Toxicology

Toxicology

BACKGROUND

We handle many materials daily that are toxic. We are often unaware of the degree to which they are toxic. For a variety of reasons, different animals respond differently to the same toxin. Some animals may be very sensitive to a toxin, whereas others are relatively resistant to its effects. Because species of animals vary, it is important to understand that what is toxic to brine shrimp may not necessarily be toxic to other kinds of animals to the same extent.

Many household items that we deal with on a regular basis are toxic materials, but we don’t usually think of them as being toxic. It can be instructive to examine several such materials to determine their toxicity.

The commonly used term to describe acute ingestion toxicity is LD50. LD means Lethal Dose (deadly amount) and the subscript 50 means that the dose was acutely lethal to 50% of the animals to whom the chemical was administered under controlled laboratory conditions. The test animals (usually mice or rats) are given specific amounts of the chemical in either one oral dose or by a single injection and are then observed for 14 days.

Since LD50 values are measured from zero up, the lower the LD50 the more acutely toxic the chemical. Therefore, a chemical with an oral LD50 of 500 would be much less toxic than a chemical with an LD50 of 5. LD50 values are expressed as milligrams per kilogram (mg/kg) which means mg of chemical per kg of body weight of the animal. Mg/kg is the same as ppm. For example, if the oral LD50 of the insecticide parathion is 4, a dose of 4 parts of parathion for every million parts of body weight would be lethal to at least half of the test animals.

BIOASSAY OF CHEMICALS ON BRINE SHRIMP

A bioassay is a toxicity test used to determine the dose or concentration of a toxin. In dealing with toxins a frequent relative danger indicator is the LD-50. For example the LD-50 for sugar in rats is 30 grams, which is out of 100 laboratory rats, 50 would be expected to die at levels of 30 grams of sugar/kg of body weight. Nicotine has an LD-50 in rats of 0.05 g, which is much more toxic. A similar measure, the LC-50, (which stands for lethal concentration) is often used. LC-50 is related to exposure rather than ingestion and is more practical for this lab activity.

In this lab we will use a small crustacean, the brine shrimp. It is normally found in brackish water and is a very hearty little organism – able to tolerate high salt concentrations.

Materials (per group)

Sharpie

5 Test tubes with caps

Test tube rack

Salt water

Disposable pipettes (1 mL)

6 Petri Dishes

Dissecting Microscope or hand lens

Net 10mL graduated cylinder

Brine Shrimp

Toxin (list what is used by your class)

250 ml clean beaker

50 ml clean beaker

Serial Dilution Method- (work in groups)

1) Use a sharpie to label 5 test tubes as follows: 1:1, 1:10, 1:100, 1:1000, 1:10,000.

2) Take 11 mL of one of the “Toxin” solutions and add it to the test tube labeled 1:1. Use 250 ml beaker to get salt water. Place 9 mL of Salt water into each of the other test tubes.

3) Pipette 1 mL of “toxic” material from the 1:1 tube into the tube labeled 1:10. Mix well.

This is called “serial dilution”

4) Pipette 1 mL from the 1:10 tube into the tube labeled 1:100. Mix well.

5) Pipette 1 mL from the 1:100 tube into the tube labeled 1:1000. Mix well

6) Pipette 1 mL from the 1:1000 tube into the tube labeled 1:10,000. Mix well.

7) Label 6 petri dishes as follows: 1:1, 1:10, 1:100, 1:1000, 1:10,000, and Control.

Be sure to label the bottom of the dish, not the cover! Transfer all the test tube contents to the dishes.

8) Put 10 mL of Salt water in the control dish.

9) Use a CLEAN 50 ml beaker to dip into the brine shrimp tank to get a sample of brine shrimp. Using a pipette, move at least 10 brine shrimp into each Petri dish and observe for 10 minutes.

10) Put any unused shrimp back into the tank for the other classes.

11) Use a dissecting microscope if needed, count the number of brine shrimp alive after 10 minutes, and then after 20 minutes. Record your data in the appropriate Table.

Table A: Toxin #1 (identify)

Concentration % Alive after 10 minutes % Alive after 20 minutes
Control
1:1
1:10
1:100
1:1000
1:10,000

Table B: Toxin #2 (Identify)

Concentration % Alive after 10 minutes % Alive after 20 minutes
Control
1:1
1:10
1:100
1:1000
1:10,000

Lab Report

Write up a lab report including the headings: Introduction, Materials and Methods, Results, and Conclusions. This lab must be typed (e.g., Microsoft Word). The draft of this lab must be turned in at the beginning of lab on the date that it is due for peer review. You must provide four (4) copies of the draft. Do not have your name on this version; use the last four digits of our student identification number instead. The final version of the lab report is due at the beginning of lab on the following week. Attach all draft copies and the peer review form to your final version. This report is worth 5% of your grade.

Introduction

This section should introduce the reader to the topic of toxicology and tie the topic to the objective(s) of your lab. This section should be no longer than ½ a page.

Material and Methods

This section should list all equipment used and procedures (in detail) followed during the lab.

Methods used to reduce error should be discussed.

This section should also include

a) an objectives statement (e.g., the objectives of this lab were to ……..).

b) Identify which part of the experiment was the control, the independent and dependent variables in your experiment. (The independent variable is typically the variable representing the value being manipulated or changed and the dependent variable is the observed result of the independent variable being manipulated).

c) State your hypothesis (A hypothesis is a proposed explanation for a phenomenon). This should be a simple one sentence statement (e.g., The hypothesis was . . . .).

Results

This section should present a typed table with your results followed by an explanation of the results observed during the lab. Graphs help visually convey the results. Results should compare both toxins and all concentrations tested and compare them to the control. The raw data must be converted to % survivorship. Do not present tables with raw data!

Conclusion

This section should be a brief statement that summarizes all results and should indicate if the hypothesis was supported by the data. Conclusion section should pertain to both toxin types and all concentrations. No more than ½ a page.

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