By Catalina Marambio Jones (NSF GK12 SEE-LA program)
Your name: Catalina Marambio Jones
Title of Lesson: Toxicology case study: Brine Shrimps
Grade Level: High School, 11 and 12 grades
Subject(s): Environmental Science, Chemistry.
Summary:
This is an introductory lesson for toxicology and risk assessment. It reviews the concepts of hazard, exposure, risk characterization, dose response curves, lethal dose 50% (LD
50
) and threshold. The lesson starts with an introductory PowerPoint reviewing about the history of toxicology including stories of famous poisoning cases, then examples of toxicology related cases in the news. Then, risk assessment procedure is explained including the concepts of hazard, exposure and dose response curve. A case study of chemical exposure of snail poison (cooper sulfate) and a herbicide on Brassica rapa (a plant in the family of mustard) is presented to the class. The teacher asks the students to hypothesis about the possible toxicity of these two chemicals on aquatic life in case the chemicals percolate to an aquatic environment. Finally, there is a laboratory activity where the students test their hypothesis, by constructing dose response curves for Brine Shrimps exposed to serial dilutions of the two chemicals. Students, determine the lethal dose of each chemical against brine shrimp and discuss about possible ways to improve the experimental design utilized.
Time Required: 2 periods of 50 min (or one of 100min)
Group Size: Depending on material availability, hopefully a maximum of 4 students per group.
Learning Goals:
After this lesson, students should be able to:
1) Understand the risk assessment process and phases (hazard identification, exposure assessment, dose-response assessment)
2) Define risk, hazard, exposure.
3) Understand dose response curves and identify from them the LD
50
and threshold.
4) Serial dilutions

Level of Inquiry:
Students hypothesize about the level of toxicity of copper sulfate and an herbicide on recently hatched brine shrimps, aquatic crustaceans utilized as a model for aquatic life .
Cost to implement:
The total cost for this lesson is $2.5 per group (considering 50 groups). If only the Brine
Shrimp laboratory is considered and the Brassica rapa experiment (which is presented for motivation) is excluded the cost is $1.8 per group. However, it needs to be noticed that most of the materials can be reused and thus with the initial investment of $124, this lesson can be performed many times (more than only 50 groups).
Material Comments air pump aquarium brine shrimp small
2 L
6 grams
Total cost ($)
13
7
6
Copper sulfate (by alpha chemicals)
Ortho Weed-B-Gone
3 mL syringe
Petri Dish, 60*15mm
Brassica rapa seeds seeders
1 pound
24 oz box 100 case 250 pack of 200
1 pack of 50
Total ($)
Number of groups (10 groups/ period x 5 periods)
Cost per group ($)
6
6
13
37
30
6
124
50
2.5
Introduction / Motivation:
Initially, teacher asks the students what is the first thing they imagine when they hear the word toxicology; likely the response will be poisons or the science of poisons. Then, using a
PowerPoint presentation a background about toxicology history is given.
Talk to the students about two important persons in the development of this science.
Paracelsus, a pioneer in the use of chemical and mineral in medicine and the first person to realize that "the dose makes the poison". Mathieu Orfila, considered the father of the toxicology; he was the first scientist to quantitatively evaluate dose response curves and had important role in forensic toxicology.
Then, teacher discuss with the class some stories of famous poisoning cases and examples of poison used as chemical or biological agents in wars. For instance, images of the execution of Socrates and the death of Cleopatra can be shown, students are then asked to comment on those famous personages, who were they? how they die?, what was the poison utilized in each case?. The teacher then presents information on the respective poisons.
Additionally, a story of the Borgia family, known for its ties to corruption and poisoning of their enemies using a poison called "Cantarella" is presented to caught students attention.
After that, examples of chemical and biological agents used in wars is exposed to the class.
For instance the used of toxic gases in that can be use are the chemicals gases used in the

