Algal Growth Inhibition Test Research Articles

Delayed fluorescence in algal growth inhibition tests

AbstractA series of 72-hour growth inhibition tests with green alga Desmodesmus (Scenedesmus) subspicatus (ISO 8692) has been performed to test the delayed fluorescence (DF) parameters as possible endpoint measurements. Sensitivity to five toxicants with direct and indirect effects on photosynthesis was tested, and the median effective concentration (EC50) values derived from the cell concentration, absorbance and DF were compared. The sensitivity of DF intensity (DFI) was comparable with the two endpoints suggested in ISO 8692 for all five toxicants: potassium dichromate, 3,5-dichlorophenol (3,5-DCP), DCMU, copper and cadmium. In the case of potassium dichromate and copper, DFI was more sensitive than the other endpoints in this study. The analysis of DF relaxation kinetics showed a specific response to the herbicide DCMU. Additionally, a 24-hour test was performed with the same toxicant concentrations (except copper), where DFI was measured 30 minutes, one hour and 24 hours after the exposure. The influence of toxicants on DFI after a 24-hour exposure was comparable with the effects after 72 hours. Only 3,5-DCP influenced DFI after 30 minutes. DF proved to be a simple, reliable and rapid measurement to assess toxicity in algal tests, which can to some extent differentiate among various toxicants.

Multi-species toxicity evaluation of a chromium-based leather tannery wastewater

This study focused on a comparative evaluation of wastewater from a chromium-based tannery in Izmir (Turkey). Two sampling campaigns included raw wastewater (R), the effluents of coagulation/flocculation process (CF), biological treatment (B) and dried sludge (DS). Samples were analyzed for their metal contents. A set of toxicity endpoints were tested by sea urchin ( Paracentrotus lividius and Sphaerechinus granularis) for the endpoints of acute embryotoxicity, developmental defects, changes in sperm fertilization success and transmissible damage from sperm to the offspring. Furthermore, algal growth ( Selenastrum capricornutum) inhibition and Daphnia magna immobilization tests were performed according to standard procedures. The sea urchin bioassay results showed that raw wastewater toxicity is enhanced by the physico-chemical treatment and is decreased by the biological treatment for both samples. Confirming the data from a previous study, a concentration-related toxicity was exerted by effluent or sludge samples according to the following rank: DS > CF > R > B. On the other hand, offspring quality was not affected by sperm exposure to any wastewater or to sludge samples. However, D. magna was immobilized for both samples by the ranking of R > CF > B while algal growth inhibition was caused in a different order for the samples. Overall results suggest submitting the CF effluent to biological treatment which absorbs the toxic content of the wastewater in leather tanning industry; however, the sludge containing toxic components must be handled very carefully.

Physicochemical substance properties as indicators for unreliable exposure in microplate-based bioassays

In the last years many efforts were made to transform standardized algal test protocols into low-cost microplate assays. While advantages were pointed out frequently, limitations are not systematically addressed, thus hindering a widespread utilisation. In this study a group of organic substances with a wide distribution of volatility (logKAW from -6.53 to -2.13) and lipophilicity (logKOW from 1.26 to 4.92) was investigated with respect to the influence of these physicochemical properties on their algal toxicity in different assays. Therefore the EC50 values were determined with a microplate assay based on ISO 8692 protocol and the results were compared with those of an established algal growth inhibition test conducted in air tight glass vessels. Using the ratio of the EC50 values, a clear connection between biological response and volatility as well as lipophilicity of test substances could be detected. Chemicals with a logKOW higher than 3 or a Henry coefficient logKAW higher than -4 were identified as less effective in the microplate assay than in the comparative assay. The loss in nominal concentration due to physicochemical properties could be shown to contribute to this using HPLC analysis. Consequently, when using microplate assay's one should be aware that lipophilic and volatile chemicals might be underestimated in their toxicity, which could be indicated from evaluating related physicochemical properties modelled from structural information prior to an experimental investigation.

