminated by 30 min vacuum. Elution was carried out with 2.5 mL of acetonitrile-dichloromethane (1:1) followed by 3.2 mL of dichloromethane.7
The C18 (200 mg, PR-COLA) and the Bond-Elut Certify (130 mg, Varian, PN: 1211-3054) cartridges were washed sequentially with 6 mL of methanol and 6 mL of bidistilled water. A 200 mL aliquot of sample was passed though the cartridge and then washed with 1 mL of water. Water residues from cartridges were eliminated by 30 min vacuum. Elution was carried out with 6 mL of methanol for the C18 cartridges and with 2 mL dichlorometane/isopropanol (8:2) with 2% ammonium hydroxide solution (make fresh daily) for the Bond-Elut Certify.
Evaporation of the solvents was performed under a stream of nitrogen. The final sample volumes were 200 µL.
Gas chromatography-mass spectrometry analysis were performed in a Hewlett Packard 5890 gas chromatography coupled to a Hewlett Packard 5972 MSD Instrument utilizing helium as carrier gas and the following conditions: fused silica column DB-5 (J & W, 30 m X 0.25 mm, df = 0.25 µm), 60 oC for 2 min to 300 oC at 6 oC min-1, splitless injection mode (time of 1 min), temperature of injector was set at 280 oC. Electron impact at 70 eV was used. All samples were analysed in Single Ion Monitoring (SIM) mode for quantification purposes of the compounds, Figure 2 (major ions corresponding to the typical fragments of the compounds were selected, Table 1).
Recoveries obtained with distilled water samples spiked with standard compounds were tested in different conditions. Three different SPE cartridges were evaluated: C18 (PR-COLA, Brazil), Bond-Elut Certify (Varian, Harbor City, CA, USA), OASIS (Waters). The water pH was also evaluated; water sample with and without addition of acetic acid (pH 4.0).
Unpolluted river water samples were also used for OASIS SPE cartridge. The compounds were spiked in 200 mL of water to give a final concentration of 1.0 µg L-1 and subsequently the water was acidified at lower pH (pH 4.0). Immediately after this operation, the water samples were extracted.
The recovery were determined by comparison of peak areas from water samples spiked with known amount of organic pollutants (1.0 µg L-1), processed according to the described method versus non-extracted pure standards which represent 100% recovery.
External calibration was used to the quantification of the extract after SPE with the standard mixture. The calibration equations for the different compounds were constructed and were linear (R2 >0.99) over the range 0.05-1.0 µg L-1. The quantification of the water was achieved using SIM with the ions indicated in Table 1. The limits of detection (LODs) were calculated by using a signal-to-noise ratio of 3 (the ratio between the peak intensity under SIM conditions and the intensity of the noise was used) and are presented in Table 2. Recoveries are presented in Table 3.
Limits of detection for OASIS cartridges
A calibration curve consisted of six concentration points from 0.01, 0.025, 0.05, 0.10, 0.20 to 0.50 µg of organic pollutants per 1 L of unpolluted river water. These spiked water samples were passed thought the OASIS cartridges. The calibration equations for the different compounds were constructed and were linear (R2 > 0.99) over the range 0.01-0.5 µg L-1. The limits of detection (LODs) were calculated by using a signal-to-noise ratio of 3 and the constructed calibration curve (Table 4).
External standard calibration was used for quantification of the extracts. Calibration graphs for SIM mode was plotted using 4-6 points in the concentration 0.05, 0.10, 0.25, 0.5, 0.75 and 1 mg mL-1. Calibration equations obtained in SIM mode and R2 values are presented in Table 1 for GC/MS.
