Title: Nicotine and Lumbriculus variegatus: Toxicity Lab



Nicotine and Lumbriculus variegatus: Toxicity Lab

Author: Ben Smith, Palos Verdes Peninsula High School

Note: Dr. Charles Drew, a zoology professor at Iowa State University, is often credited with first using Lumbriculus variegatus in basic toxicology lab investigations such as this one.

Correlation: Water Resource Management, Toxicology, Human Health, Water Pollution,

Impacts on the Environment and Human Health

This lab is one of many toxicity labs that an environmental science student could explore. Blackworms (Lumbriculus variegatus) tend to be fairly durable (compared to a test organism such as brine shrimp or daphnia) and therefore the chance of relatively high mortality even before the experiment begins is generally less than with a number of other test organisms. Blackworms typically remain alive and active in a refrigerator with 1-2 centimeters of water covering them for days and sometimes weeks. In addition, using nicotine as the potential toxin is generally of interest to students.

Purpose: Students will investigate how various concentrations of nicotine affect the behavior of Lumbriculus variegatus (California blackworms).

Student Objectives: Students will become more familiar with fundamental toxicology concepts, such as acute and chronic exposure, dose, and factors which might influence the impact(s) of a substance/chemical on Lumbriculus variegatus.

Time: This lab can be completed in one 50-minute class period, with the final observation occurring 24 hours after the completion on the “12-minute” observation and recording period.

Background information: Students will need to initially observe the worms to establish what they will consider their “baseline measurement”, or “normal behavior”. This “baseline behavior” activity level will be used in comparing worm behavior when nicotine is introduced into the test environment. Several nicotine concentrations will be used, with lab groups observing and recording worm behavior during exposure. The following activity levels will be used in quantifying worm behavior:

0 = no apparent activity

1 = not as active as “normal”

2 = “normal” activity level

3 = more active than “normal”

4 = extremely active

Normal behavior includes clinging activity in which multiple worms clump into a mass. Gently probing the worms will cause separation of clumped worms. Worms exposed to nicotine may twitch. A loss of response may be illustrated with tail-curling. Paralysis or death may occur to worms in the highest concentration. Paralysis is generally displayed by the worm stretching out and floating in the nicotine solution. Worms in the lowest concentration generally recover within 10-12 minutes, with the majority of all tested worms showing recovery in 24 hours.

Acute and Chronic Toxicity: Acute toxicity refers to a relatively high dose of a given toxin over a short time period. Chronic toxicity refers to relatively small doses of a toxin over a longer time period. Lethal Dose 50, or LD50, is commonly used in measuring acute toxicity. LD50 is the dose of a given substance which is lethal to 50% of the test organisms. The information below displays the LD50 for rats and the approximate LD50 for humans for five selected chemicals:

LD50 for selected Chemicals

Chemical Rat* LD50 Human** LD50 (Approximate)

Sucrose 29,700 2.83 liters (3 quarts)________

Ethanol 14,000 2.83 liters (3 quarts)________

Sodium Chloride 3,000 0.94 liters (1 quart)_________

Arsenic 48 1-2 teaspoons_____________

Nicotine 1 ½ teaspoon_______________

*milligrams/kilogram; **based on a 72.7 kilogram (160 lb.) person

Source of table above: Toxicology for the Citizen, Institute for

Environmental Toxicology, Michigan State University.

Intrinsic and Extrinsic Factors: Factors within as well as outside an organism’s body can influence the severity of a toxin. Intrinsic factors are those within (or of) a given organism, while extrinsic factors are beyond the morphology, physiology, etc. of an organism’s body, although these extrinsic factors may significantly influence how an organism responds to a given toxin.

Materials: Per lab group: 40 blackworms

Nicotine solution

8 Petri dishes

Spring water

“Transfer tools” to carefully move worms from dish-to-dish: cut

screening w/toothpicks, plastic spoons w/holes, modified

pipettes, and/or weighing trays with holes poked in them

graduated cylinder

beaker(s)

phone (or a watch or clock)

Procedure: (1.) Label 8 Petri dishes in the following manner:

• “Water Control”

• “Water 1”

• “Water 2”

• “Water 3”

• “Control”

• “Low Concentration” = 10 milliliters of stock nicotine solution + 190 ml water

• “Medium Concentration” = 50 ml stock nicotine solution + 150 ml water

• “High Concentration” = 200 ml nicotine stock solution

Note: The volume of nicotine solution placed in each of the Petri dishes can be discussed in class, with the class arriving at a group decision, or the volume may be selected by the teacher. Perhaps needless to say, volumes should be the same throughout the lab.

Making the Nicotine Stock Solution: (Instructors may choose to have students make the stock solution).

The nicotine stock solution can be made in about 25 minutes. Any cigarette which is regular length and strength can be used. Remove the paper wrap from two cigarettes and stir the tobacco in 500 ml of very warm water for 20 minutes. Filter the solution after soaking; 10-30 ml of nicotine solution may be lost in the filtering process.

