Inhibitory Effect of Heavy Metal Ion/s on Algae

The inhibitory effect of heavy metal Ion/s on algae

Aim: to establish the concentration of heavy metal/s that cause the EC50 effect on X/s (algae species), using a typical content of metals in wastewater (as seen in the table below for instance) as a reference to prepare different dilutions of aqueous solutions.

Typical content of metals in municipal wastewater with minor contribution of industrial wastewater "" (Henze et al.2001)

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General aspects about inhibitory experiment

  1. For accurate results, a control+ minimum of five concentrations of toxicant examined is required.
  2. Monitoring the change occurs on algae within 4-7 days.
  3. The static test is used with algae.
  4. In the control, the mean cell density must be "" cell/mL or more after 96h, with less than 20% variation between replicates.
  5. Cell density measurements used with algae is cell counts per mL, biomass, chlorophyll, absorption.

Chemicals and apparatuses needed

– Media stocks depending on the algae that will be used.

– Sufficient numbers of log-phase-growth organisms.

– Environmental chamber, incubator, or equivalent facility.

– Mechanical shaker providing orbital motion with 100 cycles/minute.

– Light meter ranging from 0-200 μE/m2/s (0-1000 ft-c).

– Water purification system.

– Analytical balance, with 0.00001 g weighing accuracy.

– Volumetric flasks and graduated cylinders class A, 10-1000 mL, borosilicate glass, for culture work and preparation of test solutions.

– Volumetric pipets with tips class A, 1-100 mL.

– Wash bottles for rinsing small glassware, instrument electrodes, and probes.

– A 250 mL borosilicate, Erlenmeyer flasks, with foam plugs or Shumadzu closures.

– 1-4 L borosilicate, Erlenmeyer flasks.

– Thermometers, glass or electronic, laboratory grade.

– Meters, pH and specific conductivity for routine physical and chemical measurements.

-Tissue grinder for chlorophyll extraction.

– UV-VIS spectrophotometer.

– Cuvettes for spectrophotometer 1-5 cm light path.

– Electronic particle counter or Coulter Counter.

– Counting chamber Sedgwick-Rafter, Palmer-Maloney, or hemocytometer.

– Centrifuge.

– Centrifuge tubes with screw-cap.

Preparation of Inoculum

The inoculum is prepared no more than 2 to 3 h prior to the beginning of the test, using Algae sp. harvested from a four- to-seven-day stock culture. Each millilitre of inoculum must contain enough

cells to provide an initial cell density of approximately 10,000 cells/mL (± 10%) in the test flasks. In the case of using 250 mL flasks, each containing 100 mL of the test solution, the inoculum must contain 1,000,000 cells/mL.

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Prepare the inoculum as follows:

1. Centrifuge 15 mL of stock culture at 1000 x g for 5 min. This volume will provide a 50% excess in the number of cells.

2. Decant the supernatant and resuspend the cells in 10 mL of control medium.

3. Repeat the centrifugation and decantation step, and resuspend the cells in 10 mL control medium.

4. Mix well and determine the cell density in the algal concentrate. Some cells will be lost in the concentration process.

5. Determine the density of cells (cells/mL) in the stock culture.

6. Calculate the required volume of stock culture as follows:

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7. Dilute the cell concentrate as needed to obtain a cell density of 1,000,000 cells/mL, and check the cell density in the final inoculum.

8. The volume of the algal inoculum should be considered in calculating the dilution of toxicant in the test flasks.

Start of the test

1-Label the test chambers with a marking pen and use the color-coded tape to identify each treatment and replicate. A minimum of five effluent concentrations and a control are used for each effluent test with replicate.

2-Randomize the position of the test flasks at the beginning of the test.

3- The test begins when the algae are added to the test flasks. Mix the inoculum well, and add 1 mL to the test solution in each randomly arranged flask. Make a final check of the cell density in three of the test solutions at time “zero” (within 2 h of the inoculation).

4- Alkalinity, hardness, and conductivity are measured at the beginning of the test in the medium,

and toxicant concentrations and control before they are dispensed to the test chambers and the data recorded on the data sheet as shown below.

Parameter

Control

Concentration

Remarks

pH

Alkalinity

Hardness

Conductivity

Temperature

Test Date

Location:

5- Test flasks are incubated under continuous illumination at 86 ± 8.6 μE/m2/s (400 ± 40 ft-c), at 25 ± 1EC, and should be shaken continuously at 100 cpm on a mechanical shaker or twice daily by hand. Flask positions in the incubator should be randomly rotated each day to minimize possible spatial differences in illumination and temperature on growth rate. If it can be verified that test specifications are met at all positions, this need not be done.

