The effects of 2,4-D herbicide and organophosphorus insecticides on growth, photosynthesis, and chlorophyll a synthesis of Chlamydomonas reinhardtii (mt +)

The effects of the herbicide (2,4-D) and six organophosphorus insecticides (Diazinon, Dimethoate, Fenitrothion, Malathion, Phenthoate and Quinalphos) on growth, photosynthesis and chlorophyll a synthesis of the fresh water green alga Chlamydomonas reinhardtii (mt +) were studied. At low concentrations (1 and 5 ppm), the herbicide and all six orgnophosphorus insecticides stimulated photosynthesis of the alga. The stimulating effects on algal growth and chlorophyll a synthesis were only observed in the presence of low concentration (1 ppm) of 2,4-D and Fenitrothion, Growth, photosynthesis and chlorophyll a synthesis of the alga were inhibited in the presence of high concentrations (10, 20 and 40 ppm) of the herbicide and all the six organophosphorus insecticides. Results also indicated that the toxicities of these organophosphorus insecticides on the tested alga were dependent on their chemical structures.     

Effects of copper, chromium and nickel on growth, photosynthesis and chlorophyll a synthesis of Chlorella pyrenoidosa 251

Individual and combined effects of three heavy metals; namely, copper, chromium and nickel, on growth, photosynthesis and chlorophyll a synthesis of the unicellular green alga, Chlorella pyrenoidosa 251, were determined. Within the concentration range of 0.1 to 1.0 mg litre(-1) of the three heavy metals tested on growth, photosynthesis and chlorophyll a synthesis of the alga, the order of toxicity of the three heavy metals was copper > chromium > nickel. The presence of one of the three heavy metals interacted synergistically with the other two heavy metals in various bimetallic combinations on growth, photosynthesis and chlorophyll a synthesis of the alga.     

Soil column experiments used as a means to assess transport, sorption, and biodegradation of pesticides in groundwater

Soil column experiments are used to investigate the fate of three pesticides of high, intermediate, and low solubility in groundwater: N- phosphonomethyl glycine (glyphosate); O,O-diethyl-S-[(ethylthio)methyl]phosphorodithioate (phorate); (2,4-dichlorophenoxy)acetic acid (2,4-D). Feed solutions are prepared by adding each pesticide (100 mg/L glyphosate, 50 micro g/L phorate, 50 mg/L 2,4-D) along with conservative tracer, KBr, in synthetic groundwater. The concentration of the pesticides in effluents is detected by ion chromatography (glyphosate, 2,4-D) and GC-FID (phorate). The Br(-) breakthrough curves are employed to estimate the dispersion coefficient and mean pore velocity in each column. Solute transport and reactive models accounting for equilibrium/non-equilibrium sorption and biodegradation are coupled with inverse modeling numerical codes to estimate the kinetic parameters for all pesticides.     

Soil column experiments used as a means to assess transport,sorption, and biodegradation of pesticides in groundwater

Soil column experiments are used to investigate the fate of three pesticides of high, intermediate, and low solubility in groundwater: N- phosphonomethyl glycine (glyphosate); O,O-diethyl-S-[(ethylthio)methyl]phosphorodithioate (phorate); (2,4-dichlorophenoxy)acetic acid (2,4-D). Feed solutions are prepared by adding each pesticide (100 mg/L glyphosate, 50 μ g/L phorate, 50 mg/L 2,4-D) along with conservative tracer, KBr, in synthetic groundwater. The concentration of the pesticides in effluents is detected by ion chromatography (glyphosate, 2,4-D) and GC-FID (phorate). The Br^? breakthrough curves are employed to estimate the dispersion coefficient and mean pore velocity in each column. Solute transport and reactive models accounting for equilibrium/non-equilibrium sorption and biodegradation are coupled with inverse modeling numerical codes to estimate the kinetic parameters for all pesticides.     

