- Email: [email protected]
Screening of pesticide toxicity in surface water from an agricultural area at Phuket Island (Thailand)
ENVIRONMENTAL POLLUTION
Environmental Pollution 102 (1998) 185±190
Screening of pesticide toxicity in surface water from an agricultural area at Phuket Island (Thailand) A. Baun*, a, N. Bussarawit b, N. Nyholm a a
Department of Environmental Science and Engineering/Groundwater Research Centre, Technical University of Denmark, Building 115, 2800 Lyngby, Denmark b Phuket Marine Biological Center, PO Box 60, 83000 Phuket, Thailand Received 15 November 1997; accepted 28 April 1998
Abstract A screening study of the toxicity of expected pesticide-contaminated surface water from an agricultural area at Phuket Island, Thailand, was carried out using standardized bioassays in combination with pre-concentration by solid phase extraction (SPE). The bioassays were an algal growth inhibition test (Selenastrum capricornutum) and a Daphnia immobilization test (Daphnia magna). Tests were run on both ®ltered water samples and samples pre-concentrated by SPE. Toxicity could be detected in nonconcentrated samples originating from areas where pesticides were applied, but pre-concentration was necessary in order to obtain full concentration±response relationships. Water collected at vegetable ®elds was very toxic. Lower toxicity was found in stream water, but the toxicity increased as the stream passed the vegetable ®elds. No toxicity was detected in an unpolluted reference sample pre-concentrated 100 times. In general, algal tests proved to be more sensitive than Daphnia tests for monitoring toxicity. It was concluded that pesticides are likely to cause toxic eects in the stream, but due to great dilution by the tide, it is not likely that the current water-borne pesticide pollution will aect the marine ecosystem. Bioassays combined with pre-concentration proved to be useful screening and monitoring tools for initial assessment of water pollution by pesticides. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Bioassays; Solid phase extraction; Pesticides; PNEC; Surface water monitoring
1. Introduction The contamination of surface water with pesticides poses a direct risk of toxic eects in the aquatic ecosystem. In contaminated agricultural areas a large variety of pesticide chemicals will often be present both as parent compounds and as their degradation intermediates. Detection and identi®cation of pesticides and their metabolites by chemical analysis can be very complicated and require sophisticated analytical techniques. Even if a compound is identi®ed, information on its aquatic toxicity may not be available. Bioassays on water samples and concentrates provide a direct functional response that relates to the overall toxic properties of the complex mixture of compounds present in a sample. Furthermore, toxicity testing with aquatic organisms is well established for environmental hazard * Corresponding author. 0269-7491/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S0269 -7 491(98)00098 -0
assessment of chemicals and chemical products and is used routinely to assess or monitor the toxicity of complex mixtures such as industrial wastewater. The detection limit of standardized biotests applied on surface water may, however, be too high to detect toxicity and hence pesticide contamination. Therefore, preconcentration of the surface water is normally necessary. In the present study the aim was to concentrate all man-made chemicals and their metabolites which could contribute to toxicity. For this purpose solid phase extraction (SPE) appears to be very eective (Galassi et al., 1992; Hendriks et al., 1994; Baun and Nyholm, 1996). A screening strategy was applied to surface water from an agricultural area near Chalong Bay, Phuket Island, Thailand (Fig. 1). In this area, vegetables (especially water spinach: Ipomoea aquatica) are grown in ¯ooded ®elds. The area is drained by a stream passing by the ®elds on its way from the mountains to Chalong Bay. The sampling locations are indicated in Fig. 1. For
186
A. Baun et al./Environmental Pollution 102 (1998) 185±190
Fig. 1. Sampling locations for screening of pesticide toxicity at Phuket Island, Thailand. S, stream; F, water spinach ®elds; BS, station for collection of a reference sample from an unpolluted area.
