Levels and Distribution of DDT in the Cinca River (Spain)
By Ormad, M P Ratia, J S; Rodriguez, L; Ovelleiro, J L
ABSTRACT: The evolution over time of the levels and distribution of dichlorodiphenyltrichloroethane (DDT) in water, surface sediments, and fish from the River Cinca (Spain), a tributary of the River Ebro, during the period 1999 to 2004, was investigated by means of gas chromatography coupled with mass spectrometry. The sampling site corresponded to a point downstream from Monzon, a heavily industrialized town with drainage into the river. This river has historically been a source of emissions of DDT and its metabolites. The highest levels were found in 1999 and 2000, although the concentrations of organic compounds in sediments and fish have decreased since then. The levels of DDT in water were below the quantification limit during the period of study. The average composition of DDT isomers measured in sediments and fish showed the prevalence of p,p’-DDE, the product of aerobic degradation of p,p’-DDT. Concentrations in fish were compared with sediment samples, and high quotients indicate that they are highly bioavailable. Water Environ. Res., 80, 464 (2008).
KEYWORDS: water pollution, sediments, fish, DDT metabolites, River Cinca.
The metabolites and isomers of dichlorodiphenyltrichloroethane (DDT) are organochlorinated compounds that are highly persistent in the environment. The DDT degrades very slowly, and its products of degradation, DDE and DDD, present chemical, physical, and toxic properties similar to the original product.
Exposure to these compounds has been associated with teratogenic effects, disruption of the endocrine system, long-term effects related to the nervous system, and hepatic dysfunctions (Sarkar et al., 1997; Willet et al., 1998). Moreover, they all possess a strong bioaccumulative tendency in living beings.
Bioavailability is a parameter that indicates the degree to which a substance is absorbed by an organism and distributed throughout a site of said organism. A substance is considered bioavailable when part of it may be incorporated by an organism present within the surrounding environment. The environment may include water, sediment, suspended particles, and food. The coefficient n-octanol/ water (K^sub OW^) is the basis for anticipating the predisposition of a compound to be absorbed by organisms. Compounds with low K^sub OW^ values are easily soluble in water, whereas those that have high K^sub OW^ values (log K^sub OW^ > 6) are hydrophobic and are more easily absorbed by the fatty tissue in biota (Van Der Oost et al., 2002). As the solubility of these substances in lipids increases, their capacity to penetrate into the body of living organisms grows. Chlorinated compounds, such as DDT and its metabolites, are very hydrophobic (see Table 1).
Dichlorodiphenyltrichloroethane was first synthesized in 1874, but it was not until 1939, when the Swiss scientist, Paul Herman Mueller, discovered its properties as an insecticide during his research studies. Mueller obtained the 1948 Nobel Prize for Medicine and Physiology for this discovery. This insecticide quickly became highly popular, and its different uses continued to expand. It was used as an agricultural and forestry insecticide, to fight lice, epidemics, malaria, and so on, until 1962, when Rachel Carson, in her book Silent Spring, opened up the debate about the toxics effects of this powerful insecticide (Deogracias Ortiz et al., 2002).
Technical DDT is a mixture of different forms of DDT-p,p’-DDT (75%), o,p’-DDT (15%), and p,p’-DDE (5%)-while the rest is a mixture of other isomers (see Figure 1). All these forms are flavorless, almost odorless, white crystalline solids. The DDT is metabolized to DDE by hydrochlorination. This reaction takes place in living beings mainly catalyzed by the enzyme dehydrochlorinase. However, it can also degrade into sediments and soils to give rise to p,p’-DDD (under anaerobic conditions) and/or p,p’-DDE. Both products of degradation have also been detected in low amounts in the technical mixture (Ballschmitter and Wittlinger, 1991).