Toxicity of surfactants to green microalgae Pseudokirchneriella subcapitata and Scenedesmus subspicatus and to marine diatoms Phaeodactylum tricornutum and Skeletonema costatum

Ecotoxicity of different commercial surfactants (six anionic, two amphoteric and one nonionic), essential constituents of cleansing hair products (shampoos), as well as ecotoxicity of eight shampoos containing different combinations of these surfactants, were tested in order to evaluate their possible toxic effects on microalgae. Specific objective of this research was to compare the sensitivity of selected freshwater and marine microalgae to these widely used surfactants and well-known pollutants in surface waters. Internationally validated methods (ISO standards) for the determination of toxic effects on the growth of planktonic freshwater green algae Pseudokirchneriella subcapitata and Scenedesmus subspicatus and marine diatoms Skeletonema costatum and Phaeodactylum tricornutum, were used. The obtained results showed that the concentrations of tested surfactants and shampoos, which resulted in 50% growth reduction of planktonic freshwater green algae, when compared to the controls without test substances (EC50), were in the range from 0.32 to 4.4 mg l(-1) for surfactants and from 2.1 to 8.5 mg l(-1) for shampoos expressed as active substance. Marine diatoms were significantly more sensitive to the tested surfactants than freshwater green algae (EC50 0.14-1.7 mg l(-1) for surfactants and 0.35-1.25 mg l(-1) for shampoos). According to the classification on the basis of environmental effects, the obtained results suggested that all tested surfactants can be classified as having toxic effects on freshwater green alga Pseudokirchneriella subcapitata. Some of them indicated that they have a very toxic effect on Scenedesmus subspicatus and marine diatoms Skeletonema costatum and Phaeodactylum tricornutum.     

Ecotoxicity characterization of dinitrotoluenes and some of their reduced metabolites

In the present study, the toxic effects of 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT) and a selection of their respective metabolites were examined and compared to 2,4,6-trinitrotoluene (TNT) using the 15-min Microtox (Vibrio fischen) and 96-h freshwater green alga (Selenastrum capricomutum) growth inhibition tests. All of the compounds tested were less toxic than TNT. Using the Microtox assay, 2,6-DNT was more toxic than 2,4-DNT and the order of toxicity for 2,6-DNT and its metabolites was: 2,6-DNT > or = 2A-6NT > 2,6-DAT; whereas that for 2,4-DNT was: 4A-2NT > 2A-4NT > 2,4-DNT > 2,4-DAT. For the algal test, 2,4-DNT was more toxic than 2,6-DNT and the order of toxicity for 2,4-DNT and its metabolites was: 2,4-DNT > 2,4-DAT approximately equal to 4A-2NT = 2A-4NT. The order of toxicity for 2,6-DNT and its reduced metabolites using the algal test was very similar to the Microtox bioassay. These results demonstrate that the reduced metabolites of 2,6-DNT tested in this study were less toxic than that of the parent compound, but certain partially reduced metabolites of 2,4-DNT can be more toxic than the parent molecule. These data put into question the general hypothesis that reductive metabolism of nitro-aromatics is associated with a sequential detoxification process.     

Toxicity of prednisolone, dexamethasone and their photochemical derivatives on aquatic organisms

Light exposure of aqueous suspensions of prednisolone and dexamethasone causes their partial phototransformation. The photoproducts, isolated by chromatographic techniques, have been identified by spectroscopic means. Prednisolone, dexamethasone and their photoproducts have been tested to evaluate their acute and chronic toxic effects on some freshwater chain organisms. The rotifer Brachionus calyciflorus and the crustaceans Thamnocephalus platyurus and Daphnia magna were chosen to perform acute toxicity tests, while the alga Pseudokircheneriella subcapitata (formerly known as Selenastrum capricornutum) and the crustacean Ceriodaphnia dubia to perform chronic tests. The photochemical derivatives are more toxic than the parent compounds. Generally low acute toxicity was found. Chronic exposure to this class of pharmaceuticals caused inhibition of growth population on the freshwater crustacean C. dubia while the alga P. subcapitata seems to be less affected by the presence of these drugs.     