practical reasons the background sample was collected in a nearby stream, with no agricultural areas upstream. Information on pesticide use in water spinach ®elds was obtained from Phuket Agricultural Oce (Phuket, Thailand), pesticide vendors, and by interviewing farmers. It turned out that a broad spectrum of pesticides are used in vegetable ®elds and for controlling insects in water spinach ®elds. Methyl parathion, lambda-cyhalothrin and endosulfan were claimed to be the most commonly used pesticide chemicals. These chemicals are classi®ed as highly toxic towards aquatic organisms. Furthermore, endosulfan and lambda-cyhalothrin have potential for bio-accumulation and are also quite persistent in the aquatic environment (Tomlin, 1994). 2. Materials and methods 2.1. Sampling Water samples were collected on three occasions in December 1996 and January 1997 in acid-washed glass containers and transported to the laboratory right after sampling. In the laboratory, samples were ®ltered through glass ®ber ®lters (Whatman GF/C). Biotests were carried out on the day of collection, or the following day after sample storage at 4 C. Solid phase extractions were initiated on the day of collection. 2.2. Bioassays Algal bioassays were carried out according to the ISO-standard for algal toxicity testing (ISO, 1989a) applying a mini-scale test version, described by Arensberg et al. (1995). Five to seven concentrations in three replicates and six controls were included in each test setup. Acid-washed 20-ml glass scintillation vials containing 2±4 ml of test solution were inoculated with an exponentially growing culture of Selenastrum capricornutum to 104 cells/ml. The vials were placed on a
microtiter plate shaker under continuous light (9010 mE mÿ2 sÿ1) and at a temperature of 292 C. Samples were incubated for 48 h and growth rates were calculated from ¯uorometric measurement of total algal pigment after 0, 24 and 48 h using the method described by Mayer et al. (1997). Samples were extracted with acetone for 24 h at 4 C in the dark, and the ¯uorescence emission at 66510 nm was measured after excitation at 43010 nm using a Jasco Spectro¯uorometer Model FP-777. Linear relationships between cell count by hemocytometer and ¯uorescence intensity were obtained in the relevant range for a 48-h toxicity test (104±2106 cells/ml). Concentration±response curves were described by the Weibull equation (Christensen and Nyholm, 1984) which was ®tted to the data using non-linear regression. Con®dence intervals around ECvalues were calculated by inverse estimation after a Taylor expansion applying a computer program developed by Andersen (1994). Acute toxicity tests on the freshwater crustacean Daphnia magna were carried out according to the ISOstandard (ISO, 1989b). Five to seven concentrations of ®ltered surface water or SPE-extract and six controls were used in each test setup. Scintillation vials (20 ml) with 5 ml of medium and ®ve animals (less than 24 h old) were used for each of four replicates per concentration (i.e. 20 animals per concentration, 30 animals in the control group). The number of immobile animals were counted after 24 h. After 48 h, problems with elevated immobilization of the controls were encountered, probably due to the high test temperature of 272 C. In accordance with the ISO standard (ISO, 1989b) only data obtained after 24 h were regarded as valid and further analyzed by Probit analysis using a standard software program (US EPA, 1988). 2.3. Solid phase extraction SPEs on ®ltered water samples were carried out as described in detail by Baun and Nyholm (1996) using Isolute ENV+ (International Sorbent Technology Ltd.) as SPE material. Isolute ENV+ consists of polystyrenebased polymers with a pore size of 850 AÊ and surface area of 1100 m2 gÿ1. Pre-packed 200 mg (1 ml) Isolute ENV+ columns in polyethylene syringes were cleaned by successive washes with methanol, n-hexane and acetone (9 ml of each solvent) and ®nally 30 ml of Milli-Q water. All solvents were analytical grade. The ®ltered surface water samples (typically 4±5 liter) were adjusted to pH 7.0 and the samples were passed through the SPE columns at ¯ow rates of 30±40 ml/min. The column euents were adjusted to pH 2.0 using conc. H3PO4 (about 1 ml/liter) and passed through precleaned Isolute ENV+ columns conditioned to pH 2.0 by acidi®ed Milli-Q water. The loaded columns were dried with atmospheric air for 5 min and then eluted
A. Baun et al./Environmental Pollution 102 (1998) 185±190
using 9 ml of n-hexane/acetone (85/15 v/v) followed by 6 ml of methanol. Each organic solvent was allowed to soak the column for 5 min before the drop-wise elution. For each water sample, the extracts obtained from the two columns (pH 2.0, 7.0) were combined and the volume was reduced using a Kuderna±Danish evaporator with atmospheric air and a temperature of 30 C. The occurrence of precipitation posed a limit for volume reduction, yielding maximum pre-concentration factors of 300±1500 times. The solvents were changed into acetone and water and prior to testing in the bioassays, the acetone was removed from the test medium by gentle aeration for 18 hours (Baun and Nyholm, 1996). 3. Results and discussion The concentrations of water samples or extracts are expressed as milliliters per liter referring back to the volume of original water sample relative to the total volume tested. Without pre-concentration the highest concentration tested was 900 ml/liter. For SPE extracts, a concentration of, for instance, 104 ml/liter corresponds to 10 times pre-concentration. Thus, if no toxicity is produced nor lost during the preparation of a SPE-extract, the results of toxicity expressed as milliliters per liter should be numerically identical.
187
pre-concentrated 100 times (corresponding to a concentration of 105 ml/liter). Water sampled at location S4 almost completely inhibited algal growth at the highest test concentrations (5104 ml/liter) and the EC50 was 3.6104 ml/liter (Table 1). Water sampled at the vegetable ®eld F2 was signi®cantly more toxic to algae than the stream water with an EC50 of 7.7103 ml/liter (Table 1). Filtered stream water samples all gave toxic responses in the algal test of less than 50% at the maximal concentration of 900 ml/liter. For the majority of the SPE extracts, inhibitions exceeded 60% with pre-concentration factors of 100 times or less and thus not only levels of `beginning toxicity' but also EC50 ®gures could be estimated appropriately. EC10 ®gures were used to represent beginning toxicity and thus used as quantitative lowest observed eect concentration (LOEC) estimates. Fig. 3 illustrates algal test results obtained with water samples tested directly and after pre-concentration, respectively. For easy comparison, these results are expressed in toxic units (TU10), which relates to EC10 as follows: TU10
1000 ; EC10 in ml=liter: EC10
Examples of the concentration±response curves obtained in the algal test are shown in Fig. 2 for the unpolluted reference sample (BS), a stream water sample (S4), and a sample from a vegetable ®eld (F2). All three samples were pre-concentrated by SPE. The water at the reference location was not toxic, even when
Results expressed in terms of TUs are more easily grasped, as a higher number means higher toxicity. TU10 can be interpreted as the number of times the water sample needed to be diluted in order to produce a 10% eect. The water samples collected at the three dierent vegetable ®elds (F1, F2, F3) were more toxic than water samples collected from the stream (Fig. 3, Table 1). The results for ®ltered water indicate that toxicity tended to increase as the water passed the vegetable ®elds (Fig. 3). From locations S1 to S2 and S3 the toxicity increased
Fig. 2. Concentration±response curves for 48 h algal growth inhibition tests with Selenastrum capricornutum. Solid phase extraction (SPE) pre-concentrated water at the reference location BS (*), a stream location S4 (~), and a ®eld location F2 (&). Error bars show standard deviations on average inhibition of triplicates.
