Lettuce Seed Bioassay

A bioassay is a test for pollution in which plants or organisms are used to assess the toxicity of a sample. It would be nearly impossible and costly to analyze a sample of unknown quality for every known toxic substance. If plants and/or organisms can grow and survive in a sample it is assumed to be non-toxic. In a sense, a bioassay tests for multiple contaminants at the same time, however if the test shows toxicity, the contaminate is not identified.

Below is a brief explanation of a lettuce seed bioassay. It is a simple, low-cost experiment that demonstrates how water contaminated with metals can be treated. Additionally, it shows how high concentrations of metals can suppress the growth of plants. Just as a bioassay test is preformed on wastewater treatment effluent, a lettuce seed bioassay can be preformed on suspected contaminated soil or water to see if it will affect the germination and growth of the seeds.

The test is rather simple to perform and it's up the individual to decide how elaborate they wish to get. In addition to showing contamination and then treatment in this experiment, different concentrations of metals can be used to find the lethal concentration at which 50% of the seeds do not germinate. If you want more information on the procedures, use your favorite Internet search engine and input "Lettuce Seed Bioassay."

In this demonstration, a copper sulfate solution will be used as the contaminant to simulate metals pollution. The polluted water will then be treated by water softener resin. By comparing a control to the treated and untreated water, the students will be able to determine if the water has been cleaned.

Materials needed:

  • Copper Sulfate
  • Two small containers to prepare the solution and treat it
  • Water softener resin - I was given some from our local water softer distributor
  • Plastic ziploc bags or petri dishes
  • Pipette
  • Butter Crunch lettuce seeds
  • Analytical Balance
  • Brown hand drying paper towels or any non-bleached paper that will not affect seed germination or growth.


Procedure:

1. Prepare the Ziploc Bags.  Copper Sulfate, Resin.  
Place two layers of brown hand drying paper cut to fit inside the bag. At least three bags will be needed for a control, treated, and untreated solution. Mark the bags respectively.

2. Mix the Copper Sulfate Solution.
Mix the copper sulfate solution by adding 1000mg to one liter of water. It's recommended using unsoftened tap water that has sat for a few hours to dissipate the chlorine. Use this same water for the control assay.

3. Clean the Softening Resin.
The water softener resin will have a light brown color that will dissolve into the water. This can be removed by cleaning. Place a heaping tablespoon of resin into a 100ml container. Add 40 to 50 mls of water. Swirl to mix and carefully pour out the water. Place the remaining resin onto some paper to absorb the last bit of water. Then place the resin back into the cup.

4. Treat the Copper Sulfate Solution.
Place 30 - 40 mls of the copper sulfate solution into the resin. (Make sure there is enough solution over the resin so a portion can be pipetted without resin.) Swirl gently for about 15 to 20 minutes allowing the resin to absorb the copper metal ions. Students will observe the blue color dissipate as the resin absorbs the copper metal cations.

5. Dose the Ziploc bags and Add Seeds.
Pipette 8mls of the control, untreated, and treated into the respective labeled ziploc bags. Then carefully add 10 butter crunch lettuce seeds to each bag. Let the assays sit in a rather darkened area for 5 days.

Photos below show the ziploc bag bioassay and a close up of germinated seeds from the 1000 mg/l copper sulfate solution. Notice the seeds germinated, but the roots didn't seem to grow with only a blackened tip on the end of the germinated shoot. (See the photo below under the "results" section to compare normal root growth and stunted growth.
Ziplock bio-assay. Closeup of seeds.

   

Results:
After 5 days, remove the assay from the darkened area. Take a sheet of graph paper and designate one line as a baseline. Remove all 10 spouts and any un-germinated seeds from the bag, then place the sprout-and-root division on the baseline. (If you look closely at the sprout you will be able to see a division between the root and the spout.) Also, place any un-germinated seeds on the baseline. By knowing the graph paper division spacing, it is easy to determine each root length. Results are recorded as root length and seed germination.

The results below show that 1000mg/l copper sulfate significantly affect root growth.

  

Treated seeds versus contaminated seeds.

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