Toxicity of chlorinated phenoxyacetic acid herbicides in the experimental eukaryotic model Saccharomyces cerevisiae: role of pH and of growth phase and size of the yeast cell population

The inhibitory effect of the herbicides 2-methyl-4-chlorophenoxyacetic acid (MCPA) and 2,4-dichlorophenoxyacetic acid (2,4-D) in Saccharomyces cerevisiae growth is strongly dependent on medium pH (range 2.5-6.5). Consistent with the concept that the toxic form is the liposoluble undissociated form, at values close to their pK(a) (3.07 and 2.73, respectively) the toxicity is high, decreasing with the increase of external pH. In addition, the toxicity of identical concentrations of the undissociated acid form is pH independent, as observed with 2,4-dichlorophenol (2,4-DCP), an intermediate of 2,4-D degradation. Consequently, at pH values above 3.5 (approximately one unit higher than 2,4-D pK(a)), 2,4-DCP becomes more toxic than the original herbicide. A dose-dependent inhibition of growth kinetics and increased duration of growth latency is observed following sudden exposure of an unadapted yeast cell population to the presence of the herbicides. This contrasts with the effect of 2,4-DCP, which essentially affects growth kinetics. Experimental evidences suggest that the acid herbicides toxicity is not exclusively dependent on the liposolubility of the toxic form, as may essentially be the case of 2,4-DCP. An unadapted yeast cell population at the early stationary-phase of growth under nutrient limitation is significantly more resistant to short-term herbicide induced death than an exponential-phase population. Consequently, the duration of growth latency is reduced, as observed with the increase of the size of the herbicide stressed population. However, these physiological parameters have no significant effect either on growth kinetics, following growth resumption under herbicide stress, or on the growth curve of yeast cells previously adapted to the herbicides, indicating that their role is exerted at the level of cell adaptation.     

Phytoplankton toxicity of the antibiotic chlortetracycline and its UV light degradation products

Two common freshwater phytoplankton species Microcystis aeruginosa and Scenedesmus obliquus were employed as test organisms to investigate the toxic effects of chlortetracycline widely used in human medicine and veterinary as antibiotic. Toxicity assays were performed into two parts: antibiotic toxicity test and antibiotic degraded products toxicity test. In general, chlortetracycline had significantly toxic effect on population growth and chlorophyll-a accumulation of two phytoplankton. Although M. aeruginosa had ability to grow after exposed to chlortetracycline at 0.5 mg L⁻¹, its photosynthesis function was also disrupted. Compared with the data in two phytoplankton species, the chlorophyceae was more sensitive than the cyanophyceae. The adverse effect on S. obliquus was stronger than that on M. aeruginosa with increasing concentrations. In addition, for M. aeruginosa, regardless of the UV light degradation time, the treated chlortetracycline also had adverse effect on population growth and chlorophyll-a accumulated. The degraded chlortetracycline under any treatment time was more toxic for S. obliquus than chlortetracycline itself excluding under 24 h. However, the correlation between the toxicity and degradation time was not clear and toxicity enhanced in fact did not follow the increase or decrease in degradation time. Our study showed that the antibiotic chlortetracycline and its degraded products had adverse effect on freshwater phytoplankton, the former has not been reported before and the latter has been overlooked in other research in the past.     

Toxicity of Heavy Metals on Scenedesmus quadricauda (Turp.) de Brébisson in Batch Cultures (7 pp)

Background Aquatic environments are often exposed to various pollutants like heavy metals that are released from industrial, agricultural and domestic wastes. Emissions of heavy metals can then enter all ecosystems and bring about severe problems in plants, especially algae depending on the concentrations of a given element. The objective of the investigation presented is to detect toxic effects due to some heavy metals in the biomass of green alga Scenedesmus quadricauda. Methods All experiments were conducted with axenic cultures of the green alga Scenedesmus quadricauda (Turp.) de Brebisson (Chlorococcales, Chlorophyta). The cultures were grown in Chu-no.10 medium, and optimum physical and chemical growth conditions were provided to get higher growth rates and lower doubling times of cells. Growth of the micro algal cultures was measured on a daily schedule by counting cultures and determination of chlorophyll-a. The sterile-filtered heavy metal concentration solutions (Cd, Pb and Cu) were prepared and added as stock solutions of their salts (CdCl2*2.5 H2O, Pb(NO3)2*H2O and CuSO4*5 H2O). Results and Discussion The growth decreased gradually with the alga exposed to Cd at 0.05, 0.1, and 0.5 mg/L in comparison with the control whereas 1 mg/L Cd2+ had a clearly destructive effect. The growth was decreased with Pb at 15, 20 and 25 mg/L gradually, while at 30 mg/L the effect was more pronounced. When Cu was used, the growth was increased gradually at 0.5, 1 and 1.5 mg/L and vice versa at 2 mg/L. Conclusion There were differences in toxic effects of the metals with different metal as well as their concentrations and the time of exposure.     