Fig. 3. Toxicity of samples tested in 48-h algal growth inhibition tests (Selenastrum capricornutum). Direct testing of ®ltered water and solid phase extraction (SPE) pre-concentrated water. Results are expressed as TU10 (=1000/EC10). Bars represent 95% con®dence intervals. N.M., not measured. Asterisk denotes no toxicity measured at highest test concentration (BS: 105 ml/liter, S4: 900 ml/liter).
3.1. Toxicity of surface water samples tested in algal test
188
A. Baun et al./Environmental Pollution 102 (1998) 185±190
Table 1 EC10, EC50, and predicted no-eect concentration (PNEC) for samples pre-concentrated by solid phase extraction (SPE) and tested in 48-h algal toxicity test (Selenastrum capricornutum) EC10 (ml/liter) [95% con®dence intervals] BS S1 S2 S3 S4 F1 F2
>105 1.8103 [8.7102; 2.0103 [7.0102; 1.7103 [1.0103; 1.4104 [9.5103; 5.9102 [4.9102; 3.2103 [2.7103;
3.6103]95% 5.9103]95% 3.0103]95% 1.9104]95% 1.1103]95% 3.9103]95%
EC50 (ml/liter) [95% con®dence intervals]
PNEC (ml/liter)
>105 1.7104 [1.2104; 2.4104]95% 2.1104 [1.4104; 3.3104]95% 1.0104 [8.2103; 1.3104]95% 3.6104 [3.2104; 4.0104]95% 2.6103 [1.0103±7.5103]95% 7.7103 [4.1103; 1.4104]95%
± 86 106 52 178 13 39
PNEC estimated from EC50 assuming an uncertainty factor of 200.
about three times, whereas the toxicity of the water seemed to decrease to below the detection limit before entering the Chalong Bay area (S4). Also, stream water samples pre-concentrated by SPE at locations S1, S2 and S3 were signi®cantly more toxic than the preconcentrated water sample at location S4. In general, TU10s of surface water tested directly as ®ltered samples were higher than the respective TU10s obtained with SPE extracts. For water samples spiked with pesticides, Baun and Nyholm (1996) showed that pre-concentration recoveries of about 85% could be obtained with pure water (Milli-Q) and about 70% recovery with stream water. Even when correcting for incomplete recovery, the direct tests of water in most cases showed higher toxicity than the tests with SPE extracts. This may be due to the presence of other compounds that contribute toxicity, but are not concentrated on the SPE columns. Due to the sorption characteristics of the SPE material (Junk et al., 1974), it is expected that the results obtained with SPE preconcentration more speci®cally re¯ect pesticide contamination in the water. 3.2. Toxicity of surface water samples tested in Daphnia test Tests carried out with D. magna generally did not reveal any toxicity during the 24 h of incubation. Even at the highest concentrations tested, the number of immobilized animals was not signi®cantly dierent from that of the control group. In general more than 90% of the animals in the control group remained mobile after 24 h. The ®eld sample F1 was, however, toxic towards D. magna after pre-concentration with an EC10 (24 h) of 2.7103 ml/liter. The sample F2 was toxic without pre-concentration (EC10 (24 h)=5.2102 ml/liter) and testing the corresponding SPE extract all animals were
immobilized at all test concentrations, even the lowest corresponding to 500 ml/liter. This was the only sample where the Daphnia test was more sensitive than the algal test. 3.3. Estimation of environmental impact For evaluation of the environmental impact in the stream and in the adjacent coastal area, the key parameter is the predicted no-eect concentration (PNEC). According to the European Commission principles for risk assessment of individual chemicals (EC, 1996) a PNEC value is derived from a set of EC50 values from short-term tests, by division of the lowest reliable EC50 available with an uncertainty factor (UF). The amount and quality of data available are two of the main parameters which determine the magnitude of the uncertainty factor. In the present study, results from two toxicity tests would lead to the use of an uncertainty factor of 1000, which is argued from a precautionary viewpoint and to encourage more elaborate testing (EC, 1996). It is, however, general experience that, in the hands of an experienced investigator, the quality of data obtained by algal testing is good. Furthermore, algal tests frequently provide the most sensitive response to complex test materials when compared to other commonly used biotests. For complex samples the interspecies sensitivity variation is assumed to be much less than that reported for pure chemicals and as suggested by Pedersen et al. (1995) in a report prepared for the Danish Environmental Protection Agency, an uncertainty factor of 200 is considered more realistic than a factor of 1000. PNEC values were, therefore, tentatively estimated from the EC50 values for algae as follows: PNEC
EC50;SPE ; EC50 in ml=liter: 200
A. Baun et al./Environmental Pollution 102 (1998) 185±190
The calculated PNECs are shown in Table 1 together with the EC10s and EC50s of the pre-concentrated samples. The water at location BS was not toxic in the concentration range tested and, therefore, no PNEC value could be estimated. The dilution factors required to safeguard against toxic eects as calculated from the PNEC are shown in Fig. 4. The water in ®elds F1 and F2 had to be diluted more than 77 and 26 times, respectively, to reach the PNEC. In the stream, acute toxic eects are likely to occur, as the PNEC corresponded to a dilution of six to 20 times. At location S4, about six times dilution is necessary to get below the PNEC. Considering the large mixing due to tidal action, this dilution factor is reached rapidly. It can, therefore, be concluded that direct toxic eects from pesticides are not likely in the marine environment at the location studied. Questions such as secondary poisoning and long-term contamination by persistent compounds have not been addressed in the present study. In order to investigate whether pesticides, pesticide metabolites, or formulation ingredients should be of any long-term concern for the marine environment, more detailed information on the amounts and types of pesticides used on Phuket Island must be collected.