Results and Discussion
Data obtained with standard compounds are presented in Table 3. Recovery (%), confidence limits (CL, %), coefficient of variation (CV, %), standard deviation (SD, %), for replicates (n = 4; 3 degree of freedom), using Student t-test (95%; ttab = 3.182) were determined. These parameters were well within the acceptance criteria of the accuracy of 70-130% and precision of 30% for organic compounds at trace level in water samples, according to the US-EPA methods general characteristics.3
The calibration curves constructed were linear over the range of interest. The correlation coefficients were in all cases higher than 0.99, indicating good performance of the chromatographic method (Table 1). Recoveries with OASIS cartridges for real water samples were around 82-119% and standard deviation were no more than 20 for pesticides standards and 56-78% and 18% for polycyclic aromatic hydrocarbons, respectively. Detection limits (Table 2) were in the range of 0.0002-0.001 µg L-1 obtained for distilled water spiked with standard compounds and percolated through the cartridges (far below 0.1 µg L-1) being good enough for trace levels determination, taking into account that 200 mL of water were percolated through the cartridges. Low recovery value was observed for acenaphthylene, 23% as it is a volatile compound and it is lost at the evaporation steep.
The choice for working with OASIS cartridges was based on the recoveries data obtained also with the other tested cartridges. Recoveries and standard deviation for distilled water spiked with the standard mixture and with C18 cartridges were 40-91% (SD = 16%) for pesticides and 14-46% (11%) for PAH, while in pH 4.0 were 58-92% and 20% for pesticides and 12-71% and 27% for PAH (Table 3). Recoveries for Bond-Elut Certify were from not detected to 147% (SD = 45%) for pesticides and nd to 62% (25%) for PAH without acetic acid addition and from nd to 87% (13%) for pesticides and nd to 75% (29%) for PAH in pH 4.0. The data for Oasis cartridges and distilled water samples spiked with standard mixture were 84-117% (27%) for pesticides and 72-105% (24%) for PAH and 95-99% (21%) for pesticides and 84-105% (25%) for PAH in pH 4.0.
It is easy to observe that worse results were obtained with Bond-Elut Certify in the conditions tested. The C18 cartridges generated worse results mainly to PAH. Oasis cartridges presented better results to both class of studied compounds: pesticides and PAH, as we were looking for a SFE cartridges that would permit to analyse both class of compounds. Data with real water sample spiked with the standard mixture showed a reduction in the recoveries values, as would be expected, although still being the better results.
Detection limits of polycyclic aromatic hydrocarbons are in the 0.010 µg L-1 range.1 In this study we observed values in the range 0.0005 up to 0.025 µg L-1 (Table 4) when unpolluted river water samples spiked with the standard mixture at different concentrations were analysed. The pesticides gave detection limits in the range 0.003-0.01 µg L-1, below the observed values in a previous paper.7
Application to a case study
Water samples from the Paraíba do Sul River were analyzed following the proposed method in six sites from two cities, in three distinct dates. The analytical protocol was applied to the analysis of real environmental samples. Analytes monitored in this study were chosen because they are commonly detected in surface waters and they are on the priority lists of the Brazilian Environmental Ministry (CONAMA no. 20/1986) and Health Ministry (regulation no. 1469/2000) and also of the United State Environmental Protection Agency (US-EPA) and European Community (e.g., Directive 76/464/EEC).
Highest reported concentrations of atrazine are associated with the first rainfall after application. The level of atrazine in river water varies seasonally with the highest concentrations found in the spring and summer months. The impact of atrazine on fish physiology occurs at concentrations as low as 3 µg L-1. Atrazine has been detected in drinking water reservoirs up to 88.4 µg L-1, and in rivers up to 100 µg L-1.4
Atrazine showed a rather constant concentration over time in two sites in Campos dos Goytacazes, in a average concentration of 0.231 µg L-1. These sites are near the sugar-cane power plants and plantations area. No detection was observed in downtown of Campos dos Goytacazes and in Resende. Figure 3 presents summaries of atrazine concentrations found in freshwater bodies around the world and in Campos dos Goytacazes, this work.
Irgarol, the herbicide 2-(methylthio)-4-(tert-buthylamino)-6-(cyclopropylamino)-s-triazine (tradename Irgarol 1051) was also detected. Irgarol 1051 is effective mostly against freshwater and seawater algae and less against animal organisms. Some studies showed that the substance is very toxic to all microalgae tested; EC50 values range from 0.45 to 2.12 mg L-1.8 This compound is used in antifouling paints as a biocide agent. The herbicide is added in paints in order to inhibit the primary growth of fouling organisms such as algal slimes and seaweeds.9,10 Data are available concerning Irgarol contamination of aquatic environment. Studies had been undertaken in Côte d'Azur,10 the southern coastal of the U.K.,11 Lake Geneva,8 seawater from Mediterranean Spanish Coast9,10 and river and coastal water from Portugal.7 Irgarol are added to ship/boat paints to prevent fouling and diffuse into the surrounding waters and contaminate environments.