Present legislation on water quality stipulates that the pollution produced by these substances in the water environment (water, sediments, and biota) downstream from their point of emission must be strictly controlled. Specifically, Directive 2006/ 11/EC (2006) includes DDT in List I pollutants. Their emission into groundwaters is totally prohibited by the application of Directive 80/68/EEC (1980). The maximum permissible concentration in industrial wastewater discharged into surface waters and the quality objectives in water, sediments, and biota in recipient watercourses are established in Directive 86/280/EEC (1986).
Article 16 of the framework Directive on water (Directive 2000/ 60/EC, 2000) establishes a strategy to combat the chemical pollution of water. As an initial measure, a list of high-priority substances was adopted (Decision 2455/2001/EC, 2001), composed of 33 substances of priority interest at a community scale, among which, DDT was not included. A proposal for a European Parliament Directive currently exists (Commission of the European Communities, 2006), the goal of which is to guarantee a high level of protection against risks for aquatic medium or via medium derived from these 33 high-priority substances and other pollutants, among which, DDT is included, by means of the establishment of environmental quality standards.
To comply with Directive 2006/11/EC (2006), the River Ebro Hydrographie Confederation (Confederacion Hidrografica del Ebro, CHE [Zaragoza, Spain]) has a monitoring network along the entire river basin, known as the Hazardous Substances Monitoring Network. This monitoring requires the sampling of water, sediments, and biota (typically fish). The goal of this network is to biomonitor the compounds in Lists I and II of Directive 2006/11/EC (2006) and to verify that the concentrations in these matrices do not significantly increase over time (the basic principle of continuous improvement, or standstill).
At present, the European Economic Commission of the United Nations (UN/EEC) (Geneva, Switzerland) allows the use of DDT in the following two cases:
(1) Protection of public health in diseases, such as malaria; and
(2) As an intermediate product in the synthesis of Dicofol, providing that the total content of DDT is below 0.1%.
DDT was widely used in Spain as a pesticide from the mid-1950s to the mid-1960s. The order prohibiting its use came into force in 1977. Historically speaking, the industrial area of Monzon has been a source of this pollutant, the reason for which this research work was carried out.
The goal of this work was to study the evolution over time, from 1999 to 2004, of the pollution caused by DDT isomers and metabolites in waters, sediments, and biota in the River Cinca downstream from Monzon and to analyze their distribution in the river’s biota and sediments.
Area Under Study. The area under study is located in the northeast of Spain, along the Cinca River. There is one station located just downstream from Monzon (Huesca) (41[degrees] 53′ N, 0[degrees] 9′ E), approximately 4 km to the south of a heavily industrialized town with a very substantial chemical industry (Figure 2). Previous studies have reported high levels of persistent organic pollutants at this site (Eljarrat et al., 2005; Raldua et al., 1997). The same stations were sampled each year.
Sampling. Water. Two replicate water samples were taken in 1-L amber glass bottles and were homogenized and refrigerated at 4[degrees]C, until their subsequent preparation and analysis. The sampling frequency during the years 1999, 2000, and 2001 was annual. From 2002 onwards, samples were taken each month.
Sediments. Two replicate surface sediments were taken at the same station where water samples were collected using a stainless-steel Van Veen grab from the riverbank, a discharge area that was chosen to carry out the sampling. Samples were repeatedly taken until obtaining a representative sample of muddy sediment of the stretch of river. The samples were kept in glass containers with a Teflon stopper and wrapped in aluminum foil (Duran, 1998). These were labeled and kept at 4[degrees]C during their transfer to the laboratory. The sampling of sediment was carried out once per year during the months of September and October.