189
immobilization tests for the samples studied. Toxic eects on aquatic organisms are likely to occur in the stream, but due to dilution by tidal action, it is not likely that the use of pesticides in the investigation area will cause toxic eects in the adjacent coastal area. It is further concluded that the strategy of using bioassays on pre-concentrated samples seems promising as screening and monitoring tools for initial assessment of pesticide pollution in surface water. Acknowledgements The present project was ®nanced by the Danida Scienti®c Cooperation Program as a part of the project: ``Qualitative and quantitative studies of natural and anthropogenic pollutants in the Andaman Sea''. Mr Sompong Panthong and Mr Somkiat Teiwtaow, Phuket Agricultural Oce, Ministry of Agriculture and Cooperative, Thailand, are thanked for their help in ®nding adequate sampling locations and for information on pesticide use and the agricultural structure of Phuket Island. References
4. Conclusions Toxicity was detected in surface water samples from an agricultural area on Phuket Island, Thailand, using standardized bioassays with algae and Daphnia. Preconcentration by SPE was necessary in order to obtain full concentration±response relationships in the toxicity tests. Water collected at vegetable ®elds was shown to be toxic in algal and Daphnia tests. Stream water was less toxic, but the toxicity increased as the stream passed the ®elds. No toxicity was detected in an unpolluted reference sample pre-concentrated 100 times. Algal tests proved to be more sensitive than the Daphnia
Fig. 4. Dilution factors needed to obtain predicted no eect concentration (PNEC) at the sampling locations. Based on EC50 values from 48-h algal tests on solid phase extraction (SPE) pre-concentrated water samples. Bars represent 95% con®dence intervals.
Andersen, H., 1994. Statistical methods for the assessment of the toxicity of wastewater. M.Sc. thesis, Institute of Mathematical Modeling, Technical University of Denmark (in Danish). Arensberg, P., Hemmingsen, V., Nyholm, N., 1995. A miniscale algal toxicity test. Chemosphere 30, 2103±2115. Baun, A., Nyholm, N., 1996. Monitoring pesticides in surface water using bioassays on XAD-2 pre-concentrated samples. Water Science and Technology 33, 339±347. Christensen, E.R., Nyholm, N., 1984. Ecotoxicological assays with algae: Weibull dose±response curves. Environmental Science and Technology 18, 713±718. European Commission (EC), 1996. Technical Guidance Documents in Support of the Commission Directive 93/67/EEC on Risk Assessment of New Noti®ed Substances and the Commission Regulation (EC) 1488/94 on Risk Assessment for Existing Substances. Brussels, Belgium. Galassi, S., Guzzella, L., Mingazzini, M., ViganoÁ, L., Capri, S., Sora, S., 1992. Toxicological and chemical characterization of organic micropollutants in River Po waters (Italy). Water Research 26, 19±27. Hendriks, A.J., Maas-Diepeveen, J.L., Noordsij, A., Van der Gaag, M.A., 1994. Monitoring response of XAD-concentrated water in the Rhine Delta: a major part of the toxic compounds remains unidenti®ed. Water Research 28, 581±598. International Organization for Standardization (ISO), 1989a. Water qualityÐfresh water algal growth inhibition test with Scenedesmus subspicatus and Selenastrum capricornutum. ISO 8692:1989. Geneva, Switzerland. International Organization for Standardization (ISO), 1989b. Water qualityÐdetermination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea). ISO 6341:1989. Geneva, Switzerland. Junk, G.A., Richard, J.J., Grieser, M.D., Witiak, D., Witiak, J.L., Arguello, M.D., Vick, R., Svec, H.J., Fritz, J.S., Calder, C.V., 1974. Use of macroreticular resins in the analysis of water for trace organic contaminants. Journal of Chromatography 99, 745±762.
190
A. Baun et al./Environmental Pollution 102 (1998) 185±190
Mayer, P., Cuhel, R., Nyholm, N., 1997. A simple in vitro ¯uorescence method for biomass measurements in algal growth inhibition tests. Water Research 31, 2525±2531. Pedersen, F., Damborg, A., Kristensen, P., 1995. Guidance Document for Risk Assessment of Industrial Waste Water. Danish Environmental Protection Agency, Project no. 298. Copenhagen, Denmark.
Tomlin, C., 1994. The Pesticide Manual. Incorporating the Agrochemical Handbook, 10th Edition. Crop Protection Publication. British Crop Protection Council and the Royal Society of Chemistry, Cambridge. United States Environmental Protection Agency (US EPA), 1988. EPA Probit Analysis Program for Calculating EC values, version 1.4. Cincinnati, Ohio.
