What is toxicology?

- Study of toxic substances →

- Study of poison →

What is xenobiotic?

- Foreign substance to an organism

● May produce useful results (eg. pharmaceuticals) or be toxic (lead)

What is anthropogenic?

- Caused or produced by humans

● Used to describe origin of a compound which otherwise would not occur naturally

TOXINS VS. TOXICANTS

Toxicants → poisonous agent → produces adverse biological effects

● E.g. metals , PAHs, PCBs, etc.

Toxin → toxic substance produced by living organism

- E.g. snake venom, mycotoxin, tetrodotoxin, etc

History of toxicology

● Father of toxicology → Paracelsus (born 1493/1494)

- Believed that it was not the substance that was toxic but the amount that was toxic

- Paracelsus’ basic tenant of modern toxicology “All substances are poisons: there is none

that is not a poison. The right dose differentiates a poison and a remedy
Bernardino Ramazzini (born 1633)

- showed that occupational exposures could be involved in the causation of disease

- Book “Diseases of workers” listed illnesses/disease in many different occupations

ENVIRONMENTAL TOXICOLOGY

Environmental toxicology

- The study of the fate and effects of chemicals in the environment

Exposure and effect

● Must have exposure to have an effect

● No exposure =

For this to occur, the toxicants must

1. Be released into the environment

2. Be exposed to the target organism

3. Be taken up by target organism → modification?

4. Causes a response in the target organism

Classes of environmental toxicants

1. Radiation → e.g. Japan's Fukushima Daiichi nuclear plant re;ease

2. Inorganic → metals and ammonia

3. Organic → dioxins, furans, polycyclic aromatic hydrocarbons (PAHs)

4. Pesticides → insecticides, herbicides, fungicides

5. Complex effluents → e.g. STP effluent, mine effluent, or pulp and paper effluent
HISTORICAL EXAMPLES

● Chimney sweeps

- 18th century

- Chimney soot was removed by chimney sweeps

- Sweeps had an increase in scrotal cancer → Percival Pott (legge, 1955)

- Attributed to the PAHs formed from incomplete combustion

● Mad Hatters

- Hat makers (hatters) would sometimes have used solutions in hat making

- Solutions contained mercury (Hg)

- Hatters worked in poorly ventilated rooms and inhaled mercury vapor

- Caused neuropathology on the hatters (Koertge, 1965) → “Mad as a hatter.”

HISTORICAL NEED FOR ECOTOXICOLOGY

A. Changes in human behavior

- Pre-industrial revolution

● Small, rural-based communities

● Receiving environment able to “cope with” the wastes produced

- Post - industrial revolution

● Developing countries shifted from rural communities to industrialized

civilizations

● Receiving environment can no longer “cope with” the wastes produced

 B. Pollution

- Substance in the environment that produces adverse effects

- Pollutants are not necessarily toxicants

● E.g. phosphorus → eutrophication

● But many are → PAHs, PCBs, oil, metals, etc

C. Paradigm shift

● Dilution paradigm

- “The solution to pollution is dilution”

● Boomerang paradigm

- “What you throw away can come back and hurt you”

D. Relationship to other Sciences

- Toxicology draws from several scientific disciplines and it contributions have become

increasingly important to several disciplines

ELEMENTS OF A TOXICITY EVENT

Introduction:

- Recall, a toxic event involves the following:

1. Generations of a contaminant

2. Release of the contaminant

3. Movement of the contaminant to a receptor

4. Exposure at a high enough contaminant level for long enough amount of time

5. A response
Exposure and Response :

● Once in the environment need to have exposure to a toxicant to get a response in an organism

- 10 Rainbow trout fingerlings

- In clean water →

- Add a toxicant →

Exposure Routes:

- It is possible for organisms to be exposed to a toxicant obtained in their diet, or in the air, water,

or soil

Animals can be exposed to a toxicant via the following exposure routes:

1. Oral → administration of toxicant by mouth or diet

● Organism is given a specific dose (amount) of toxicant

● Units of dose is expressed as weight of toxicant/weight of organism

 2. Injection → organism injected with toxicant

- Exposure to the dose can be done via intravenous, subcutaneous or intraperitoneal

injection

3. Topical or surface → toxicant/drug is applied to the skin in an applicable dose

4. Respiratory

- Exposure from air →

- Exposure from water →

- Organism is exposed to a specific concentration of toxicant

- Units for concentration is expressed as weight of toxicant/volume of air or water

MEASURING TOXICITY

A. Measurement

Question: How do we measure toxicity?