Fish. Three examples of two different species were captured in each fish sample. This selection was carried out in accordance with the following criteria: the fish belonged to the most abundant species in the area, were easy to capture, and were not listed as protected species. Individuals of approximately the same age were captured, to compare results from one year to another. The age of fish is determined by counting the growth lines in the otoliths or in the scales, or by means of their size, because each age presents approximately the same size. The latter criterion was the one used in this study, as it is considered to present less error, as indicated in the guidelines of the U.S. Government program Biomonitoring of Environmental Status and Trends (U.S. Geological Survey, Reston, Virginia). The size established in the sampling was 24 to 45 cm for Barbus graellsii and 15 to 25 cm for Alburnus alburnus. Sampling was carried out by means of electric fishing. The captured fish were wrapped in aluminum foil and transported to the laboratory in refrigerated bags. Once there, they were ground in a mincer until obtaining as homogeneous a pool as possible. This pool was composed of three examples of each species. The resulting mixture was kept in labeled glass bottles that were kept frozen at – 20[degrees]C until their subsequent treatment.
The sampling of fish was carried out once per year coinciding with the sampling of sediment. This was done in September or October mainly for two reasons. First, the lipid content of the fish, which is an important reservoir for organic pollutants, presents a maximum during this period. second, the water level is typically lower, which means that the taking of sediment samples and electric fishing was both easier and safer to carry out (Calvo, 2004).
Sample Preparation and Extraction Procedure. The aqueous samples underwent solid-liquid extraction using an AUTOTRACE Workstation (Zymark, Barcelona, Spain) automatic extractor before their analysis by gas chromatography coupled with mass spectrometry (GC/MS) (EPA method 525.2; U.S. EPA, 1995). During the solid-liquid extraction, 900 mL of sample were made to pass through cartridges filled with solid ENV+ (a hydroxylated polystyrenedivinylbenzene copolymer, ISOLUTE). The DDT contained in the sample was retained in the solid phase and dried under nitrogen gas for 10 minutes. Then, the samples were eluted by passing 10 mL of ethyl acetate (SDS [Barcelona, Spain], for pesticide analysis) through the cartridge, thus facilitating the passage of these compounds from the water phase to an organic phase. The extracts so obtained were concentrated under a nitrogen gas flow until an approximate volume of 1 mL was obtained, after which, 3 mL of hexane were added (SDS, for pesticide analysis), to carry out a change of solvent. The extract was then concentrated until obtaining an approximate volume of 0.5 mL. Then, 10 [mu]L of a solution of 10 mg/L (10 ppm) of anthracene deuterade D10 (Supelco, Barcelona, Spain) were added to each extract for subsequent quantification of the DDT present in the samples. These extracts are analyzed by GC/MS.
For the determination of DDT in sediments and biota, extraction was performed on an Ultra-Turrax homogenizing disperser (Ika-Werke, Yellow Line, Barcelona, Spain). For the extraction, 20 g of sample were taken to mix with 30 mL of acetone (SDS, for pesticide analysis); 30 mL of petroleum ether (SDS, for pesticide analysis) and 30 mL of dichloromethane. The phases were separated by centrifugation, and a 50-mL aliquot of the resulting extract was taken and then concentrated in a rotary evaporator, until obtaining 10 mL (URS S.L., 2004).
The concentrated extract was purified using a gel permeation chromatography permeation column and concentrated to 0.5 mL of hexane. This extract was purified once again using a Florisil column (Supelco). The obtained extract was concentrated under a nitrogen flow until a final volume of 0.5 mL hexane was reached. The internal standard used in this work was anthracene deuterade DlO (Supelco). Approximately 10 [mu]L of a solution of 10-mg/L (10-ppm) anthracene deuterade D10 was then added to this final extract.
The percentage recovery of these procedures was up to 80% for each DDT relative, and the detection limit for each isomer was 0.01 ng/L for water samples, 1.0 [mu]g/kg of dry weight for sediments, and 1.0 [mu]g/kg of wet weight for fish.