Environmental Pollution 102 (1998) 185±190
Screening of pesticide toxicity in surface water from an agricultural area at Phuket Island (Thailand) A. Baun*, a, N. Bussarawit b, N. Nyholm a a
Department of Environmental Science and Engineering/Groundwater Research Centre, Technical University of Denmark, Building 115, 2800 Lyngby, Denmark b Phuket Marine Biological Center, PO Box 60, 83000 Phuket, Thailand Received 15 November 1997; accepted 28 April 1998
Abstract A screening study of the toxicity of expected pesticide-contaminated surface water from an agricultural area at Phuket Island, Thailand, was carried out using standardized bioassays in combination with pre-concentration by solid phase extraction (SPE). The bioassays were an algal growth inhibition test (Selenastrum capricornutum) and a Daphnia immobilization test (Daphnia magna). Tests were run on both ®ltered water samples and samples pre-concentrated by SPE. Toxicity could be detected in nonconcentrated samples originating from areas where pesticides were applied, but pre-concentration was necessary in order to obtain full concentration±response relationships. Water collected at vegetable ®elds was very toxic. Lower toxicity was found in stream water, but the toxicity increased as the stream passed the vegetable ®elds. No toxicity was detected in an unpolluted reference sample pre-concentrated 100 times. In general, algal tests proved to be more sensitive than Daphnia tests for monitoring toxicity. It was concluded that pesticides are likely to cause toxic eects in the stream, but due to great dilution by the tide, it is not likely that the current water-borne pesticide pollution will aect the marine ecosystem. Bioassays combined with pre-concentration proved to be useful screening and monitoring tools for initial assessment of water pollution by pesticides. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Bioassays; Solid phase extraction; Pesticides; PNEC; Surface water monitoring
1. Introduction The contamination of surface water with pesticides poses a direct risk of toxic eects in the aquatic ecosystem. In contaminated agricultural areas a large variety of pesticide chemicals will often be present both as parent compounds and as their degradation intermediates. Detection and identi®cation of pesticides and their metabolites by chemical analysis can be very complicated and require sophisticated analytical techniques. Even if a compound is identi®ed, information on its aquatic toxicity may not be available. Bioassays on water samples and concentrates provide a direct functional response that relates to the overall toxic properties of the complex mixture of compounds present in a sample. Furthermore, toxicity testing with aquatic organisms is well established for environmental hazard * Corresponding author. 0269-7491/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S0269 -7 491(98)00098 -0
assessment of chemicals and chemical products and is used routinely to assess or monitor the toxicity of complex mixtures such as industrial wastewater. The detection limit of standardized biotests applied on surface water may, however, be too high to detect toxicity and hence pesticide contamination. Therefore, preconcentration of the surface water is normally necessary. In the present study the aim was to concentrate all man-made chemicals and their metabolites which could contribute to toxicity. For this purpose solid phase extraction (SPE) appears to be very eective (Galassi et al., 1992; Hendriks et al., 1994; Baun and Nyholm, 1996). A screening strategy was applied to surface water from an agricultural area near Chalong Bay, Phuket Island, Thailand (Fig. 1). In this area, vegetables (especially water spinach: Ipomoea aquatica) are grown in ¯ooded ®elds. The area is drained by a stream passing by the ®elds on its way from the mountains to Chalong Bay. The sampling locations are indicated in Fig. 1. For
186
A. Baun et al./Environmental Pollution 102 (1998) 185±190
Fig. 1. Sampling locations for screening of pesticide toxicity at Phuket Island, Thailand. S, stream; F, water spinach ®elds; BS, station for collection of a reference sample from an unpolluted area.
practical reasons the background sample was collected in a nearby stream, with no agricultural areas upstream. Information on pesticide use in water spinach ®elds was obtained from Phuket Agricultural Oce (Phuket, Thailand), pesticide vendors, and by interviewing farmers. It turned out that a broad spectrum of pesticides are used in vegetable ®elds and for controlling insects in water spinach ®elds. Methyl parathion, lambda-cyhalothrin and endosulfan were claimed to be the most commonly used pesticide chemicals. These chemicals are classi®ed as highly toxic towards aquatic organisms. Furthermore, endosulfan and lambda-cyhalothrin have potential for bio-accumulation and are also quite persistent in the aquatic environment (Tomlin, 1994). 2. Materials and methods 2.1. Sampling Water samples were collected on three occasions in December 1996 and January 1997 in acid-washed glass containers and transported to the laboratory right after sampling. In the laboratory, samples were ®ltered through glass ®ber ®lters (Whatman GF/C). Biotests were carried out on the day of collection, or the following day after sample storage at 4 C. Solid phase extractions were initiated on the day of collection. 2.2. Bioassays Algal bioassays were carried out according to the ISO-standard for algal toxicity testing (ISO, 1989a) applying a mini-scale test version, described by Arensberg et al. (1995). Five to seven concentrations in three replicates and six controls were included in each test setup. Acid-washed 20-ml glass scintillation vials containing 2±4 ml of test solution were inoculated with an exponentially growing culture of Selenastrum capricornutum to 104 cells/ml. The vials were placed on a