Growth Stimulation and Inhibition Effects of 4-Hydroxybenzoic Acid and Some Related Compounds on the Freshwater Green Alga Pseudokirchneriella subcapitata

4-hydroxybenzoic acid (4-HBA) exhibited low algal toxicity with the 72-h median inhibition concentration (IC50) of 9.9 mmol/L in the standard growth inhibition test using the freshwater green alga Pseudokirchneriella subcapitata. In contrast, it stimulated the algal growth at lower concentrations ranging from 0.1 to 1.0 mmol/L. Comparative studies with benzoic acid and 2- and 3-hydroxybenzoic acids (2-HBA and 3-HBA) indicated that 2-HBA was the most toxic, giving a 72-h IC50 of 0.172 mmol/L, and 4-HBA was the least toxic and that only 4-HBA had the pronounced growth stimulation activity. In a semicontinuous exposure to 4-HBA (0.15 and 0.3 mmol/L), algae maintained increased cell growth compared with controls during up to 10 times consecutive batch cultures, without any indication of adaptive responses to the growth enhancing effect of 4-HBA. Return to the clean standard medium of the exposed cells resulted in the quick recovery from the stimulant effect. Furthermore, 4-HBA (0.3 mmol/L) was found to diminish the toxicity of 2-HBA (<1.0 mmol/L). The presence of such compounds in the complex mixture might interact with other toxicants to alter the whole toxicity when assessed by the algal growth inhibition test. The effects of 4-HBA on P. subcapitata growth observed in the present study are not expected for planktonic algae in the aquatic environments, because known environmental concentrations are far below the effective concentration range.

Inhibition of calcium and magnesium-containing scale by a new antiscalant polymer in laboratory tests and a field trial

Membrane scaling is caused by the deposition of unsoluble salts as feedwater converted to brine, affectting the operation of seawater RO plants. Membrane scaling at the Dalian RO desalination plant was analyzed using Dalian seawater. Based on the characteristics of Dalian seawater, a new environmentally friendly, biodegradable antiscalant PAP-1 was synthesized in the laboratory. The performance of PAP-1 to inhibit calcium and magnesium-containing scale was studied both in the laboratory and at the Dalian seawater RO desalination plant. It was found that PAP-1 is effective in inhibiting scale formation. The experimental results in the laboratory indicate that the proper dose is 3–5 ppm when the concentration of calcium is 1000 mg/L and of magnesium is 2800 mg/L. The pH of the feed water was in the range of 3–9. The efficiency of calcium scale inhibition of PAP-1 is higher than 97.24% and magnesium scale inhibition is higher than 98.63% with 9 ppm PAP-1. The test performed at the Dalian seawater RO plant confirmed the scale inhibition ability of PAP-1 compared with the Nalco PermaTreat 191 scale inhibitor. The results from both antiscalants were similar. The average scale formation rates of calcium and magnesium of 14 samples was 2.822 g/h and 5.562 g/h separately with the addition of PAP-1 in each membrane unit; PAP-1 was more effective than Nalco PermaTreat 191. At the same time, the algae growth inhibition test showed that the addition of PAP-1 does not affect the growth of algae, which is an indication that PAP-1 has no toxicity and is a suitable and environmentally friendly antiscalant.

Effects of antibacterial agents, levofloxacin and clarithromycin, on aquatic organisms

Contamination of surface waters by pharmaceutical chemicals is an emerging environmental problem. This study evaluated the toxic effects of the antibacterial agents levofloxacin (LVFX) and clarithromycin (CAM), which are widely used in Japan, on aquatic organisms. Ecotoxicity tests using a bacterium, alga and crustacean were conducted. Microtox test using a marine fluorescent bacterium showed that LVFX and CAM have no acute toxicity to the bacterium. From the results of the Daphnia immobilisation test, LVFX and CAM did not show acute toxicity to the crustacean. Meanwhile, an algal growth inhibition test revealed that LVFX and CAM have high toxicity to the microalga. The phytotoxicity of CAM was about 100-fold higher than that of LVFX from a comparison of EC50 (median effective concentration) value. From the Daphnia reproduction test, LVFX and CAM also showed chronic toxicity to the crustacean. Concentrations of LVFX and CAM in the aquatic environment were compared with PNEC (predicted no effect concentration) to evaluate the ecological risk. As a result, the ecological risk of LVFX is considered to be low, but that of CAM is higher, suggesting that CAM discharged into an aquatic environment after therapeutic use may affect organisms in the aquatic environment.

Phytotoxicity of grey wastewater evaluated by toxicity tests

An algal growth inhibition test and a short-term acute assay for willows were used in order to evaluate the phytotoxicity of grey wastewater from different sources. Bathroom grey wastewaters were toxic towards algae (EC10 = 36 – 375 mL/L), whereas kitchen and laundry wastewaters were found to be toxic to both organisms (EC10 = 55 – 198 mL/L). The investigation showed that untreated grey wastewater could pose an environmental hazard if discharged into small-size receiving waters. Discharge to willow evaporation beds is problematic and toxic effects may be encountered, but, compared to direct discharge to surface water, the risks of toxic effects are much less pronounced.

