DISCLAIMER:
The
user assumes all responsibilities for the safe and proper handling and
application of chemicals listed and described herein. The author (CDD) assumes no liability
relating to the use and effects of these listed chemicals, or any others. Before handling any chemical, users should
obtain a Material Safety Data Sheet
(MSDS) and/or International Chemical Safety Card for each chemical
from: http://hazard.com/msds/
These sheets should be made available to all users in
the laboratory. Before performing
experiments, users should carefully follow warnings and instructions on all
labeling of consumer products. Mention
herein of any product brand names in no way represents the author’s favorable
or unfavorable endorsement of these products.
Charles D. Drewes
EEOB Department, 339 Science II
Web site: http://www.eeob.iastate.edu/faculty/DrewesC/htdocs
Email:
cdrewes@iastate.edu
Phone: (515) 294-8061
OBJECTIVES
Toxicology
is the study of the adverse effects
of chemicals on organisms. Though formal
training in toxicology may not begin until professional school, student
interest and inquiry in this area may begin much earlier. A motivated high school student, for example,
may study toxicity effects for a biology research project or science fair. This student faces an array of important
questions. What scientific problem will
be studied? Can simple yet meaningful
experiments be done? What organism,
chemical, materials, methods, and safety precautions will be used? How will experiments be designed and how will
results be interpreted?
When
undertaking a toxicology project, a student may quickly discover that general
biology texts, school and public libraries, or the world wide web provide
little practical guidance or tutorial assistance. Faced with this lack of guidance, the result
for many students is frustration and haphazard results due to flawed research
design. However, student research in
toxicology need not be an unguided or haphazard experience.
At
the outset, I want to strongly emphasize that my aim is not to popularize and promote toxicology experimentation at
pre-college levels. Rather, it is to
provide young, ambitious, and careful students -- who have already decided to
attempt a toxicology project -- with a “toxicology primer” that contains useful
suggestions and practical guidance that enhance the quality, meaningfulness,
and safety of their experiments.
Hopefully, such information should be valuable to biology teachers and
other research mentors, at all educational levels.
Ideas
in this booklet derive from my research experience and mentoring activities in
ecotoxicology and neurotoxicology, mostly involving aquatic and terrestrial
oligochaete worms. So, I admit to a
strong “worm bias” and the content of this article reflects that bias. However, I think most toxicologists would
agree that these organisms offer good potential for toxicity studies in both
science education and professional research.
Many other invertebrate species, of course, may be equally or more
useful, depending on one’s objectives and interests. I hope the ideas here will be of general use,
as a conceptual template, regardless of species. Since the vocabulary of
toxicology may be alien to students, I have included a glossary of common
toxicological terms.
For
economic, ethical, and pedagogical reasons I do not recommend that any
pre-college student undertake a toxicology project that uses vertebrate
animals. Also, I do not recommend that
students use hazardous chemicals or attempt a toxicology project unless they
have approval and supervision from a qualified teacher/mentor. Maximizing safety and minimizing human health
risks are paramount concerns for all toxicology investigations! Nevertheless, given a little creative and
careful thinking, I believe that many novel, safe, and scientifically valuable
investigations can be done in toxicology by novice researchers, even when faced
with limited resources. Above all,
research students should remember the “double-K.I.S.S.” guidelines:
first: Keep It Scientifically Sound!
second: Keep It Simple and Safe!
“BIOLOGICAL SMOKE DETECTORS”
Recently, I met with several middle school
students and their teacher to discuss their proposed student research project
in environmental toxicology. One of
these students asked me why anyone would study effects of a toxicant on
invertebrate organisms, such as freshwater oligochaete worms. I tried to explain and justify using the old
analogy of a “canary in the coal mine.”
However, based on the group’s quizzical reaction, I realized that the
analogy was alien and from a by-gone era.
Obviously, I needed an updated analogy to emphasize how living organisms are used as “early warning
systems,” or sentinels, for detecting harmful substances in the environment and
for studying their biological effects. One warning device that comes to my mind is a
“smoke detector”... thus the title, “Biological Smoke Detectors.” When a smoke detector goes off it doesn’t
necessarily mean there’s a fire or that anyone will be harmed, but it does
indicate that there is a potential problem that requires close and immediate
inspection. The same is true for toxicity tests that signal adverse
biological effects on living organisms.
Fig. 1. The
canary was once used as sensitive bio-indicator for the presence of poisoness gas
in underground mine shafts. Today, the
term “canary in a coal mine” is an antiquated metaphor used in reference to
many types of “early warning systems.”