Answer:

- Designed to determine the effects of a toxicant on an organism/group of organism

- E.g. lethality bioassay

● Used to determine if the chemical is toxic and how toxic is it relative to other

chemicals

- There are many types of bioassays used in toxicology

- E.g. Rainbow trout (water), Daphnia magna (water), Hyalella azteca (water, sediment),

earthworm (soil), plants (air, soil or water)

Important Bioassay Parts:

- Besides the test organism, need to have

1. Exposure time

● Time to allow for response to occur at the dose/concentration of toxicant

2. Dose/Concentration of the toxicant

● Amount of toxicant to develop a response withing a given time

Classifying Toxicity Responses

- There are two types of toxicity responses observed based on the degree of the response

1. Lethal →

2. Sublethal

- Response under the level that directly causes death

- Can be at the inf=dividual, species or community level

a. Individual → changes include:

- Biochemical → enzyme inhibition, e.g. acetylcholinesterase

inhibition by organophosphate pesticides

- Hormonal → changes in cortisol levels due to stress

- Physiological → changes in growth or reproduction

b. Species → changes in population structure

c. Community → changes in the diversity and abundance of a species

There are 5 types of toxicity responses based on the timing of the response

1. Acute

- A severe stimulus that quickly brings about a response in the target organism

- Short term →

- Acute responses are a common endpoint in routine toxicity testing

● E.g. rainbow trout or Daphnia magna lethality bioassay

2. Chronic

- The stimulus slowly brings about a response

- Exposure occurs for a long period of time

- It is a long -term test → at least 10% of the organisms lifespan

3. Subacute

- Less severe stimulus than acute

- Response takes longer to develop

4. Cumulative

- Response occurs because the stimulus has been repeated several times

- Exposure may be identical as previous or may be different

5. Delayed

- Response does not emerge until well after the exposure to the stimulus

- E.g. cancer
MODULE 2: DOSE RESPONSE RELATIONSHIPS AND TOXICITY TEST METHODS

Introduction

● Recall, a toxic event involves the following

1. Generation of a contaminant

2. Release of the contaminant

3. Movement of the contaminant to a receptor

4. Exposure at a high enough containment level for a long enough amount of time

5. A response

- Need to have exposure to get a response → different possible exposure routes

- Can use a bioassay to measure toxicity → recall it is key to know exposure time and

dose/concentration of the toxicant

Dose (or concentration) - Response Relationships

- Dose-response curves show the relationship between the dose of a toxicant and the observe

effects/response in the target/test organism

- It is a graphical representation of the observed responses

The sigmoidal shape is the most common shape of a dose-response curve, n=but by no means do all

curves look like this

Bioassay Results:

- Need a way to measure the results of the bioassay

- Can report the observed results as follows:

1. Keep the time constant and vary the dose or concentration - can report

● LC50 →

- Concentration causing lethality in 50% of the test organism in a given time

- E.g. 96 hours LC50 for rainbow trout as determined to be →

● LD50 →

- Dose causing lethality in 50% of the test organisms in a given time

 2. Keep the dose or concentrations constant and vary the time - can report:

● LT50 →

- Time to cause lethality in 50 % if the test organisms at a given dose/concentration

- E.g. the LT50 for _________________ exposed to fathead minnow was determined to be

76.8 hours

3. Can also measure endpoint that do not result in death →

- E.g. enzyme induction → cytochrome P450

- E.g. inhibition of growth, metabolism or an enzyme

- Expressed as EC50, ED50, ET50

- Where E =

Toxicologists transform the dose response data

- Linearize by converting to log dose /Concentration versus Probit (probability unit) for percent

response
Dose-Response Terminology

- There are several terms used to describe the level of response after assaying a toxicant

MODIFYING FACTORS

What are modifying factors?

- Modifying factors can adjust the toxicity of a given chemical to an organism

- Want to keep all conditions instant during the bioassay → other than toxicant concentration

- There are 2 types of modifying factors:

a. _________________ → Species, sex, age, size, nutrition

b. _________________ → exposure route, partitioning, pH, O2, temperature, light

TOXICITY TEST METHODS

A. Test methods

- There are a variety of toxicity test methods that can be divided into

1. Whole organism tests that assess

● Acute → endpoint is generally survival

● Chronic → endpoint may be growth or reproduction

● Multi-species → survival or growth of multiple species

2. Biomarker tests that assess

● Endpoints that are biochemical or molecular

● AhR induction, enzyme inhibition, mutations, etc.