Two determinations were performed per each type of sample (water, sediment, and fish)
Analysis. Analytical determination was performed by GC/MS in electronic mode by injection to a Varian 3800-Saturn 2000 apparatus (Varian, Barcelona, Spain) equipped with an ion trap detector. A DB- 5 MS capillary column (30 m, 0.25 mm, 0.25 [mu]m; Cromlab, Barcelona, Spain) was used for analyses of DDT. Approximately 1 [mu]L of each sample was injected to a splitless model. The oven temperature was increased from 80[degrees]C (held for 1 minute) to 150[degrees]C at a rate of 8[degrees]C/min, followed by an increase to 300[degrees]C (held for 15 minutes) at a rate of 4[degrees]C/ min. The temperatures of the injector and detector were 250 and 300[degrees]C, respectively. High-pure Helio (N55) (Air Liquid, Zaragoza, Spain) was used as the carrier gas.
Table 2 shows the base peaks and retention times in the chromatographic column for each of the analytes under analysis.
As the metabolites p,p’-DDD and o,p’-DDT with the same m/z appear in the same residence time, they cannot be separately quantified.
Results and Discussion
The results are expressed in nanograms per liter for water samples, micrograms per kilogram dry weight for sediments, and micrograms per kilogram wet weight for fish (a pool per year of three examples of each species).
Evolution of Pollution by DDT in Waters of the River Cinca. Table 3 shows the concentration of DDT in the waters of the River Cinca from 1994 to 2004, with total DDT being understood as the sum of the concentrations of pp’-DDE, pp’-DDD, op’-DDT, and pp’-DDT.
As seen in Table 3, the total concentration of DDT metabolites and their isomers during the period under study is lower than the quantification limit of the analytical method (Q.L.= 0.01 ng/L)
Evolution of Organic Pollutants in Sediments of the River Cinca. Figure 3 shows the results obtained in the analysis of pp’-DDE, pp’- DDD, op’-DDT, and pp’-DDT, in the sediments of the River Cinca during the period 1999 to 2004 inclusive. The most abundant pollutant in the sediments of the River Cinca in the first year of sampling was pp’-DDE, with a concentration up to 673 ng/g. From 2000 onwards, however, a sharp drop in concentration was observed, until reaching a concentration of 33.6 ng/g in 2004. Although isolated increases can be observed in some years, no meaningful increase is detected over time, as stipulated in Directive 86/280/EEC (CHE, 2002, 2004a, 2004b, 2005).
The concentration levels of DDT measured in this study were compared with those obtained in other rivers in the world (Table 4). One of the most recent studies was carried out in the River Haihe, one of northern China’s largest rivers (Yang et al., 2005). This river flows through the city of Tainjin, one of the rivers most polluted with DDT in China, as a result of continual discharges over decades. As can be seen in Table 4, the concentration levels in this river range between 0.32 and 80.18 ng/g. If the concentration obtained in the River Cinca is compared with that of other rivers, it can be seen that the concentration of DDT in the River Cinca in 1999 (757 ng/g) is similar to that of the Mataniko River (750 ng/ g), while the concentration obtained in 2004 is similar to that of the remaining rivers.
Evolution of Organic Pollutants in the Biota of the River Cinca. As can be seen in Figures 4 and 5, the pollutant that accumulates most in the sampled fish is also p,p’-DDE, reaching concentration levels in 2000 of 6530 ng/g in Barbus graellsii and 3240 ng/g in Albumus albumus. However, the concentrations of p,p’-DDE have decreased drastically over the years, until reaching 1290 and 290 ng/ g, respectively, in 2004 (CHE, 2002, 2004a, 2004b, 2005). As occurs with sediments, the concentration obtained in the sampled fish of pp’-DDD, op’-DDT, and pp’-DDT is minor compared with the concentration of pp’-DDE.