first and second world wars and the gases also used during the Halabja massacre during the Iran-Iraq war in 1988. As the last part of the introduction a review of recent toxicology-related news can be presented.
At the end of the presentation it needs to be noted that most of the examples and news of toxicology are related to poisons used for bad purposes and is probably because of that that the first word that came to their minds in the beginning was poison. However, toxicology is indeed intended for good purposes such as pharmacology , forensic toxicology and the prevention and amelioration of toxic effects of chemical, biological and physical agents on humans and the environment. Here the more current definition of toxicology can be introduced.
Background Concepts for Teachers:
There are several websites where background of toxicology and risk assessment can be reviewed; this section gives just a very succinct background of the concepts required for the lesson but is highly advisable to review the resources presented in the references for more information.
-Toxicology definitions:
The old definition of toxicology and many times taught in K12 curriculum is: " Toxicology is the science of poisons"; a newer and more correct definition is the one given by the Society of toxicology (SOT), i.e., "Toxicology is the study of the adverse physicochemical effects of chemical, physical or biological agents on living organisms and the ecosystem, including the prevention and amelioration of such adverse effects."
The following paragraphs were obtained from the Society of Toxicology website, http://www.toxicology.org/AI/NEWS/news-riskassess.asp
-------"Risk assessment is a process by which scientists evaluate the potential for adverse health or environmental effects from exposure to naturally occurring or synthetic agents.
These agents include (1) chemicals such as those that occur in food naturally, food additives, drugs, and environmental contaminants, and (2) physical agents, such as radiation or electromagnetic fields. Risk assessment typically includes an estimate of the probability of harm, such as the probability of liver toxicity after use of a particular drug or the effect that a chemical in the environment may have on wildlife, and a clear description of the various assumptions and uncertainties that go into the risk assessment."
"As described by the National Research Council of the National Academy of Sciences, risk assessment involves four components: Hazard identification—an evaluation of the adverse health effects the agent is capable of causing. Examples might include the capacity of an agent to cause liver or nervous system damage or to cause cancer. Dose—response
assessment—a determination of how much of an agent is required to cause a toxic effect, and prediction of exposure levels at which risk is likely to be negligible or nonexistent.

Exposure assessment—a determination of how much of an agent people might be exposed to under various conditions such as use of a drug or a consumer product, environmental exposure at a hazardous waste site. Risk characterization—an integration of the pertinent information from the preceding steps to characterize the risks to the exposed population—e.g., what is the likelihood that there will be an increase in cancer in a population exposed to a particular contaminant in drinking water? What is the likelihood of liver toxicity if an individual uses a particular drug?"--------
Other information relevant for the lesson:
Hazard: Agent that has the potential to cause harm.
Risk: Is the probability of harm happening when exposed to the hazard.
Dose-response: Is a mathematical model that relates the amount or dose of toxicant and its associated effect or response. For example a typical S-shaped dose response curve looks as the following picture. As the dose increases the response increases as well. Different studies would evaluate different types of response, for example population mortality o population fraction showing either a deleterious effect because of a toxicant (Ex. % of population with cancer) or a beneficial effect in case of a remedy (Ex. % of population showing pain relief).
The highest dose at which no response is observed is called the threshold, and in case that a toxicant is being under studied, it will represents the highest dose that would be consider safe. When the response is the population mortality the dose that caused 50% of population death is called lethal dose 50% (LD
50
) and this value is associated with the potency of a toxicant. If comparing between two agents typically the one with the lowest
LD
50
would be consider the most toxic. If the response analyzed is a beneficial effect due to a remedy, for instance headache relief due to aspirin consumption then dose that produces the effect in 50% of the population will be the effective dose 50%.
50%
Threshold
LD
50 or ED
50
0%
20 40 60 80 100 110 120 140 160 180 200
Dose
( for example mg/Kg)
LD
50 Therapeutic index: For a medication, this index is defined as the . The highest this
ED
50 index the safer is the drug because is more difficult to get poisoned by mistake. For example for aspiring the typical ED to relief headache on adults is about 2 pills (200 mg each) and the corresponding LD
50 is about 200 pills. Therefore, the therapeutic index is
100, this means that one should accidentally ingest 100 times the recommended dose for a

