Procedure:

1. Spread seeds on damp paper towel, cover with several paper towels, and store in a warm,
    dark place for 3 days until they sprout.

2. Make stock solutions of 0.1M CuSO4, 0.05M CuSO4 and 0.025M CuSO4. For 0.1 M, add
    15.96 g CuSO4 and fill with distilled water to the 1 Liter mark on the volumetric flask. Use
    the same procedure for 0.05 M by adding 7.98 g CuSO4 and for 0.025 M by adding 3.99 g
    and filling the 1 Liter volumetric flasks with the distilled water to the line.

3. Prepare 26 growth chambers by cutting off the top third of each plastic bottle. The top piece
    is then inverted,  placed on top of the chamber and taped to the base.  Heat a cork borer and
    use it to punch a 1 centimeter port hole in the bottle approximately 1 inch above the bottom of
    the bottle  The port hole will serve to remove water samples.

4. Add equal amounts of soil to each growth chamber. Be sure to consider control
    chambers using only regular potting soil (without plants) and autoclaved soil (without
    plants).  Seedlings should have grown into paper towel. Cut small squares containing 25 seedlings each and plant in the growth chambers,. Use 3 growth chambers for each of  the three types of seeds: lettuce, radish and mustard.  In the remaining bottles, set up 9 chambers with regular soil, 6 with autoclaved soil and 2 with paper towel (no soil). This will make a total of 26 chambers.

5. Allow the seedlings to grow in soil for three days. The first day, add 50 mL distilled water
    to all of the growth chambers. The second day, water one set of bottles (1 mustard plant, 1
    radish, 1 lettuce, 2 autoclaved, 2 plain soil ) with 50 mL of .1M CuSO4, the second set with
    50 mL of 0.05M CuSO4 and the third set with 50 mL of 0.025M CuSO4.  As an additional
    control, water 3 bottles containing regular soil and two bottles with paper towel only with distilled
    water each time the other chambers are watered.

 
 Schematic of set up.
6. The third day, water one set of bottles (1 mustard plant, 1 radish, 1 lettuce, 2 autoclaved,
    2 plain soil ) with 50 mL of 0.1M CuSO4, the second set with 50 mL of 0.05M CuSO4 and
    the third set with 50 mL of 0.025M CuSO4.  Also, water the 3 bottles containing plain soil and two bottles with paper towel
    and no soil with distilled water as  controls. Repeat the overall watering process for a total of four times or 200 mL of
    solution.

7. On the fourth day, remove a small sample of the filtrate (water which has drained through the
    soil), dilute to 1000 mL and test for copper using the test kit. Compare with  the quantity of copper found in the stock
    solutions . Stock solutions will need to be similarly diluted.  All solutions are diluted to permit testing in the range of the
    copper test kit.

8. The results obtained from the copper test are in mg/L or parts per million (ppm). The copper test kit utilized is a color
   comparison test; therefore results may vary.

   To convert the Molar solutions used to water the plants, use the following:
 
    Example:  For 0.1M CuSO4 has a molecular weight of 159.6 g/m:

     0.1M CuSO4 *  63 g Cu (CuSO4) *  1000 mg  = 6300 mg/L or 6300 ppm
          Liter                mole                        gram

9. To determine the amount of copper in the plants, remove the plants from the soil and dehydrate them in a microwave for 5
    minutes. CAUTION: Watch them as they dehydrate because the plants may combust.  Remove the plants, mass them, and
    record.

10. Place each dried plant sample  in a 5 mL of a 1M solution of HCl.  Grind the plants in the solution using the mortar and
    pestle. CAUTION: Be careful not to slosh HCl while grinding the plant material. Let it set overnight in a glass container.

11. The next day take 1 mL of the HCl/plant solution to 100 mL distilled water.  Use the copper test kit on the sample.

12. Record and graph results.
Graph 1: Filtrate copper concentrations:

 Graph of molar concentrations vs copper in ppm.
 
Graph 2: Relative accumulation of copper in biomass:

copper concentration in plant biomass.

Conclusions

             The initial observation of the results was that there was higher copper concentration in the filtrate than was originally administered to the plants.  It does appear that the concentration of copper administered made a difference in the results. Because lettuce had the least amount of copper in its filtrate, relative to initial concentration, it was assumed that lettuce was the most efficient bioaccumulator. However, relative to the other plants, the mustard accumulated the most copper in its biomass .
            Strengths found in this inquiry activity were: the extensive research experiences; development of problem-solving strategies; development of a method for germinating seeds on  paper towel which creates a convenient growth "mat" making plant harvesting easy; developing a method for testing copper in the plants; and a useful, multi-purpose plant growth apparatus. Weaknesses include: a lack of plant replicates; quantification difficulties with comparing molar units to ppm; the spectrophotometer wave length for copper sulfate was an unknown at the time of testing; and there was no publicized technique for analyzing copper in the plants. Consequently, a technique to determine copper in plant tissue was developed too late; therefore, there were no established controls. It is suggested that these weaknesses be remediated prior to repeating this inquiry experience.
            While, there was no clear explanation for the results, two possible factors may have contributed to the data.  First, solutions may have evaporated, increasing copper concentration in the filtrate. Second, copper is pH sensitive. Plant roots lower the pH of a soil system. Therefore, differing concentrations of copper might  precipitate in the soil and not be present in the filtrate.
 

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