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6- Temperature and pH are measured at the end of each 24-h exposure period in at least one test flask at each concentration and in the control.

Test duration

The test is terminated 96 h after initiation. The algal growth in each flask is measured by one of the following methods: (a) cell counts, (b) chlorophyll content, or (c) turbidity (light absorbance).

1- Cell counts

1-1 Automatic Particle Counters

Several types of automatic electronic and optical particle counters are available for use in the rapid determination of cell density (cells/mL) and mean cell volume (MCV) in μm3/cell. When the Coulter Counter is used, an aliquot (usually 1 mL) of the test culture is diluted 10X to 20X with a l% sodium chloride electrolyte solution, such as ISOTON®, to facilitate counting. The resulting dilution is counted using an aperture tube with a 100-μm diameter aperture. Each cell (particle) passing through the aperture causes a voltage drop proportional to its volume. Depending on the model, the instrument stores the information on the number of particles and the volume of each, and calculates the mean cell volume. The following procedure is used:

1. Mix the algal culture in the flask thoroughly by swirling the contents of the flask approximately six times in a clockwise direction, and then six times in the reverse direction; repeat the two-step process at least once.

2. At the end of the mixing process, stop the motion of the liquid in the flask with a strong brief reverse mixing action, and quickly remove 1 mL of cell culture from the flask with a sterile pipet.

3. Place the aliquot in a counting beaker, and add 9 mL (or 19 mL) of electrolyte solution (such as

Coulter ISOTON®).

4. Determine the cell density.

1-2 Manual microscope counting method

Cell counts may be determined using a Sedgwick-Rafter, Palmer-Maloney, hemocytometer, inverted microscope, or similar methods. This method has the advantage of allowing for the direct examination of the condition of the cells.

2- Chlorophyll Content

1. Adjust the “blank” reading of the fluorometer using the filtrate from an equivalent dilution of toxicant filtered through a 0.45 μm particle retention filter.

2. Mix the contents of the test culture flask by swirling successively in opposite directions (at least three times), and remove 1 mL of culture from the flask with a sterile pipet.

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3. Place the aliquot in a small disposable vial and record the fluorescence as soon as the reading stabilizes. (Do not allow the sample to stand in the instrument more than 1 min).

4. Discard the sample.

3-Turbidity (Absorbance)

A rapid technique for growth measurement involves the use of a spectrophotometer to determine the turbidity, or absorbance, of the cultures at a wavelength of 750 nm. Because absorbance is a complex function of the volume, size, and pigmentation of the algae, it would be useful to construct a calibration curve to establish the relationship between absorbance and cell density.

The algal growth measurements are made as follows:

1. A blank is prepared as described for the fluorometric analysis.

2. The culture is thoroughly mixed as described above.

3. Sufficient sample is withdrawn from the test flask with a sterile pipet and transferred to a 1- to 5-cm cuvette.

4. The absorbance is read at 750 nm and divided by the light path length of the cuvette, to obtain an “absorbance-per-centimetre” value.

5. The 1-cm absorbance values are used in the same manner as the cell counts.

Summary of test conditions and test criteria

1. Test type: Static non-renewal

2. Temperature: 25 ± 1EC (recommended) Test temperatures must not deviate (i.e., maximum minus minimum temperature) by more than 3°C during the test

3. Light quality: “Cool white” fluorescent lighting

4. Light intensity: 86 ± 8.6 μE/m2/s (400 ± 40 ft-c or 4306 lux)

5. Photoperiod: Continuous illumination

6. Test chamber size: 125 mL or 250 mL

7. Test solution volume: 50 mL or 100 mL

8. Renewal of test solutions: None

9. Age of test organisms: 4 to 7 days

10. Initial cell density in test chambers: 10,000 cells/mL.

11. No. replicates chambers per concentration: 4

12. Shaking rate: 100 cpm continuous, or twice daily by hand

13. Aeration: None

14. Dilution water: Algal stock culture medium, enriched uncontaminated source of receiving or other natural water, synthetic water prepared using MILLIPORE MILLI-Q® or equivalent deionized water and reagent grade chemicals, or DMW.

NOTE: before conducting this test or any other experimental work, growth assays are required for the algae species will be used during our study.

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