microtiter plate shaker under continuous light (9010 mE mÿ2 sÿ1) and at a temperature of 292 C. Samples were incubated for 48 h and growth rates were calculated from ¯uorometric measurement of total algal pigment after 0, 24 and 48 h using the method described by Mayer et al. (1997). Samples were extracted with acetone for 24 h at 4 C in the dark, and the ¯uorescence emission at 66510 nm was measured after excitation at 43010 nm using a Jasco Spectro¯uorometer Model FP-777. Linear relationships between cell count by hemocytometer and ¯uorescence intensity were obtained in the relevant range for a 48-h toxicity test (104±2106 cells/ml). Concentration±response curves were described by the Weibull equation (Christensen and Nyholm, 1984) which was ®tted to the data using non-linear regression. Con®dence intervals around ECvalues were calculated by inverse estimation after a Taylor expansion applying a computer program developed by Andersen (1994). Acute toxicity tests on the freshwater crustacean Daphnia magna were carried out according to the ISOstandard (ISO, 1989b). Five to seven concentrations of ®ltered surface water or SPE-extract and six controls were used in each test setup. Scintillation vials (20 ml) with 5 ml of medium and ®ve animals (less than 24 h old) were used for each of four replicates per concentration (i.e. 20 animals per concentration, 30 animals in the control group). The number of immobile animals were counted after 24 h. After 48 h, problems with elevated immobilization of the controls were encountered, probably due to the high test temperature of 272 C. In accordance with the ISO standard (ISO, 1989b) only data obtained after 24 h were regarded as valid and further analyzed by Probit analysis using a standard software program (US EPA, 1988). 2.3. Solid phase extraction SPEs on ®ltered water samples were carried out as described in detail by Baun and Nyholm (1996) using Isolute ENV+ (International Sorbent Technology Ltd.) as SPE material. Isolute ENV+ consists of polystyrenebased polymers with a pore size of 850 AÊ and surface area of 1100 m2 gÿ1. Pre-packed 200 mg (1 ml) Isolute ENV+ columns in polyethylene syringes were cleaned by successive washes with methanol, n-hexane and acetone (9 ml of each solvent) and ®nally 30 ml of Milli-Q water. All solvents were analytical grade. The ®ltered surface water samples (typically 4±5 liter) were adjusted to pH 7.0 and the samples were passed through the SPE columns at ¯ow rates of 30±40 ml/min. The column euents were adjusted to pH 2.0 using conc. H3PO4 (about 1 ml/liter) and passed through precleaned Isolute ENV+ columns conditioned to pH 2.0 by acidi®ed Milli-Q water. The loaded columns were dried with atmospheric air for 5 min and then eluted
A. Baun et al./Environmental Pollution 102 (1998) 185±190
using 9 ml of n-hexane/acetone (85/15 v/v) followed by 6 ml of methanol. Each organic solvent was allowed to soak the column for 5 min before the drop-wise elution. For each water sample, the extracts obtained from the two columns (pH 2.0, 7.0) were combined and the volume was reduced using a Kuderna±Danish evaporator with atmospheric air and a temperature of 30 C. The occurrence of precipitation posed a limit for volume reduction, yielding maximum pre-concentration factors of 300±1500 times. The solvents were changed into acetone and water and prior to testing in the bioassays, the acetone was removed from the test medium by gentle aeration for 18 hours (Baun and Nyholm, 1996). 3. Results and discussion The concentrations of water samples or extracts are expressed as milliliters per liter referring back to the volume of original water sample relative to the total volume tested. Without pre-concentration the highest concentration tested was 900 ml/liter. For SPE extracts, a concentration of, for instance, 104 ml/liter corresponds to 10 times pre-concentration. Thus, if no toxicity is produced nor lost during the preparation of a SPE-extract, the results of toxicity expressed as milliliters per liter should be numerically identical.
187
pre-concentrated 100 times (corresponding to a concentration of 105 ml/liter). Water sampled at location S4 almost completely inhibited algal growth at the highest test concentrations (5104 ml/liter) and the EC50 was 3.6104 ml/liter (Table 1). Water sampled at the vegetable ®eld F2 was signi®cantly more toxic to algae than the stream water with an EC50 of 7.7103 ml/liter (Table 1). Filtered stream water samples all gave toxic responses in the algal test of less than 50% at the maximal concentration of 900 ml/liter. For the majority of the SPE extracts, inhibitions exceeded 60% with pre-concentration factors of 100 times or less and thus not only levels of `beginning toxicity' but also EC50 ®gures could be estimated appropriately. EC10 ®gures were used to represent beginning toxicity and thus used as quantitative lowest observed eect concentration (LOEC) estimates. Fig. 3 illustrates algal test results obtained with water samples tested directly and after pre-concentration, respectively. For easy comparison, these results are expressed in toxic units (TU10), which relates to EC10 as follows: TU10
1000 ; EC10 in ml=liter: EC10
Examples of the concentration±response curves obtained in the algal test are shown in Fig. 2 for the unpolluted reference sample (BS), a stream water sample (S4), and a sample from a vegetable ®eld (F2). All three samples were pre-concentrated by SPE. The water at the reference location was not toxic, even when
Results expressed in terms of TUs are more easily grasped, as a higher number means higher toxicity. TU10 can be interpreted as the number of times the water sample needed to be diluted in order to produce a 10% eect. The water samples collected at the three dierent vegetable ®elds (F1, F2, F3) were more toxic than water samples collected from the stream (Fig. 3, Table 1). The results for ®ltered water indicate that toxicity tended to increase as the water passed the vegetable ®elds (Fig. 3). From locations S1 to S2 and S3 the toxicity increased
Fig. 2. Concentration±response curves for 48 h algal growth inhibition tests with Selenastrum capricornutum. Solid phase extraction (SPE) pre-concentrated water at the reference location BS (*), a stream location S4 (~), and a ®eld location F2 (&). Error bars show standard deviations on average inhibition of triplicates.