The observed concentration of Irgarol in downtown of Campos dos Goytacazes water (138 ng L-1) was only three times lower than the lowest EC50 reported for algae (450 ng L-1). Figure 4 presents summaries of Irgarol concentrations found in water bodies around the world and in Campos dos Goytacazes, this work. Figure 5 shows mass fragmentogram in SIM mode for atrazine (Figure 5A; m/z 200) and Irgarol (Figure 5B; m/z 182) in river water samples from downtown of Campos dos Goytacazes and from standard compounds spiked at a final concentration of 0.20 µg L-1 in an unpolluted river water sample (Figures 5D and 5E).
Polycyclic aromatic hydrocarbons
Benzo[a]pyrene was detected only at 0.255 µg L-1 in Resende (July/2001), near the Presidente Dutra highway. This is illustrated in Figure 5C, while Figure 5F shows the m/z 252 mass fragmentogram from the corresponding benzo[a]pyrene standard spiked at a final concentration of 0.20 µg L-1 in an unpolluted river water sample. PAHs were not detected in the water samples from Campos dos Goytacazes. In a previous work regarding PAHs in the Paraíba do Sul River, concentration values of benzo[a]pyrene were from 0.03 µg L-1 up to 1.2 µg L-1 near the CSN industry (National Siderurgical Company, the most important industry in this area with metallurgical activities), in the Volta Redonda city12 (see map).
The Brazilian regulations (CONAMA no 20/1986) specify that benzo[a]pyrene detection should not exceed 0.01 µg L-1 in drinking water, while Brazilian Health Ministry (regulation no. 1469/2000) specify a value of 0.70 µg L-1. Lethal concentration (LD50) down to less than 10 µg L-1 for total PAHs (åPAHs) has been reported for various organisms and is used as a threshold value.13 Imperative and maximum admissible concentration of polycyclic aromatic hydrocarbons (total of 6 compounds; fluoranthene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, benzo[ghi]perylene and indeno[1,2,3-cd]pyrene) in surface water for drinking water purposes is 0.2 µg L-1 in the EC regulations.1
Total parent PAH concentrations (ng L-1) in sub-surface water from various marine sites in the world were: 0.300-0.594 ng L-1 in the Baltic Sea, 4-36 ng L-1 in Seine River and Estuary (France), 106-945 ng L-1 in Western Xiamen Bay14 (China), 4228-29325 ng L-1 in Daya Bay13 (China).
Precision, accuracy, linearity, specificity and limits of detection were determined for four pesticides and six polycyclic aromatic hydrocarbons investigated in this study using Oasis cartridge solid phase extraction followed by GC/MS analysis. This methodology was applied to real river water samples from the Paraíba do Sul in two distinct cities where the main pollution would be from industrial and agricultural activities. The detected compounds were atrazine and Irgarol (triazines pesticides) in downtown of Campos dos Goytacazes and benzo[a]pyrene in Resende. The detected concentration values were around 0.2 µg L-1.
Among the organic pollutants determined in this study, atrazine is caused by agricultural contamination, mainly from sugar-cane crops; Irgarol is used in boating paints and benzo[a]pyrene has an fossil fuel origin.
Although this analytical method were optimised and applied for analysing 75 organic compounds, mainly pesticides, halogenated compounds and phenols, in river water samples from Portugal in a previous paper,7 in the present one we extended the method to the detection of polycyclic aromatic hydrocarbons and applied for analysing Brazilian river water samples.
E. Gerchon acknowledges the grant from CNPq (PIBIC) and FAPERJ is acknowledged for financial support to this project (E-26/170.619/2001) and the grant to E.O. Reis (E-26/152.409/2000).
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Received: February 19, 2003
Published on the web: March 30, 2004