PURPOSE OF INVERTEBRATE
TOXICITY TESTING
Toxicity
testing involves the discovery and analysis of chemical effects on organisms. Extensive toxicity testing, using many
species, is needed to understand the full spectrum of biological effects of
chemicals and to decrease the health risks that chemical effects pose for
humans and ecosystems. Toxicity testing
is crucial for a wide variety of chemicals that are present in drugs,
cosmetics, pesticides, food additives, cleansers, solvents, and industrial
wastes. Invertebrates are used in
toxicity testing for one of two reasons:
(1) Some invertebrate species may have
considerable relevance to the
environment. For example, earthworms
are ecologically beneficial to soil ecosystems and zooplankton are key links in
aquatic food chains. Some invertebrates
may also have environmental relevance, not because they are beneficial, but
because they are pests. In either case,
it is important to know if and how the presence of a chemical in the
environment might affect any of these organisms. Laboratory testing under controlled
experimental conditions is an important approach to understanding and
predicting possible effects of chemicals in ecosystems.
(2) Invertebrates may provide useful insights to understanding chemical effects
on human health. This is because
invertebrates share some of the same biochemical and physiological processes
that exist in nearly all animals, including humans. For example, many nerve cell functions are
common to worms, insects, fish, rats, humans, etc. Therefore, invertebrate toxicity testing may
be a useful tool for understanding and detecting biological effects of
chemicals at molecular, cellular, or behavioral levels in many organisms.
LETHAL AND
SUBLETHAL EFFECTS
Toxicity testing in the past 20 years has
moved away from lethality (or LC50) studies and toward studies of sublethal effects (see Glossary). This is because sublethal effects occur at
concentrations below those for lethality and thus are more sensitive indicators
of toxicity.
Fig. 2. Comparison of lethal and sublethal treatment
effects. Treatment with high chemical
concentrations may be lethal in some or all treated organisms. Lethal effects are easy to recognize and
tabulate. Treatment with lower
concentrations may produce sublethal effects which are sometimes difficult to
discern but, nevertheless, important from a behavioral or ecological
standpoint.
One hurdle for a student toxicology project is deciding what sublethal effects to study. This is a challenge because such effects seldom have been described in invertebrates and there are few standard methods for measuring them. Thus, it is advisable to begin by carefully observing an organism’s normal behavior prior to any toxicity testing. Then, during preliminary testing, look for obvious effects, such as changes in an organism’s color, posture, or spontaneous movements... or perhaps changes in its reaction to stimuli such as light, touch, or body inversion. Some effects may only be seen under magnification, such as rhythmic movements of the organism’s heart or respiratory system. To systematically study behavioral effects, students may need to design and build simple devices for handling, observing, or testing organisms. In addition, they may need to develop criteria for scoring or measuring effects. From such observation and testing, students will likely gain new insights about the biology and behavior of normal as well as treated organisms.
SOME WORMY IDEAS FOR
TOXICITY TESTING
Freshwater organisms, such as aquatic
oligochaetes are good choices for toxicity testing because: (1) they are important
parts of aquatic ecosystems and food chains, (2) they are exposed to many
chemicals that contaminate water and sediments, and (3) certain freshwater
species, such as Lumbriculus variegatus
(the blackworm or mudworm), have been used previously for studying toxicity
effects. Lumbriculus is cheap (commercial or field sources), easily cultured
in the lab (asexual reproduction), and simple to handle (Drewes, 1996b). Most important, there are interesting aspects
of this worm’s biology that may be useful indicators of toxicity (Rogge and
Drewes, 1993; Drewes, 1997; Lesiuk and Drewes, 1999).
[Note
that the genus Lumbriculus is very
different from the mud-dwelling genus Tubifex. Lumbriculus
displays unique locomotor behaviors, such as helical swimming and reversal
(Drewes, 1999; Drewes and Cain, 1999). Tubifex are much less acrobatic and
display neither of these behaviors.]
Fig. 3. The segmented oligochaete, Lumbriculus variegatus, is widely
distributed throughout the
SUBLETHAL
CHEMICAL EFFECTS IN LUMBRICULUS
Possible
sublethal effects in blackworms
include changes in body shape or
behaviors such as swelling, coiling, rigidity, convulsions, limpness,
paralysis, ataxia, hyperactivity, constrictions, or segment autotomy (body
fragmentation). Some chemicals may cause
changes in body color due to
circulatory effects such as blood pooling or blood loss in different body
regions, especially the tail end. Also,
there may be important and interesting effects on other body functions that are
only evident with more detailed inspection and testing of treated and normal
worms.