(2.) Place 10 worms in each of the following Petri dishes:

Water Control, Water 1, Water 2, and Water 3

Plastic spoons, plastic weigh boats with holes in them, or other similar

tools can be used to transfer worms from the stock of worms. Some

attention should be given to minimizing the fluid transferred with the

worms.

(3.) Observe the worms “normal” behavior, with and without probing.

Students should become familiar with the 0-4 activity level scale.

(4.) Move the worms from the Petri dishes indicated in the “Left Column”

to the Petri dishes in the “Right Column” below:

“Left Column” “Right Column”

Water Control ( Control

Water 1 ( Low Nicotine Concentration

Water 2 ( Medium Nicotine Concentration

Water 3 ( High Nicotine Concentration

5. (5.) Observe and note worm behavior at the following time intervals:

Time 0 (immediately after the 10 worms per petri dish have been

moved.)

4 minutes after the transfer

8 minutes after the transfer

12 minutes after the transfer

(6.) Move the worms back into the petri dishes in the “Left Column”

(original dishes) for the “recovery” period.

(7.) Again observe and note worm behavior at Time 0, 4, 8, and 12 minutes.

(8.) Worms will be left in their respective “recovery” Petri dishes until the next class meeting (24 hours later), when final observations/notes will be made.

Environmental Science AP Peninsula High School Instructor: Ben Smith

Nicotine and Lumbriculus variegatus: Toxicity Lab Data Sheet

Activity Rating: Rate the worm activity at each time interval.

• No Apparent Activity = 0

• Not as Active as Normal = 1

• Normal Activity = 2

• More Active than Normal = 3

• Extremely Active = 4

_________________________Exposure____________________

Behavior Control Low Nicotine Medium Nicotine High Nicotine

Rating 0 minute 0 minute 0 minute 0 minute

Please Circle

Number 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4

________________________________________________________________________________

4 minutes 4 minutes 4 minutes 4 minutes

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4

________________________________________________________________________________

8 minutes 8 minutes 8 minutes 8 minutes

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4

_______________________________________________________________________________

12 minutes 12 minutes 12 minutes 12 minutes

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4

_______________________________________________________________________________

Notes: 0 Minutes 0 minutes 0 minutes 0 minutes

Curled,

Clumped,

Straight, Etc. _______________________________________________________________________________

4 minutes 4 minutes 4 minutes 4 minutes

_______________________________________________________________________________

8 minutes 8 minutes 8 minutes 8 minutes

________________________________________________________________________________

12 minutes 12 minutes 12 minutes 12 minutes

_________________________________________________________________________________

Recovery: Record the most appropriate letters applicable to the majority of

worms in each Petri dish at each time interval.

_Behavior Control Low Nicotine Medium Nicotine High Nicotine

CR = Completely 4 min 4 min 4 min 4 min

Recovered ______________________________________________________________________________

PR = Partially 8 min 8 min 8 min 8 min

Recovered ______________________________________________________________________________

NR = Not 12 min 12 min 12 min 12 min

Recovered ______________________________________________________________________________

24 hours 24 hours 24 hours 24 hours

____________________________________________________

Lab Questions:

1. Acute toxicity tests are a relatively high, one-time exposure for a brief duration. Chronic toxicity tests typically involve a persistent and generally longer exposure, although this can vary greatly due to variation in life span for different organisms. Which type of toxicity test did you carry out in this lab?

2. Explain a possible benefit of using an acute toxicity test.

3. Is this lab investigation a controlled experiment? Explain how it is or is not:

4. Using the data from this lab, design a chronic toxicity test that could be conducted on blackworms.

5. Describe three intrinsic factors which could affect toxicity and discuss how each might influence the impact(s) of nicotine or another toxin.

6. Identify an extrinsic factor which might influence the severity of a toxin in a given organism and briefly explain how this factor could be involved in how adversely the organism is impacted by a toxin.

7. Briefly discuss how lifestyles/lifestyle choices are involved in assessing risk in terms of human health and the potential toxins which humans may be exposed to:

8. Discuss whether the effects of nicotine exposure were irreversible or reversible at each concentration used.

9. Can your test results in this lab be applied to humans or to other vertebrates?

Please explain:

Information:

a) relevant resources and references: lab handout; many online resources regarding

toxicology.

Drewes, Charles D. 1996. “Those Wonderful Worms.” Carolina Tips, Carolina Biological Supply Company, Burlington, NC.

“Chemicals & Human Health Website”, Southwest Environmental Health Sciences Center & the Biology Project, the University of Arizona.

b) important terms: LD50, acute effect, acute exposure, chronic effect, chronic

exposure, controlled experiment, threshold dose-response model, non-threshold

dose-response model, risk assessment, synergistic interactions, toxicity,

persistence

................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download