● Can be done in vitro with prokaryotic (ex. Ames Test) or eukaryotic

target

B. Points to consider

- Once a target organism (in vivo) or cell (in vitro) has been chosen there are other important points

to consider such as:

● Duration of exposure such as acute, subacute, sub-chronic, chronic

● Route of exposure such as oral, dermal, inhalation, subcutaneous injection

● Toxic endpoint/outcome measured such as lethality, altered target organ or

physiological/biochemical function, irritation, mutagenicity, etc.

C. Standardized toxicity tests

- There are several standardized toxicity tests (environment canada, US EPA, ASTM, OECD) such

as:

● Acute lethality tets using fish (e.g. rainbow trout) or invertebrates (e.g Daphnia spp._

● Reproduction and survival test using Ceriodaphnia dubia

● Survival, growth, and reproduction in sediment and water using Hyalella aztec

● Inhibition of growth using macrophyte Lemna minor

● Test for growth in contaminated soil using terrestrial plants, earthworm tests, lettuce seed

germination root elongation]

● In vitro skin irritation with reconstructed human epidermis

● Various rodent test (oral, inhalation, dermal) with acute, short term / 90-day, prenatal

development, genotoxicity, neurotoxicity

EXAMPLE OF TOXICITY TEST METHODS

1. Rainbow Trout acute test

- A core standardized aquatic toxicity test

- Uses swim up fry or fingerling rainbow trout

- Main test parameters:

● No renewal of test solutions during the test →

● 10 fish/test solutions

● 96 hour test where lethality is the endpoint →

● Reference toxicant → phenol and /or zinc sulfate.

2. Daphnia acute test:

- Another core standardized aquatic toxicity test

- Uses neonates of D. magna or D. pulex

- Main test parameters:

● No renewal →

● 10 daphnids/test solution

● 48 hour test where lethality is the endpoint →

● Reference toxicant → zinc sulfate, sodium chloride, or potassium dichromate

3. Hyalella aztec test

- A sediment bioassay used for standardized toxicity testing

- Uses 7 to 9 day old H. azteca

- Main test parameters:

● 42 -day whole sediment toxicity test

● Static renewal of overlying water

● 20 amphipods/test vessel

● At end of test record # of surviving male and female adults and young

● Reference toxicant → copper sulfate, cadmium chloride

4. Growth inhibition of Lemna

- A routine toxicity test assessing the growth of an aquatic macrophyte (aquatic plant)

- Uses Lemna minor from 7 to 10 day old culture

- Main test parameter:

● 7-day static test or static renewal (where solution is renewed every 3 days)

● Two 3-frond plants/replicate

● Growth based on increased number of fronds during test and dry weight at end

● Reference toxicant → nickel or potassium chloride

 5. Ames Mutagenicity test

- The Ames test (developed by Bruce Ames) assess the mutagenicity of a chemical using

mutations in bacteria as an endpoint

- Uses a strain of salmonella that has a defect in the histidine biosynthesis pathway →

this strain will not grow in absence of histidine

- Bacteria are mixed with homogenate extract from PCB-induced rate liver (called the

S-9 fraction) → this homogenate mimin=cs the mammalian metabolic pathways that

could biotransform parent compound into a mutagenic metabolite

- Count the # of his+ revertants after incubation in minimal histidine → suggest

chemical caused mutation if they reverted to His+

MODIFYING FACTORS

1. INTRODUCTION

- Modifying factors can adjust the toxicity of a given chemical to an organism

- Want to keep all conditions constant during the bioassay → other than toxicant

concentration

- Recall there are 2 types of modifying factors →

2. BIOTIC MODIFYING FACTORS:

- Test organism → there are variations between species

- Life stage, size → early developmental stages are more susceptible whereas larger/older

animals are less sensitive

 - Health and nutrition → deceases state of health or decreased nutrition serve as a

potential for added stress

- Test acclimation /acclimatization → changes in biological functions that occurs over

time

● Usually increased in tolerance

● Acclimatization associated with lab studies

● Acclimatization associated with field studies

- Sex differences → endocrine disruptors impact reproductive systems differently

- Genetics → different populations of same species can have variable sensitivity

3. ABIOTIC MODIFYING FACTORS:

● Temperature or low dissolved O2 → can be an added stress to the organism

● pH → changes in pH alter toxicity

- NH3 toxicity increases with pH

- Metal toxicity decreases with pH

● Water hardness → measures as CaCO3

- Metals more toxic in soft water

- Calcium inhibits metal uptake by fish

- Metal - CO3 complexes can prevent uptake

● Binding and sorption → suspended materials can complex with or chelate contaminants

- Metals chelated by DOC (humic/fulvic acids)

- Organics bind to sediments

● Light → increases toxicity of aromatics, especially PAHs

- UV radiation adds a second reactive oxygen stress