Interpretation of Results
The evolution of the distribution of the different isomers and metabolites of DDT from 1999 to 2004 in the samples of sediments and biota from the River Cinca is shown in Figures 6 and 7. The relative concentrations of the principal compound and its metabolites can be used as indicative indices to determine the possible causes of pollution. Because DDT may be biodegraded under aerobic conditions to DDE and under anaerobic conditions to DDD (Kalantzy et al., 2001), a quotient of (DDE + DDD)/DDT > 0.5 means that the DDT has degraded into its principal metabolites (Zhou et al., 2006). Moreover, if this quotient is practically zero, this would indicate the presence of a recent discharge of DDT into the river (Phuong et al., 1998). Figures 6 and 7 show that, in the majority of samples of surface sediments and fish obtained from the River Cinca, the degraded metabolites make up a highly significant proportion of total DDT, with the most abundant metabolite being p,p’-DDE. The (DDE+DDD)/DDT quotients in the sediments range between 0.75 and 1 and are even higher in fish, between 0.9 and 1, with all being above 0.5. Furthermore, it may be deduced that there has been no discharge of DDT during the period under analysis, as confirmed by the analysis of the water samples. The ODD/DDE quotients are all below 0.12, which means that DDT is mainly degraded in the river via aerobic mechanisms.
As can be seen in Table 3, no concentration of DDT was detected in the waters throughout the entire period under study. However, analysis of the sediments and biota revealed high concentrations of DDT, especially at the start of the sampling period (1999 to 2000). This gives an idea of the persistence of these compounds and the complex mechanisms of distribution between water and biota or water and sediment existing over time, as a result of the physical- chemical characteristics of DDT.
When the concentrations of DDT in biota are compared with the concentrations measured in the sediments of the River Cinca, quotients are obtained that give a rough idea of where these compounds tend to accumulate in the aquatic medium. The influence of the lipid content in biota when carrying out these comparisons is uncertain. Some studies obtain these quotients by previously measuring the lipid content of the biota, because that is where pollutants preferentially accumulate (Bierman, 1990; Eljarrat et al., 2005). However, other research studies have not determined any correlation between fat content and the concentration of DDT in biota (Zhou et al, 1999).
Tables 5 and 6 show these quotients for the two classes of analyzed fish over time. Of all the metabolites, p,p’-DDE is the one with the greater tendency to accumulate in biota instead of in sediment. The remaining metabolites present lower quotients. Moreover, if the quotients obtained for the two species under study are compared, it can be seen that DDT tends to accumulate, to a greater extent, in Barbus graellsii than in Albumus albumus. Conclusions
This study investigated the pollutant status of DDT during the period 1999 to 2004 in the water, surface sediments, and fish of the River Cinca in Spain. The following conclusions can be drawn:
(1) The levels of DDT in water are below the quantification limit.
(2) DDT is present in the sediments and fish collected from the River Cinca, although they were not detected in the water.
(3) The levels of these compounds have decreased drastically in sediments and fish, despite their high persistence.
(4) The predominant species among DDT is p,p’-DDE. The composition analyses and the low level of p,p’-DDT indicates that there has been no recent discharge into the River Cinca.
(5) DDT is bioavailable and tends to accumulate more in fish than in sediments. The most bioavailable metabolite is p,p’-DDE.
In addition, this study has led to an improved understanding of the pathways of DDT in the River Cinca. Further work is needed to determine the bioaccumulation of DDT in the food network and the associated risks to ecosystems and human health.
The authors thank the Water Quality Area and the Water Laboratory of the River Ebro Hydrographie Confederation (Zaragoza, Spain) for their technical support and the Spanish Ministry of Education and Science (Madrid, Spain) for the financial support on the project “Aplicacion de tecnicas de oxidacion avanzada en la potabilizacion de aguas naturales de la cuenca del Ebro” (project ctm2005-04585/ tecno).
Submitted for publication July 18, 2007; revised manuscript submitted October 17, 2007; accepted for publication October 22, 2007.
The deadline to submit Discussions of this paper is August 15, 2008.
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M. P. Ormad*, J. S. Ratia, L. Rodriguez, J. L. Ovelleiro
Department of Chemical Engineering and Environmental Technology, University of Zaragoza, Spain.
* Department of Chemical Engineering and Environmental Technology, University of Zaragoza, Spain; e-mail: email@example.com.
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