Fig. 3. Toxicity of samples tested in 48-h algal growth inhibition tests (Selenastrum capricornutum). Direct testing of ®ltered water and solid phase extraction (SPE) pre-concentrated water. Results are expressed as TU10 (=1000/EC10). Bars represent 95% con®dence intervals. N.M., not measured. Asterisk denotes no toxicity measured at highest test concentration (BS: 105 ml/liter, S4: 900 ml/liter).
3.1. Toxicity of surface water samples tested in algal test
188
A. Baun et al./Environmental Pollution 102 (1998) 185±190
Table 1 EC10, EC50, and predicted no-eect concentration (PNEC) for samples pre-concentrated by solid phase extraction (SPE) and tested in 48-h algal toxicity test (Selenastrum capricornutum) EC10 (ml/liter) [95% con®dence intervals] BS S1 S2 S3 S4 F1 F2
>105 1.8103 [8.7102; 2.0103 [7.0102; 1.7103 [1.0103; 1.4104 [9.5103; 5.9102 [4.9102; 3.2103 [2.7103;
3.6103]95% 5.9103]95% 3.0103]95% 1.9104]95% 1.1103]95% 3.9103]95%
EC50 (ml/liter) [95% con®dence intervals]
PNEC (ml/liter)
>105 1.7104 [1.2104; 2.4104]95% 2.1104 [1.4104; 3.3104]95% 1.0104 [8.2103; 1.3104]95% 3.6104 [3.2104; 4.0104]95% 2.6103 [1.0103±7.5103]95% 7.7103 [4.1103; 1.4104]95%
± 86 106 52 178 13 39
PNEC estimated from EC50 assuming an uncertainty factor of 200.
about three times, whereas the toxicity of the water seemed to decrease to below the detection limit before entering the Chalong Bay area (S4). Also, stream water samples pre-concentrated by SPE at locations S1, S2 and S3 were signi®cantly more toxic than the preconcentrated water sample at location S4. In general, TU10s of surface water tested directly as ®ltered samples were higher than the respective TU10s obtained with SPE extracts. For water samples spiked with pesticides, Baun and Nyholm (1996) showed that pre-concentration recoveries of about 85% could be obtained with pure water (Milli-Q) and about 70% recovery with stream water. Even when correcting for incomplete recovery, the direct tests of water in most cases showed higher toxicity than the tests with SPE extracts. This may be due to the presence of other compounds that contribute toxicity, but are not concentrated on the SPE columns. Due to the sorption characteristics of the SPE material (Junk et al., 1974), it is expected that the results obtained with SPE preconcentration more speci®cally re¯ect pesticide contamination in the water. 3.2. Toxicity of surface water samples tested in Daphnia test Tests carried out with D. magna generally did not reveal any toxicity during the 24 h of incubation. Even at the highest concentrations tested, the number of immobilized animals was not signi®cantly dierent from that of the control group. In general more than 90% of the animals in the control group remained mobile after 24 h. The ®eld sample F1 was, however, toxic towards D. magna after pre-concentration with an EC10 (24 h) of 2.7103 ml/liter. The sample F2 was toxic without pre-concentration (EC10 (24 h)=5.2102 ml/liter) and testing the corresponding SPE extract all animals were
immobilized at all test concentrations, even the lowest corresponding to 500 ml/liter. This was the only sample where the Daphnia test was more sensitive than the algal test. 3.3. Estimation of environmental impact For evaluation of the environmental impact in the stream and in the adjacent coastal area, the key parameter is the predicted no-eect concentration (PNEC). According to the European Commission principles for risk assessment of individual chemicals (EC, 1996) a PNEC value is derived from a set of EC50 values from short-term tests, by division of the lowest reliable EC50 available with an uncertainty factor (UF). The amount and quality of data available are two of the main parameters which determine the magnitude of the uncertainty factor. In the present study, results from two toxicity tests would lead to the use of an uncertainty factor of 1000, which is argued from a precautionary viewpoint and to encourage more elaborate testing (EC, 1996). It is, however, general experience that, in the hands of an experienced investigator, the quality of data obtained by algal testing is good. Furthermore, algal tests frequently provide the most sensitive response to complex test materials when compared to other commonly used biotests. For complex samples the interspecies sensitivity variation is assumed to be much less than that reported for pure chemicals and as suggested by Pedersen et al. (1995) in a report prepared for the Danish Environmental Protection Agency, an uncertainty factor of 200 is considered more realistic than a factor of 1000. PNEC values were, therefore, tentatively estimated from the EC50 values for algae as follows: PNEC
EC50;SPE ; EC50 in ml=liter: 200
A. Baun et al./Environmental Pollution 102 (1998) 185±190
The calculated PNECs are shown in Table 1 together with the EC10s and EC50s of the pre-concentrated samples. The water at location BS was not toxic in the concentration range tested and, therefore, no PNEC value could be estimated. The dilution factors required to safeguard against toxic eects as calculated from the PNEC are shown in Fig. 4. The water in ®elds F1 and F2 had to be diluted more than 77 and 26 times, respectively, to reach the PNEC. In the stream, acute toxic eects are likely to occur, as the PNEC corresponded to a dilution of six to 20 times. At location S4, about six times dilution is necessary to get below the PNEC. Considering the large mixing due to tidal action, this dilution factor is reached rapidly. It can, therefore, be concluded that direct toxic eects from pesticides are not likely in the marine environment at the location studied. Questions such as secondary poisoning and long-term contamination by persistent compounds have not been addressed in the present study. In order to investigate whether pesticides, pesticide metabolites, or formulation ingredients should be of any long-term concern for the marine environment, more detailed information on the amounts and types of pesticides used on Phuket Island must be collected.