The Prestige oil spill: A laboratory study about the toxicity of the water-soluble fraction of the fuel oil

The Prestige oil spill caused severe effects on the coastal fauna and flora due to direct contact of organisms with the fuel oil. However, the water soluble fraction (WSF) of the fuel oil can also provoke deleterious effects in the long term and even in regions not directly affected by the spill. Our objective was to determine the toxicity of the WSF using a battery of laboratory toxicity tests. To obtain a WSF in the laboratory, a sample of the spilled fuel was mixed with adequate medium, sonicated, agitated and filtered. No cytotoxic effects were detected in RTG-2 cells exposed to the WSF. In an algae growth inhibition test (OECD test guideline 201) the WSF did not affect the growth of Chlorella vulgaris. Furthermore, acute and reproductive toxicity tests (OECD test guideline 202) carried out using Daphnia magna did not indicate any deleterious effect of the WSF. In a bioassay designed in our laboratory, D. magna were fed with algae previously exposed to the fuel, but no toxic effects were detected. However, the WSF was able to induce a dose-dependent increase of ethoxyresorufin-O-deethylase activity in RTG-2 cells, indicating the presence of chemicals that could cause sub-lethal effects to organisms. After chemical analyses it was established that the final total quantity of polyaromatic hydrocarbons dissolved in medium was approximately 70 ng/ml. These low concentrations explain the observed lack of toxicity.

A novel method for evaluating bioavailability of polycyclic aromatic hydrocarbons in sediments of an urban stream

Hydrophobic organic pollutants in urban wet weather discharges can accumulate in the sediments of receiving waters and may have adverse effects on the ecological system, especially on benthic organisms. Here, a novel method is developed for evaluating the bioavailability of such hydrophobic organic pollutants by considering the digestive guts in deposit-feeding polychaetes. We compared the amount of polycyclic aromatic hydrocarbons (PAHs) extracted by an organic solvent and by sodium dodecyl sulfate (SDS) solution (as a hypothetical digestive gut fluid of polychaetes) and interpreted the ratio of the two values as bioavailability. The sediment extracts were applied to bacterial acute toxicity tests and algal growth inhibition tests. Sediment samples were collected from an urban stream system receiving wet weather discharges. The bioavailability of the total amount of 12 PAHs in the sediments was in the range 14-38% based on the results from the GC/MS determination of the two different extracts. Lower molecular PAHs showed higher bioavailability compared to the higher molecular ones. The sediment extracts were shown to be toxic towards both algae and bacteria. The SDS extracts showed similar or higher toxicity in the two biotests compared to the organic solvent extracts in spite of their lower PAHs content.

TBT-contaminated Sediments: Treatment in a Pilot Scale (9 pp)