One
function that that may be useful and relevant to both ecological and medical
toxicity testing is the pulsation rate of the worm’s dorsal blood vessel (Fig.
3A). Just as in humans, pulsation rates
in worms may speed up or slow down as a result of toxicant exposure. Lesiuk and Drewes (1999) describe methods for
measuring pulsations rates in the dorsal blood vessel before, during, and after
exposure to common pharmacological agents such as nicotine and caffeine.
Other
functions that may be potentially affected by toxicants include locomotor
behaviors such as swimming (Fig. 3B), crawling, and body reversal -- all
behaviors that are easily evoked and readily measured (Drewes, 1999; Drewes and
Cain, 1999). These functions have
special environmental relevance because they relate to the worm’s ability to
move about within its environment and escape from predators.
Another
biological process that is easily studied and measured is regeneration of head
and tail segments (Drewes, 1996a).
Regeneration of lost segments is a key developmental process that has
great adaptive significance to worms. It
is a means for restorative growth following loss of segments, which frequently
occurs in nature as a result of predatory attack or spontaneous fragmentation;
the latter is a normal mechanism for asexual reproduction in these worms.
These are only suggestions for sublethal effects. Many other effects (physiological, biochemical, and behavioral) likely occur which may also be amenable to study, but there has been very little research study or publication of any such effects. This should be viewed as a great opportunity and source of motivation for students to make novel and significant contributions using such toxicity assays.
SELECTING THE
CHEMICAL(S)
When
selecting a chemical for toxicity testing, consider its relative safety in
handling, availability, and relevance to “real-world” ecological or medical
situations. A few chemicals that meet
these criteria are listed below, along with brief descriptions of their use and
relevance. Most are available in either
pure-form or diluted commercial formulations.
A source for pure-form chemicals is Sigma Chemical Company (
When using any chemical, carefully consult and comply with all information given on the MSDS Sheet and International Chemical Safety Card for each chemical. This information is available at the following world wide web sites:
http://www.cdc.gov/niosh/ipcs/icstart.html
To see a sample MSDS
Sheet and a sample International Chemical Safety Card for a compound
such as “d-limonene” (one chemical listed below in the “LIST OF POSSIBLE TEST CHEMICALS”), see the following sites:
http://hazard.com/msds/f/bwn/bwnws.html
http://www.cdc.gov/niosh/ipcsneng/neng0918.html
Fig. 4. Chemical structures of some compounds that
cause significant and interesting sublethal effects on Lumbriculus variegatus.
LIST OF POSSIBLE TEST CHEMICALS
boric acid: An inorganic
salt (H3BO3). An insecticidal powder
used indoors for cockroach and ant control.
Occurs in nature as the mineral, sessolite. Used for weatherproofing wood and fireproofing
fabrics. Used externally on humans as an
antiseptic, eye ointment, and antibacterial agent. Used extensively in industry for cements,
glass, leather products, carpet, soaps, cosmetics, dyeing, printing, painting,
and photography. If ingested by humans,
may cause many toxic effects: vomiting, cramps, skin lesions, circulatory
collapse, speeding up of the heart, and convulsions. Known to cause reproductive and developmental
toxicity effects in mammals. A good
candidate chemical for toxicity effects on worm regeneration. Very soluble in water.
caffeine: An alkaloid that occurs naturally in tea and
coffee leaves and cola nuts. Known to
stimulate many nervous system functions, heart rate, respiration, and urine
flow in mammals. Present in caffeinated
soft drinks. Active ingredient in many over-the-counter anti-sleep drugs. Very soluble in water. (cf., Lesiuk and
Drewes, 1999).
capsaicin: Main active ingredient in red pepper, or
chili pepper (genus Capsicum). Known to affect nervous system functions and
development of sensory neurons. Creates
stinging, burning sensation on skin or mucus membrane. Used in some cat/dog repellents. Nearly insoluble in water. Freely soluble in ethanol. Example of a commercial source is red pepper
powder.
carbonic acid: Dissolved CO2 in water =
carbonated water = seltzer water. Toxic
to aquatic invertebrates, such as worms.
Sometimes used by microscopists to narcotize invertebrates prior to
chemical preservation.
chlorinated water: Chlorinated water contains chlorine, a
purifying agent for drinking water.
Power plant effluents produce high chlorine levels in marine and fresh
waters. Chlorine has short-term
stability in water (hours or days).