Treatment of 2,4-D, mecoprop, and dicamba using membrane bioreactor technology

Phenoxyacetic and benzoic acid herbicides are widely used agricultural, commercial, and domestic pesticides. As a result of high water solubility, mobility, and persistence, 2,4-dichlorophenoxyacetic acid (2,4-D), methylchlorophenoxypropionic acid (mecoprop), and 3,6-dichloro-2-methoxybenzoic acid (dicamba) have been detected in surface and waste waters across Canada. As current municipal wastewater treatment plants do not specifically address chronic, trace levels of contaminants like pesticides, an urgent need exists for an efficient, environmentally friendly means of breaking down these toxic herbicides. A commercially available herbicide mix, WeedEx, containing 2,4-D, mecoprop, and dicamba, was subjected to treatment using membrane bioreactor (MBR) technology. The three herbicides, in simulated wastewater with a chemical oxygen demand of 745 mg/L, were introduced to the MBR at concentrations ranging from 300 μg/L to 3.5 mg/L. Herbicides and biodegradation products were extracted from MBR effluent using solid-phase extraction followed by detection using high-performance liquid chromatography coupled with mass spectrometry. 2,4-D was reduced by more than 99.0 % within 12 days. Mecoprop and dicamba were more persistent and reduced by 69.0 and 75.4 %, respectively, after 112 days of treatment. Half-lives of 2,4-D, mecoprop and dicamba during the treatment were determined to be 1.9, 10.5, and 28.3 days, respectively. Important water quality parameters of the effluent such as dissolved oxygen, pH, ammonia, chemical oxygen demand, etc. were measured daily. MBR was demonstrated to be an environmentally friendly, compact, and efficient method for the treatment of toxic phenoxyacetic and benzoic acid herbicides.     

Removal of 2,4-dichlorophenol by suspended and immobilized Bacillus insolitus

In this experiment, Bacillus insolitus was isolated and selected from a mixed culture that have been acclimated to chlorophenols. Decomposition of chlorophenolic compounds will be studied using this pure culture in both suspended and immobilized form. The results are: at lower initial concentrations of 2,4-dichlorophenol (10-50 mg/l), immobilized Bacillus insolitus shows a higher removal of 2,4-dichlorophenol than Bacillus insolitus in suspended growth. When the 2,4-dichlorophenol concentration becomes higher (50-200 mg/l), both immobilized and suspended Bacillus insolitus have approximately the same efficiency for removal of 2,4-dichlorophenol. Higher concentrations of 2,4-dichlorophenol are inhibitive to the growth of either suspended or immobilized Bacillus insolitus. At lower concentrations of 2,4-dichlorophenol, immobilized mixed culture may have the same removal efficiency of 2,4-dichlorophenol as immobilized pure culture of Bacillus insolitus. But with regard to the overall 2,4-dichlorophenol removal efficiency, immobilized pure culture is considered to be superior to immobilized mixed culture.     

Mixture toxicity of the antifouling compound irgarol to the marine phytoplankton species Dunaliella tertiolecta

This study examined the toxicity of irgarol, individually and in binary mixtures with three other pesticides (the fungicide chlorothalonil, and the herbicides atrazine and 2,4-D), to the marine phytoplankton species Dunaliella tertiolecta. Standard 96-h static algal bioassays were used to determine pesticide effects on population growth rate. Irgarol significantly inhibited D. tertiolecta growth rate at concentrations > or = 0.27 micro g/L. Irgarol was significantly more toxic to D. tertiolecta than the other pesticides tested (irgarol 96 h EC50 = 0.7 micro g/L; chlorothalonil 96 h EC50 = 64 micro g/L; atrazine 96 h EC50 = 69 micro g/L; 2,4-D 96 h EC50 = 45,000 micro g/L). Irgarol in mixture with chlorothalonil exhibited synergistic toxicity to D. tertiolecta, with the mixture being approximately 1.5 times more toxic than the individual compounds. Irgarol and atrazine, both triazine herbicides, were additive in mixture. The toxicity threshold of 2,4-D was much greater than typical environmental levels and would not be expected to influence irgarol toxicity. Based on these interactions, overlap of certain pesticide applications in the coastal zone may increase the toxicological risk to resident phytoplankton populations.     