Dedicated to Prof. Dr. Ulrich Forstner on his 65th birthday Sediments in harbours and nearby shipyards demonstrate widespread contamination with tributyltin (TBT). Therefore, reuse and relocation of dredged material from these locations are prohibited. Even if the International Marine Organization (IMO) convention concerning TBT-based paints is ratified (Champ, 2003) the TBT problem in sediments will continue to remain for many years due to the persistence of TBT. An electrochemical process has been developed to treat polluted sediments. Dredged materials with high and low TBT-contents were studied on a technical and a pilot scale. The treatment process was assessed by chemical analysis and a biotest battery. Additionally, an economic analysis was performed to check the economic feasibility of the process to treat dredged material from two different locations at different operating conditions. Furthermore an up-scaling estimation was performed to evaluate treatment costs at a larger scale, i.e. for a plant having a capacity of 720 000 t/a. Butyltin species and polycyclic aromatic hydrocarbons (PAH) were decomposed due to electrochemically-induced oxidation, while the treatment did not alter heavy metal and PCB concentrations. The bacteria luminescence test indicated a reduced toxicity after the electrochemical treatment, while the algae growth inhibition test and bacteria contact test did not confirm these results. Based on a small consumer price of €0.12/kWh, treating the high-contaminated sediment in the pilot plant would cost €21/m3 and €31/m3 for the low contaminated sediment, respectively. Assuming an industrial consumer price of €0.06/kWh for electricity in an up-scaled process with a capacity of 720 000t/a, the total treatment costs for the low contaminated sediment would be €13/m3. The results of treating dredged material from Bremerhaven and the fine-grained fraction from the METHA plant show that the effectiveness of the process performance is more related to the binding form and sediment composition than to the initial concentration of TBT. The electrochemically treated material complies with chemical criteria for relocation of dredged material, but post-treatment, e.g. washing and/or reduction of remaining oxidants with Fe-II-salts, is needed to fulfil ecotoxicological criteria for relocation. Economic investigations have shown that the electrochemical treatment might be a technical option to treat TBT contaminated, dredged material. However, the technique is not fully developed and cannot remove all chemicals of concern, e.g. heavy metals. The decision as to whether this technique can be applied is site-specific and should be taken based on the case-by-case approach. Since biotests integrate the effects of all contaminants present in a sample, process-accompanying evaluation procedures need additional assessment methods such as TIE (toxicity identification evaluation) as a second tier following ecotoxicological tests, in which the reasons for the effects are identified. For reasons of sustainability, a much more effect-oriented and long-term cost effective approach should be applied in future to avoid the release of harmful substances into the environment. Life cycle assessment should be carried out to identify and quantify impacts of sediment treatment processes in order to take into account both the distant effects of local actions and local effects of distant actions.

Bioassay selection, experimental design and quality control/assurance for use in effluent assessment and control.

In the UK Direct Toxicity Assessment Programme, carried out in 1998-2000, a series of internationally recognised short-term toxicity test methods for algae, invertebrates and fishes, and rapid methods (ECLOX and Microtox) were used extensively. Abbreviated versions of conventional tests (algal growth inhibition tests, Daphnia magna immobilisation test and the oyster embryo-larval development test) were valuable for toxicity screening of effluent discharges and the identification of causes and sources of toxicity. Rapid methods based on chemiluminescence and bioluminescence were not generally useful in this programme, but may have a role where the rapid test has been shown to be an acceptable surrogate for a standardised test method. A range of quality assurance and control measures were identified. Requirements for quality control/assurance are most stringent when deriving data for characterising the toxic hazards of effluents and monitoring compliance against a toxicity reduction target. Lower quality control/assurance requirements can be applied to discharge screening and the identification of causes and sources of toxicity.

下水処理水中に含まれるレボフロキサシン，クラリスロマイシンの分析と藻類生長への影響

In recent years, antibiotics resident in sewage and in the water environment have become an emerging public concern in many developed countries. However, limited knowledge is available on the occurrence of antibiotics in sewage and discharge from wastewater treatment plants (WWTPs) in Japan. Moreover, little is known on the significance of their occurrence in the water environment from the viewpoints of biological adverse effects. The objectives of this research were, therefore, to determine the occurrence of selected antibiotics, namely levofloxacin (LVFX) and clarithromycin (CAM). That are commonly used in Japan, in discharge from WWTPs and then to evaluate their possible effects on algal growth. Therefore, we developed a novel analysis method for LVFX and CAM in wastewater by LC/MS/MS whose detection limits and recovery ratios are 2-3ng·l-1 and 53-87%, respectively. We also conducted algal growth inhibition tests using Pseudokirchneriella subcapitata, and results showed that the EC50s of LVFX and CAM are 1200μg·l-1 and 11μg·l-1, LOECs are 630μg·l-1 and 6.3μg·l-1, and NOECs are 310μg·l-1 and 3.1μg·l-1, respectively, LVFX and CAM concentrations in secondary effluent of five WWTPs that use the activated sludge process ranged from 152-323ng·l-1 and 303-567ng·l-1, respectively, which indicates that the PEC/PNEC ratio of LVFX is less than one but that of CAM exceeds two at the maximum secondary effluents if a safety factor of ten is considered. This suggests a possibility of algal growth inhibition due to CAM in WWTP discharge in the case of insufficient dilution of the receiving waters.

Algal growth inhibition test: does shading of coloured substances really matter?