fatal outcome, what is not very likely. However, there are other medications whose therapeutic index may be very small thus would be easy to have an accidental overdose.
This type of medication are typical prescribed only under close medical supervision.
Introduction
First part of slide show, presenting the introduction described above. Students are provided with worksheet 1 to fill out during presentation.
Lecture's Body:
1. Second part of slide show. The phases of risk assessment are introduced and the concepts related to dose response are presented. After explaining the dose response graph, an example is presented to the students on the whiteboard, where the teacher introduces the therapeutic index concept through an example comparing the index for an over-thecounter medication and a prescription medication (High versus low therapeutic index).
2. Students work for 3 min in a think pair activity (slides 32 and 33).
3. Discussion of think pair share activity and clarification of any concepts that students may have trouble with.
Laboratory Section:
Teacher shows the class the result of the toxicology experiment performed on
Brassica rapa. This experiment consisted in growing from seeds 2 sets of these plants. The first set was watered with a decreasing serial dilution of cooper sulfate and the second with a serial dilution of Ortho Weed-B-Gon Weed Killer. Students will observe something like this:
Copper sulfate
Herbicide
Dilution
1 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 factor
`
Teacher introduces the lab. In the lab students will evaluate the toxicity of copper sulfate and the herbicide against brine shrimp by constructing dose response of each chemical. First students forms groups (hopefully no larger than 4). Then Students make hypothesis of the toxicity of the chemicals to the brine shrimp. For example what chemical would be more toxic to the shrimps.
Laboratory procedure:

Students prepare 6 to 8 serial dilutions of each chemical:
1.
Prepare chemical concentrated solution. For copper, first prepare a 20 or 30 g/L copper sulfate solution. Each group needs only 11 mL of it but a larger volume can be prepared in advance and be share with entire class. For the herbicide use chemical directly from container.
2.
Label the containers from 1 to 8 for each containers set (one set for copper sulfate and the other for the herbicide.
3.
Pour 10 mL of corresponding concentrated chemical solution in container 1 for each set.
4.
Add 10 mL of water from container 2 to 8 in each set.
5.
Pour 1 mL of corresponding concentrated chemical solution in container 2, mix well by gently moving container.
6.
Transfer 1 mL of solution in container 2 to container 3, mix well by gently moving container.
7.
Transfer 1 mL of solution in container 3 to container 4, mix well by gently moving container.
8.
Repeat for all remaining containers.
9.
When finish mixing container 8, take 1 mL from it and discharge in the waste disposal container.
10.
At this point all 8 containers will have 10 mL and from one container to the next the concentration will be diluted by a factor of 10.
6
7
8
Container
3
4
1
2
5 m m m m m
11.
Add 5 mL of brine shrimp suspension to each container.
12.
Count number of alive shrimp at the beginning (only the ones moving)
13.
Wait 10 min.
14.
Count remaining alive brine shrimp.
15.
Students must fill the following table as they work on the lab.
Table: Raw data for chemical ___________________________________.
Final concentration chemical Initial number of alive shrimps
Final number of alive shrimps
% dead shrimps at the end

Notes:
 % dead shrimps


# alive shrimps

# beginning alive
  shrimp
# alive end
 shrimps end


100
 Final chemical concentration needs to include dilution caused by the addition of 5mL brine shrimp
16.
Using the Final chemical concentration column and the % of dead shrimp students draw the dose response curves and extract the values for LD50 and threshold (even if they can only present ranges, for example LD50 must be higher than... or LD50 must be lower than.....
17.
Using this data student check if their hypothesis was correct or not.
18.
Students comment on possibilities to improve the experimental design. For example, where 10 min exposure time enough? if not, how much time should we exposed the shrimps next time? Was the initial concentration high enough? Was the number of serial dilutions enough?
Materials List
Each group will need:
 16 transparent containers (at least 20 mL each), halves of 6cm petri dishes work well.
 10 mL graduate cylinder
 two 1mL syringe for transfer (1 for copper sulfate, one for herbicide), or micropipettes if available.
 One 5mL syringe to transfer the brine shrimps (or micropipette if available).
 Magnifying glasses are desired but no essential.
 Globes
 4 cups o small beakers (one to hold copper sulfate concentrated solution, one for herbicide and one for water and one for brine shrimps)
To share with the entire class:
 Ortho Weed-B-Gon Weed Killer
 Copper sulfate solution (at least 20 g/L)
 Brine Shrimps recently hatch (To growth them follow instructions of supplier, you will need a transparent container (small aquarium or soda bottle) about 1 litter of non chlorinated water or aged tap water, a pinch of baking soda and 1.5 table spoon/litter of non iodinated table salt, and air pump). There are several videos in youtube that shows how to growth them.