Light regime, riboflavin, and pH effects on 2,4-D photodegradation in water

A laboratory study was conducted to determine the effects of light regime, riboflavin, and pH on photodegradation of 2,4-D in aqueous solution. In controlled-environment chamber experiments, riboflavin sensitized 2,4-D photolysis in a concentration-dependent manner under both attenuated UV (-UV) and enhanced UV (+UV) light regimes. The photolysis half-life of 2,4-D in solutions containing 10 mg L-1 riboflavin was 9.7 and 12.5 h when exposed to +UV and -UV, respectively, compared to no photolysis in the absence of riboflavin. In contrast, the extrapolated half-life of 2,4-D in solutions containing 2.5 mg L-1 riboflavin was 46 h under +UV and 72 h under -UV. The rate of 2,4-D photolysis in the presence of riboflavin increased under both light regimes as initial pH of the solution was decreased from 7.5 to 4.5. The half-life of 2,4-D in the presence of 10 mg L-1 riboflavin at pH 4.5 and exposed to +UV was 1.6 h. Lumichrome, a principal photoproduct of riboflavin, did not photosensitize 2,4-D. Concentrations of 2,4-dichlorophenol formed as a result of riboflavin-sensitized 2,4-D photolysis were higher under the -UV than the +UV regime. These results indicate that riboflavin concentration, solution pH, and light regime are interacting factors that may be manipulated to enhance rates of aqueous 2,4-D photolysis.     

Toxicity of the herbicide glyphosate to Chordodes nobilii (Gordiida, Nematomorpha)

Nematomorpha (horsehair worms) is a poorly known group of worm-like animals similar to nematodes. Adults are free-living and reproduction takes place in freshwater environments, where preparasitic larvae undergo development. All species have a parasitic juvenil stage and infection may result in the host's death, insects being the most frequent host. Most of the life cycle occurs in freshwater environments, which are often contaminated by different pollutants. Based on the lack of information on the toxicity of herbicides to horsehair worms, the objective of this study is to evaluate the effect of different concentrations of glyphosate (technical grade and formulated product) on Chordodes nobilii (Gordiida, Nematomorpha). Bioassays were performed with embryos and larvae (preparasitic stages), and adults (postparasitic stage). Test organisms were exposed for a short period of time to concentrations ranging between 0.1 and 8 mga.e.l(-1) of glyphosate (technical and formulated). Although embryo development was not inhibited, there was a significant decrease in the infective capacity of larvae derived from eggs that had been exposed to >or= 0.1mg/l. Similar results were obtained for directly exposed larvae. No differences in toxicity were detected between the active ingredient and formulated product. Adult exposed for 96 h to 1.76 mgl(-1) formulated Gly shown a mortality of 50%. Results indicate that C. nobilii is affected at glyphosate concentrations lower than those expected to be found in freshwater environments and those specified in the legislation.     

Hardness corrections for copper are inappropriate for protecting sensitive freshwater biota

Toxicity testing using a freshwater alga (Chlorella sp.), a bacterium (Erwinnia sp.) and a cladoceran (Ceriodaphnia cf. dubia) exposed to copper in synthetic and natural freshwaters of varying hardness (44-375 mg CaCO3/l), with constant alkalinity, pH and dissolved organic carbon concentration, demonstrated negligible hardness effects in the pH range 6.1-7.8. Therefore, the use of a generic hardness-correction algorithm, developed as part of national water quality guidelines for protecting freshwater biota, is not recommended for assessing the toxicity of copper to these, and other, sensitive freshwater species. Use of the algorithm for these sensitive species will be underprotective because the calculated concentrations of copper in water that cause a toxic effect will be higher.     

Spiking solvent, humidity and their impact on 2,4-D and 2,4-DCP extractability from high humic matter content soils

The 2,4-dichlorophenoxyacetic acid (2,4-D) is a hormone-like herbicide widely used in agriculture. Although its half life in soil is approximately two weeks, the thousands of tons introduced in the environment every year represent a risk for human health and the environment. Considering the toxic properties of this compound and its degradation products, it is important to assess and monitor the 2,4-D residues in agricultural soils. Furthermore, experiments of phyto/bioremediation are carried out to find economic and environmental friendly tools to restore the polluted soils. Accordingly, it is essential to accurately measure the amount of 2,4-D and its metabolites in soils. There is evidence that 2,4-D extraction from soil samples seriously depends on the physical and chemical properties of the soil, especially in those soils with high content of humic acids. The aim of this work was to assess the variables that influence the recovery and subsequent analysis of 2,4-D and its main metabolite (2,4-dichlorophenol) from those soils samples. The results showed that the recovery efficiency depends on the solvent and method used for the extraction, the amount and kind of solvent used for dissolving the herbicide and the soil water content at the moment of spiking. An optimized protocol for the extraction and quantification of 2,4-D and its main metabolite from soil samples is presented.     