Coloured substances are often assumed to behave differently in the algal growth inhibition test due to shading of the algae. We have investigated EU-notifications of new substances and found no significant differences in algal test results between colours and non-colours. A modified method to differentiate between shading and toxicity is not satisfactory, because the method used to determine the shading effect is incorrect. Due to the shaking of the test flasks in the algal test intermittent light conditions are achieved which lead to a higher growth rate than expected from proposed tests with constant reduced light conditions. This will result in overestimation of the shading effect and thus to underestimation of chemical toxicity. Furthermore, we showed in experiments with the reference substance potassium dichromate that reduced light can reduce the measurable toxicity of chemicals. Comparing our results with approaches proposed in the literature, a simple approach to reduce effects of undesired shading when testing coloured substances is to achieve light saturation for the algae by increasing illumination at least to the upper limit given in test guidelines (120 μE s −1 m −2) and, optionally, to reduce the light path by choosing smaller test volumes.

The F684/F735 chlorophyll fluorescence ratio: a potential tool for rapid detection and determination of herbicide phytotoxicity in algae

The use of herbicides constitutes the principal method of weed control but the introduction of these compounds into the aquatic environment (primarily through runoff) may have severe consequences for non-target plants. In this study, we describe a sensitive and inexpensive method for detection of photosynthesis-inhibiting herbicides, based on chlorophyll (Chl) fluorescence emission. Algae exhibited a Chl fluorescence signature with two maxima around 684 and 735 nm, correlated with the total Chl content of the algal suspension. The ratio of these two maxima (i.e. F684/F735) can be used as an indicator of stress in the photosynthetic apparatus, and thus represents a very simple method for in vivo evaluation of the health status of algae. Determination of the F684/F735 fluorescence ratio revealed the presence and phytotoxicity of atrazine, metribuzin, terbuthylazine, diuron, DCPMU, DCPU and paraquat. The toxic effect of these pollutants was estimated by monitoring the increase in the F684/F735 value, which reflects photosystem II and photosystem I photochemistry. We observed a drastic increase in the magnitude of this ratio, correlating quantitatively with herbicide concentration and corresponding to a decline in algal photosynthetic activity. For the tested herbicides affecting photosynthetic electron transport, the magnitude of the effect was as follows: diuron=DCPMU>metribuzin>atrazine>terbuthylazine>paraquat>DCPU. The F684/F735 Chl fluorescence ratio thus gives toxicity responses which compare favourably with tests such as the algal growth inhibition test, and could therefore be used to detect the presence and phytotoxicity of herbicides in aquatic environments.

藻類増殖阻害試験の簡易化と埋立処分場浸出水の有害性評価への適用

To evaluate the total hazard existing in effluent water, three different bioassays, namely, the in vitro basal cytotoxicity test using a human cell line, bioluminescence inhibition test using luminescent bacteria and algal growth inhibition test, were applied to landfill leachate samples. To simplify the algal growth inhibition test, a new method using 96-well microplates and a microplate fluorometer was developed. Based on the results obtained using the bioassays, scoring of the total hazard existing in the samples was attempted. While the scores obtained with the basal cytotoxicity and bioluminescence inhibition tests showed similar patterns, those obtained with the algal growth inhibition test showed a different pattern. Nevertheless, the total scores of the samples indicate that the total hazard existing in raw leachate was significantly reduced by a wastewater treatment process. Thus, this type of scoring of the total hazard existing in landfill leachate may be useful for priority setting for the risk management of landfill sites.

Acute toxicity of fatty acids to the freshwater green alga Selenastrum capricornutum.

The acute toxicity of fatty acids (C14 to C18) commonly found in wood was determined by the standard algal growth inhibition test using the freshwater green alga Selenastrum capricornutum. Toxicity, quantified as IC50 values, varied depending on the number of total carbons and double bonds. Of the tested acids, oleic (cis-9-octadecenoic) acid showed the highest toxicity (72-h IC50 = 0.47 mg/L) to the alga, and triolein, a triglyceride of oleic acid, showed no apparent toxicity. Further examination of a series of C18:1 acids with a double bond at the 6, 11, or 12 position revealed that both double-bond position and cis or trans configuration affected toxicity. The 72-h IC50 data for these fatty acids and related compounds seemed to correlate well with the melting point (mp), showing two separate linear relationships: at mp < 35 degrees C toxicity increased with increasing melting point, and at mp > 40 degrees C toxicity decreased with melting point.

Toxicity assessment of wood preservatives

The article describes the influence of wood preservatives on water bioceonosis. The “battery” of toxicology tests was used to cover luminescent bacteria test, algae growth inhibition test, crustacean and fish lethal tests. The test samples used were pure wood preservative and water after the wood preservative leaching from wood sample. It was observed that the wood preservative is leaching from the wood sample in 10% within 1 month of exposition. Achieved results indicate the necessity of toxic assessment of wood preservative after wood element protection.

