Lettuce Seed Ecotoxicology

• Set up a properly designed environmental toxicology experiment using lettuce seeds as a model organism 

• Create a column graph to display the influence of NaCl concentration on root growth in lettuce seeds

• Use ANOVA and Tukey's post-hoc tests to analyze data from your lettuce seed environmental toxicology experiment

• Write the Lab Title on a new page on the right-hand side of your notebook. *Remember to include the Lab Date!*

• Write the Background, Aim and your Hypothesis for the lab

• Draw Summary Data Table 1  (See Data Collection )

• Add an entry for this lab in your Table of Contents

• Model Organism:  Lactuca sativa L. var. Buttercrunch Lettuce

• Experimental Questions: 

  • How well do lettuce seeds grow when exposed to a range of salt (NaCl) concentrations? 

    • At what salt (NaCl) concentration is root growth of lettuce seeds significantly different from the control? 

    • At what salt (NaCl) concentration(s) is root growth inhibited? 

    • Do lettuce seeds show a linear change in root length as the NaCl concentration increases?

    • Is there a concentration level after which growth is completely inhibited?

• Independent Variable: NaCl concentration

  • • Control Group: 0 mM NaCl      

    • Experimental groups: 200 mM NaCl, 100 mM NaCl, 75 mM NaCl,  50 mM NaCl, 25 mM NaCl

• Dependent Variable: Root length (mm)

I hypothesize that as the concentration of NaCl increases, root growth in lettuce seeds will  (choose) significantly increase in a linear way / significantly decrease in a linear way / will not significantly change in a consistent way.  I predict that root growth will be significantly impacted starting at  (choose) 25mM, 50mM, 75mM, 100mM, or 200 mM NaCl.

Environmental toxicology

(Modified from Environmental Inquiry, Cornell University and Penn State University 2009 )

 Toxicology is the study of the effects of chemicals on living things. We tend to think of some chemicals as toxic and others as nontoxic, but in fact, any chemical is toxic at high enough concentrations.

The food we eat, the water and beverages we drink, and the air we breathe all are made up of complex mixtures of chemicals. Many of these chemicals are beneficial to our health when we consume them in small amounts but would be harmful at larger doses. For example, vitamin D is an important part of the human diet, but it also is a highly toxic chemical. In tiny amounts it is good for you, but taking higher than the recommended doses can cause serious health problems including kidney stones, high blood pressure, deafness, and even death.

Environmental toxicology is the study of the effects of chemicals on plants, animals, and the ecosystems in which they live. Scientists use experiments called bioassays to determine the effects of chemicals on living things.

Bioassays & Lettuce Seeds as a model organism

(Modified from Environmental Inquiry, Cornell University and Penn State University 2009 )

A bioassay involves use of a biological organism to test for chemical toxicity. The idea behind these bioassays is that the test organism will react in a predictable way to various types of environmental contaminants. 

Lettuce seeds have been used as a model organism by scientists for a variety of environmental testing purposes to identify contaminated areas and design pollution clean-up strategies. Lettuce seed bioassays have proven to be an easy and inexpensive means of testing the toxicity of some types of contaminants of concern in water and sediments, including heavy metals and some pesticides and other organic toxicants.  Toxicity is determined by measuring growth of the radicles, or embryonic roots,  of seeds exposed to various concentrations of the chemical. Today your group will set up a standard lettuce seed bioassay to determine the toxicity of a series of salt (NaCl) solutions. Your group will perform the growth measurements next week prior to lab.

1. Create an NaCl Dilution Series with your benchmates

2. Prepare your Lettuce Seeds

1. Obtain a packet/dish of lettuce seeds. Carefully count out 110 lettuce seeds.

2. Obtain a small beaker and add a small amount of 10% bleach solution. Place your lettuce seeds in a 10% bleach solution for 15 minutes (Set a timer!). This kills fungal spores that can interfere with seed germination. 

*NOTE: While you are waiting, set up your petri dishes and filter papers. (See below)

3. After 15 minutes, place your seeds in a small screen in a funnel over the bleach rinse beaker. Take care not to lose any seeds! 

4. Rinse the seeds in the funnel using distilled water from a squirt bottle.  Rinse for 1-2 minutes to ensure the bleach is removed. Take care not to lose any seeds!. 

3. Prepare your Petri Dishes

1. Obtain 18 petri dishes for your bench group experiment. You have 6 NaCl treatments and you will have 3 replicate dishes for each NaCl treatment.

2. Label the dish bottoms (not the tops!) with the treatment name, replicate  (A, B, or C), and your group name. For example, “200 A TP&TK” ; “200 B TP&TK” ; “200 C TP&TK”

3. Place a filter paper or unbleached nontoxic paper towel in each petri dish. 

4. To each petri dish, add 2 ml of the appropriate test solution to fully wet the paper in the dish. 

4. Set up your Lettuce Seed Bioassay

1. Add 5 prepared and rinsed lettuce seeds to each dish. Space the 5 seeds evenly on the filter paper so that they do not touch each other or the sides of the dish.

2. Cover the dishes. Place the dishes in a plastic Ziploc bag and seal it to retain moisture. 

3. Incubate the seeds in the dark at constant room temperature (preferably 24.5 degrees C) for 5-7 days total.

Lettuce Seed Bioassay Data Collection (5-7 days after Set-up)

You will need to measure the growth of your plants 5-7 days after the experimental set up. Coordinate with your benchmates to divide up the work.  Use the data sheet provided to record:

• The date of your measurements

• The number of seeds out of 5 that have germinated (aka sprouted) in each dish

• The radicle length to the nearest mm for each sprout. The radicle is the embryonic root; Look carefully at the plants to make sure you are measuring just the root, not the shoot and leave as well. For example, in the picture below, you would measure just the part between the two dotted lines, not the seed case, shoot, and embryonic leaves to the left.

• Calculate the average root length of the seeds for each dish (Calculated from the 5 root length measurements in each dish).

• Compile all data with your benchmates and complete Summary Table 1.

Calculating Mean & SD in Excel

Use formulas in Excel to calculate Mean and Standard Deviation for root length in each NaCl treatment.

Column Graph

You will use Excel to create a column graph to display mean & SD  root length in each NaCl treatment.

See instructions and guidelines  for creating properly formatted column graphs and captions.

ANOVA

You will use an online calculator to perform ANOVA tests to examine significant differences among your NaCl concentrations. The ANOVA test will tell you  if any significant differences exist and (2) the Post-Hoc tests will tell you what specific pairs of treatments  significantly differ from each other.

See detailed instructions for the ANOVA test and for how to incorporate ANOVA results in your figures and results paragraphs