189
immobilization tests for the samples studied. Toxic eects on aquatic organisms are likely to occur in the stream, but due to dilution by tidal action, it is not likely that the use of pesticides in the investigation area will cause toxic eects in the adjacent coastal area. It is further concluded that the strategy of using bioassays on pre-concentrated samples seems promising as screening and monitoring tools for initial assessment of pesticide pollution in surface water. Acknowledgements The present project was ®nanced by the Danida Scienti®c Cooperation Program as a part of the project: ``Qualitative and quantitative studies of natural and anthropogenic pollutants in the Andaman Sea''. Mr Sompong Panthong and Mr Somkiat Teiwtaow, Phuket Agricultural Oce, Ministry of Agriculture and Cooperative, Thailand, are thanked for their help in ®nding adequate sampling locations and for information on pesticide use and the agricultural structure of Phuket Island. References
4. Conclusions Toxicity was detected in surface water samples from an agricultural area on Phuket Island, Thailand, using standardized bioassays with algae and Daphnia. Preconcentration by SPE was necessary in order to obtain full concentration±response relationships in the toxicity tests. Water collected at vegetable ®elds was shown to be toxic in algal and Daphnia tests. Stream water was less toxic, but the toxicity increased as the stream passed the ®elds. No toxicity was detected in an unpolluted reference sample pre-concentrated 100 times. Algal tests proved to be more sensitive than the Daphnia
Fig. 4. Dilution factors needed to obtain predicted no eect concentration (PNEC) at the sampling locations. Based on EC50 values from 48-h algal tests on solid phase extraction (SPE) pre-concentrated water samples. Bars represent 95% con®dence intervals.
Andersen, H., 1994. Statistical methods for the assessment of the toxicity of wastewater. M.Sc. thesis, Institute of Mathematical Modeling, Technical University of Denmark (in Danish). Arensberg, P., Hemmingsen, V., Nyholm, N., 1995. A miniscale algal toxicity test. Chemosphere 30, 2103±2115. Baun, A., Nyholm, N., 1996. Monitoring pesticides in surface water using bioassays on XAD-2 pre-concentrated samples. Water Science and Technology 33, 339±347. Christensen, E.R., Nyholm, N., 1984. Ecotoxicological assays with algae: Weibull dose±response curves. Environmental Science and Technology 18, 713±718. European Commission (EC), 1996. Technical Guidance Documents in Support of the Commission Directive 93/67/EEC on Risk Assessment of New Noti®ed Substances and the Commission Regulation (EC) 1488/94 on Risk Assessment for Existing Substances. Brussels, Belgium. Galassi, S., Guzzella, L., Mingazzini, M., ViganoÁ, L., Capri, S., Sora, S., 1992. Toxicological and chemical characterization of organic micropollutants in River Po waters (Italy). Water Research 26, 19±27. Hendriks, A.J., Maas-Diepeveen, J.L., Noordsij, A., Van der Gaag, M.A., 1994. Monitoring response of XAD-concentrated water in the Rhine Delta: a major part of the toxic compounds remains unidenti®ed. Water Research 28, 581±598. International Organization for Standardization (ISO), 1989a. Water qualityÐfresh water algal growth inhibition test with Scenedesmus subspicatus and Selenastrum capricornutum. ISO 8692:1989. Geneva, Switzerland. International Organization for Standardization (ISO), 1989b. Water qualityÐdetermination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea). ISO 6341:1989. Geneva, Switzerland. Junk, G.A., Richard, J.J., Grieser, M.D., Witiak, D., Witiak, J.L., Arguello, M.D., Vick, R., Svec, H.J., Fritz, J.S., Calder, C.V., 1974. Use of macroreticular resins in the analysis of water for trace organic contaminants. Journal of Chromatography 99, 745±762.
190
A. Baun et al./Environmental Pollution 102 (1998) 185±190
Mayer, P., Cuhel, R., Nyholm, N., 1997. A simple in vitro ¯uorescence method for biomass measurements in algal growth inhibition tests. Water Research 31, 2525±2531. Pedersen, F., Damborg, A., Kristensen, P., 1995. Guidance Document for Risk Assessment of Industrial Waste Water. Danish Environmental Protection Agency, Project no. 298. Copenhagen, Denmark.
Tomlin, C., 1994. The Pesticide Manual. Incorporating the Agrochemical Handbook, 10th Edition. Crop Protection Publication. British Crop Protection Council and the Royal Society of Chemistry, Cambridge. United States Environmental Protection Agency (US EPA), 1988. EPA Probit Analysis Program for Calculating EC values, version 1.4. Cincinnati, Ohio.