Phytotoxicity of coloured substances: is Lemna duckweed an alternative to the algal growth inhibition test?

Coloured substances cause problems when interpreting algal tests, because effects due to light absorption can interact with potential toxicity. The Lemna Duckweed growth inhibition test can complement the algal test, on condition that the test is performed on a black, not reflecting surface. On white surfaces, test solution colour can strongly impact Lemna growth. For example, average control sample growth rate of is much higher on white surfaces (0.362 d(-1)) than on black surfaces (0.284 d(-1)). We found that 10 mg l(-1) of the dyestuff "Brilliant Blue R spezial" inhibited average Lemna growth rate about 22% on white surfaces but did not inhibit growth on black surfaces. The reason for this difference stems from the difference in amount of light reflected from below the test beakers. With Brilliant Blue on white surfaces, the test solution colour reduces utilizable light and causes a deterioration of light conditions, whereas on a black surfaces, reflected light is absent a priori, and thus no inhibiting effect was measured. Of particular importance is the choice of test parameter. With Brilliant Blue, a LOEC for average growth rate, based on frond numbers, of 320 mg l(-1) was determined. However, when average growth rate was calculated using dry weights of the plants, the LOEC decreased clearly to 1.0 mg l(-1). In this study, the Lemna test was much more sensitive than the algal test. We recommend Lemna tests be used in addition to algal tests, because doing so may significantly improve the assessment of phytotoxicity of chemicals and sewage.

Combination Effect of Light and Toxicity in Algal Tests

The sensitivity of Scenedesmus subspicatus against potassium dichromate is positively correlated to the photon flux density during the algal growth inhibition test. Low photon flux densities led to significantly reduced maximum effects and higher EC50 levels. To improve the testing of colored substances, we distinguished between the toxic effect (chemical part, represented by potassium dichromate) and the shading effect (physical part, simulated by reduced light intensities during the test) of a hypothetical light absorbing substance. The contribution of these single effects to the total inhibition varied greatly. At high concentrations of potassium dichromate (1.6 and 3.2 mg L(-1)) the physical part never exceeded 25% of the total inhibition, not even at strongest light reduction, while at low concentrations (0.2 and 0.4 mg L(-1)) the physical effect became more prominent when halving the amount of available light. Further, the combination effect of the chemical and the physical effect could be calculated well only by using the concept of independent action. Thus, if chemical and physical effects are measured in combination, as is the case in tests with dye-stuffs, the current test protocol for the algal growth inhibition test may lead to incorrect estimations of the toxic potential.

Screening of physical–chemical methods for removal of organic material, nitrogen and toxicity from low strength landfill leachates

Physical–chemical methods have been suggested for the treatment of low strength municipal landfill leachates. Therefore, applicability of nanofiltration and air stripping were screened in laboratory-scale for the removal of organic matter, ammonia, and toxicity from low strength leachates (NH 4-N 74–220 mg/l, chemical oxygen demand (COD) 190–920 mg O 2/l, EC 50=2–17% for Raphidocelis subcapitata). Ozonation was studied as well, but with the emphasis on enhancing biodegradability of leachates. Nanofiltration (25 °C) removed 52–66% of COD and 27–50% of ammonia, the latter indicating that ammonia may in part have been present as ammonium salt complexes. Biological pretreatment enhanced the overall COD removal. Air stripping (24 h at pH 11) resulted in 89% and 64% ammonia removal at 20 and 6 °C, respectively, the stripping rate remaining below 10 mg N/l h. COD removals of 4–21% were obtained in stripping. Ozonation (20 °C) increased the concentration of rapidly biodegradable COD (RBCOD), but the proportion of RBCOD of total COD was still below 20%, indicating poor biological treatability. The effect of the different treatments on leachate toxicity was assessed with the Daphnia acute toxicity test ( Daphnia magna) and algal growth inhibition test ( Raphidocelis subcapitata). None of the methods was effective in toxicity removal. By way of comparison, treatment in a full-scale biological plant decreased leachate toxicity to half of the initial value. Although leachate toxicity significantly correlated with COD and ammonia in untreated and treated leachate, in some stripping and ozonation experiments toxicity was increased in spite of COD and ammonia removals.