For Brassica rapa experiment:
 Seed starter pots
 soil
 Brassica rapa seeds (www.fastplants.org)
 water
 copper sulfate
 Herbicide
Prepare two sets of 15 pots, one for copper one for herbicide, by filling them with soil. Plant two or three seeds in each pot at about 1 cm below surface. Label the containers from 1 to 15. Every day, water the containers with the same volume (20 mL) of decreasing concentrations of the chemicals. Container 1 will be watered with the highest concentration and container 15 with the lowest concentration. Start the plants 2 weeks before the lesson. One week will do it too but plants will be very small.
To prepare the watering solutions make serial dilutions with a factor of 1/2.
Container 1 will have the highest solution (20 g/L for copper sulfate), then container 2 will be watered with 10 g/L, container 3 with 5 g/L, container 4 2.5 g/L and so on.
20 mL
Step 1
40 mL 20 mL
Concentrated solution Water
20 mL
=
40 mL
Step 2
40 mL 20 mL
=
20 mL 40 mL
Same procedure for remaining solutions .
End
20 mL 20 mL 20 mL 20 mL 20 mL 20 mL

Safety Issues:
 Use globes and goggles
 When done rinse hand with water and soap.
 Do not ingest any of the solutions
 Dispose chemicals in agreement with local regulations
Lesson Closure:
Teacher guides a class discussion, where students comment about their findings. What chemical was more toxic? Was this expected from the results observed in the plants?
Where the students able to draw dose response curves? Was the experimental design adequate? How the design could have been improved in terms of exposure time, number of serial dilutions, maximum concentration?
If the procedure was follow as described above students will find that this particular herbicide did not seem to be toxic to the shrimps for the 10 min exposure time and thus
LD
50 can be reported as higher than the commercial herbicide concentration. The solution of copper sulfate would result more toxic to the shrimps than the herbicide, what may be surprising for some students because in the case of the plants the herbicide was more toxic.
The teacher, may conclude the lesson with this in mind. Different organisms may react different to the same toxicant and thus toxicologist need to make sure to choose the correct models to represent the situation they want to study. For example, if a scientist need to develop dose response curve for products destined for human consumption, is very important to choose the animal that will react in a similar way than a human. For instance another mammal should have a more closer response to a human than a bird or a reptile.
Assessment:
Activity beginning assessment:
Ask questions in the beginning an through the presentation. What is toxicology? what makes a poison a poison? What typical poisons the students can name?
Activity Embedded Assessment:
Use think pair share to check how students are doing, circulate and assist during the lab, ask the students to explain on their words what are they doing.
Post-Activity Assessment:
During wrap up check what students understood by asking of their results and analysis.
Also you may want to use the worksheets that the students completed during the lesson and lab to check their understanding after the lesson.

References:
The following reference was use to choose the subject of study and general laboratory procedure. http://www.cvs.k12.mi.us/klolich/APES/Pollution/Handouts/19/Brine%20shrimp%20an d%20LD50%20activity.pdf
The following resources were used as general references to prepare the presentation: http://toxicology.org/ http://www.epa.gov/iris/ http://www.epa.gov/spc/pdfs/rchandbk.pdf (section 1.2) www.riskbites.com
Attachments:
 Worksheet 1
 Worksheet 2, lab section
 Power point: Toxicology brine shrimp lab
List CA Science Standards addressed:
From the college board curriculum for AP environmental science:
-Toxicology: Assessing chemical hazards
-Water pollution