Effects of herbicides on two submersed aquatic macrophytes, Potamogeton pectinatus L. and Myriophyllum sibiricum Komarov, in a prairie wetland

Clopyralid, picloram, 2,4-D and a mixture of 2,4-D plus picloram, (Tordon 202C) were added to the water of 1 m square enclosures in a prairie wetland in Saskatchewan, Canada to produce concentrations of 0.01 and 0.1 mg active ingredient litre(-1). Effects on the submersed macrophytes, Potamogeton pectinatus and Myriophyllum sibiricum, were monitored by taking repeated measurements of plant weight, flower and tuber production and inspecting for injuries at 30 and 60 days after application. Clopyralid did not inhibit weight gain (growth) in either species, but stimulated growth and flowering by M. sibiricum at 0.01 mg litre(-1) and tuber production by P. pectinatus at both rates. The low rate of 2,4-D stimulated flowering by M. sibiricum and tuber production by P. pectinatus, whereas the high rate inhibited growth of M. sibiricum and injured both species. Picloram did not affect growth of either species, but injured M. sibiricum at both concentrations and inhibited flowering at 0.1 mg litre(-1). Tordon 202C at 0.1 mg litre(-1) caused reduced growth and flowering in M. sibiricum and injured both species; 0.01 mg litre(-1) also injured M. sibiricum. Mortality resulted only from Tordon 202C and 2,4-D. Field data are lacking to assess the extent to which submerged macrophytes in prairie ponds are exposed to harmful concentrations of herbicide from aerial spraying, drift from ground application, runoff or wind erosion of soil.     

Influence of glyphosate and its formulation (Roundup) on the toxicity and bioavailability of metals to Ceriodaphnia dubia

This study examined the toxicological interaction between glyphosate (or its formulation, Roundup) and several heavy metals to a freshwater cladoceran, Ceriodaphnia dubia. We demonstrated that all binary combinations of Roundup and metals (Cd, Cu, Cr, Ni, Pb, Se and Zn) exhibited "less than additive" mixture toxicity, with 48-h LC50 toxic unit > 1. Addition of glyphosate alone could significantly reduce the acute toxicity of Ag, Cd, Cr, Cu, Ni, Pb and Zn (but not Hg and Se). The ratio between glyphosate and metal ions was important in determining the mitigation of metal toxicity by glyphosate. A bioaccumulation study showed that in the presence of glyphosate the uptake of some metals (e.g. Ag) was halted but that of others (e.g. Hg) was increased significantly. Therefore, our study strongly suggests that glyphosate and its commercial formulations can control the toxicity as well as the bioavailability of heavy metals in aquatic ecosystems where both groups of chemicals can co-occur.     

Degradation of PCDD, PCDF, PAH, PCB and chlorinated phenols during the destruction-treatment of landfill seepage water in laboratory model reactor (UV, Ozone, and UV/Ozone)

Seepage water of toxic waste landfills is polluted with high concentrations of toxic organic compounds. The concentrations in the seepage water we applied for chlorinated phenols are between 2 μg/1 and 1 mg/l, for PCB between 800 pg/l and 250 ng/l, for PAH between 200 ng/l and 12 μg/l and for PCDD and PCDF between 20 pg/l and 1 ng/l. Usual purification methods produce highly contaminated residues, which have to be treated by pyrolysis or are deposited again at a landfill. A better way is to destruct these contaminants by UV/ozone treatment. The treatment of seepage water by UV-irradiation, ozone and UV/ozone is compared. Results show no significant effect during all treatments for PCB and PCDD/PCDF. The chlorinated phenols and PAH were mostly destroyed by UV/ozone treatment more than 90 %. The pH value has no influence on the UV/ozone treatment of seepage water.