Methodology for toxicity testing of effluent using algae, duckweed, and mungbean seed

College

Gokongwei College of Engineering

Department/Unit

Chemical Engineering

Document Type

Conference Proceeding

Source Title

ARRPET-DLSU 2nd National Workshop 2005

First Page

74

Last Page

75

Publication Date

10-28-2005

Abstract

The Asian Regional Research Programme on Environmental Technology Phase I - De La Salle University (ARRPET I - DLSU) is targeting the development of a sequential chemical-biological treatment of residual PCBs in water. The chemical process is carried out using the UV/ HO, system where the reaction of ultraviolet light (photolysis) with hydrogen peroxide (H202) generates hydroxyl radicals (-OH that are ultimately responsible in the oxidation of organic pollutants such as PCBs. The biological process involves a three-phase fluidized bed reactor utilizing the biodegrading capacity of the biofilm adsorbed on cement particles. The critical segment of this scheme is the transition from the outlet of the chemical treatment to the inlet of the biological treatment. The effluent from the first phase of the treatment may contain unknown chemical compounds produced as a result of the UV/HO, treatment. To examine the toxicity of the effluent, three types of toxicity tests will be employed: an algal, a duckweed, and mungbean seed germination test. For the duckweed toxicity test, the acquired duckweed starter culture is acclimatized to the test environment for at least 2 weeks before the start of a toxicity test. The duckweed is grown in a suitable container such as an aquarium or a stainless steel basin. To prepare the culture medium, the formulation recommended by the American Public Health Association is used; as prescribed, the medium does not require autoclaving. A water depth of at least 40 mm or more is recommended. A constant cool-white fluorescent light (2,150 to 4,300 lux at the water surface) is provided. Diluted (1/4 strength) culture solution is added weekly to make up for vaporization losses. Stock culture is transferred monthly to a freshly prepared nutrient solution. Prior to the definitive tests, both a screening test and a range-finding test are done as preliminary activities. For the screening test, a predetermined concentration (e.g., 100% effluent) is used to determine if a sample is inhibitory in comparison with the control water. If the test sample is inhibitory by more than 10%, further range-finding test is required. For the range-finding test, at least three concentrations, usually at ratios of 0.1 (e.g., 10%, 1%, and 0.1%), are examined for inhibitory effects relative to the control water. For the definitive test, five concentrations of effluent are used. Ideally, a series of concentrations are prepared in which the midpoint concentration produces an inhibition effect of approximately 50% and the highest and lowest inhibition concentrations produce approximately 90% and 10% inhibition. Three to four replicates of each test treatment and the controls (a negative control containing only duckweed nutrient medium and a positive control containing 20 mg/L of potassium chromate) are required. Glass petri dishes are used as test vessels. The same amount of nutrients should be added to all control and test samples. Twelve duckweed fronds are placed in each test vessel. Frond counts are done daily to determine any immediate toxic effects. Test duration is 96 hours. Sample toxicity is expressed as percent inhibition relative to the control. The concentration-inhibition relationship is reported as well as the confidence limit of test results. For the seed germination test using mungbean seed, distilled-deionized water is used as dilution water and control water. As with the duckweed test, both a screening test and a range-finding test precede the definitive test. All tests are performed at least in triplicate or quadruplicate. For each test, 100 × 15-mm petri dishes are used as the test vessels. A 10 mL test solution is pipetted into each test vessel containing 10 to 15 seeds. The seeds should not be in contact with each other or with the sides of the culture dishes. All data on root and shoot elongation of germinated seeds in each dish is recorded. For validation of the experimental run, the seed root in the controls should have reached at least 20 mm at the end of the test. A seed is counted as having germinated if the radicle reaches a length of 5 mm or longer. The root elongation is determined by measuring the length of the primary root from the transition point of the hypocotyl to the tip of the root. Abnormal appearances such as discoloration, stunted growth and chlorosis are reported. The sample toxicity is expressed in percent inhibition relative to the control. The concentration-inhibition relationship is reported as well as the confidence limit of test results. The static algal growth inhibition test is used for testing the toxicity of the effluent to algal species. In principle, exponentially growing cultures of selected algae are exposed to various dilutions of the effluent and the inhibition of growth relative to a control culture is determined over a fixed period of time. The recommended test algae species must be a fast-growing species that is convenient for culturing and testing. Several species are recommended, including Selenastrum capricornutum, Scenedesmus subspicatus, and Chlorella vulgaris. Chlorococcum humicola and Scenedesmus bijugatus will be used in this study, as both species are relatively fast growing. The range of effluent dilutions in which effects are likely to occur is determined on the basis of the results from range-finding tests, wherein at least five dilutions arranged in a geometric series will be selected. Afterwards, the range of test concentrations is narrowed in order to determine the concentration that reduces the algae growth parameter to 50% that of the control. This requires that two of the concentrations tested fall on each side of the concentration that inhibited the growth parameter to 50%. The lowest concentration tested should have no observed effect on the growth of the algae while the highest concentration should inhibit growth by at least half relative to the control, or preferably, stop growth completely. The algal bioassay definitive test begins by preparing test cultures containing the desired effluent dilutions and the desired quantity of algal inoculum. First, algal medium is used to dilute the effluent to the desired concentrations. An algal inoculum is taken from an exponentially growing algae culture and added to the test solution to obtain an initial cell density of the order of 10* cells/mL. Triplicate vessels for each concentration are prepared. Negative controls are included for each triplicate. The culture flasks are shaken and placed in a shelf. The flasks are shaken manually twice or thrice a day and are maintained at room temperature. Continuous cool-white fluorescent lighting at 4,304 lux ‡ 10% is recommended. The flasks are repositioned daily within the environmental chamber to minimize possible effect of spatial differences in illumination and temperature on growth. The algal cell density is estimated by relating the turbidity (absorbance) of an algal suspension with direct cell counts. The resulting calibration curve is used to correlate the spectrophotometric reading with the cell density. The turbidity is measured at a wavelength of 750 nm and divided by the light path length of the cuvette to obtain an "absorbance per centimeter value." The algae biomass is measured on a regular basis. The cell concentration in each flask (including the control) is determined at least at 24, 48, and 72 hours after the start of the test. Algal medium is used as a blank when using spectrophotometers. The pH is measured at the beginning of the test and after 72 hours. The pH should not normally deviate by more than one unit during the test.

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Disciplines

Environmental Sciences

Keywords

Toxicity testing; Polychlorinated biphenyls

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