Chlorinated water also contains varying amounts of chloramine, formed by
the reaction of ammonia with chlorinated water.
Chloramine also has disinfectant and sanitizing properties but has
longer stability in water than chlorine.
Chlorine and chloramine in water are extremely toxic to aquatic
organisms, including invertebrates and fish.
Data regarding the chlorine concentration (and concentrations of other
constituents) in municipal water supplies are normally available to the public
from water treatment personnel.
CMA (calcium magnesium acetate):
Used for de-icing highways.
Believed to be less toxic to aquatic life than NaCl. Effects on many aquatic organisms are
unknown. Commercial formulation of CMA
is Chevron Ice-B-Gon Deicer. Water
soluble.
ginseng: Extracts from roots of ginseng plants (genus Panax) contain ginsenosides (types of
saponins). Used in oriental medicine as
a tonic. Claimed to enhance circulation,
heart contraction and revitalization.
Believed to reduce stress and fatigue in humans. Very water soluble. Commercial source: Panax Ginseng Extract, available in oriental food stores, consists
of a water extract from red ginseng roots that is nicely packaged as ten
separate 10-ml vials intended for full-strength human consumption or dilution
in other drinks. This commercial
extract, when diluted to 1/50th full strength, appears to be a potent disrupter
of locomotor reflexes in Lumbriculus. Ginsenosides, obtained from water extracts
from actual ginseng roots, have potent effects on Lumbriculus blood vessel pulsation rates (S. Wong, personal
communication).
limonene: A naturally occurring substance in lemon,
orange, caraway and dill. Constitutes
about 98% of orange peel oil by weight.
Used as an insecticide and insect repellent. Widely used for control of fleas, lice, mites
and ticks. Virtually non-toxic to
warm-blooded animals, but can cause skin sensitivity and irritation. Pleasant lemon-like odor. Practically insoluble in water but miscible
with ethanol. Example of commercial
source: “Natures Answer Flea and Tick
Dip” contains 78.2% d-limonene and the label recommends diluting the product at
a ratio of 3 parts product to 256 parts water (= 0.9%) and then applying
directly to the pet. Major effects,
including neural and behavioral toxicity, rapidly occur in Lumbriculus at 0.009%, or less.
This is ≤1/100th
of the recommended concentration for pets and serial dilutions can be made from
this concentration. (see Karr et al., 1990
nicotine: A highly toxic alkaloid. Principal active ingredient in tobacco
products and a controlled substance. Formerly used extensively as an
insecticide for home, farm, and orchard.
Nicotine in liquid form is readily absorbed through the skin (example =
nicotine patch). Effects occur at many
sites within the central and peripheral nervous systems of vertebrates and invertebrates. Mimics the action of the neurotransmitter, acetylcholine.
Symptoms of toxicity in humans include salivation, abdominal cramping,
headache, loss of coordination, and respiratory failure. Very water soluble. An aqueous extract, made by soaking the
tobacco contents of one cigarette in 100 ml of water, will provide a
potent stock solution from which serial
dilutions can be made. Short-term
treatment with these solutions will have major effects on Lumbriculus locomotion and blood pulsations (Lesiuk and Drewes,
1999). CAUTION: The aqueous extracts
from even one cigar or cigarette may cause serious adverse effects in humans if
ingestion or prolonged contact with the skin occurs.
pyrethrum: An extract from flowers of a chrysanthemum
grown in Africa and South America that contains several closely related
insecticidal compounds (= pyrethrins).
Dried and crushed flower heads were used as a louse powder in the
Napoleonic Wars. Pyrethrins act on
insects and other invertebrates with phenomenal speed, causing temporary paralysis (knock-down) but not always
death. Formulated as household
insecticidal sprays and dusts for use on vegetables. Considered generally safe to humans and
domestic animals. Not very toxic if
ingested by humans because pyrethrins are hydrolyzed in the gastrointestinal
tract. Skin contact may cause
dermatitis. Synthetic pyrethrin-like
compounds (= pyrethroids) are used in many commercial insecticide formulations
because they may be more stable and more active than natural pyrethrins. Pyrethroids are potent neurotoxins that
modify function of voltage-gated sodium
channels in neuronal membranes and induce repetitive firing of action
potentials. Practically insoluble in
water but very soluble in ethanol.
Example of commercial source: “Scratchex Power Dip For Dogs and Cats,”
designed to kill fleas and ticks on contact.
Scratchex contains 0.54%
pyrethrins. The label recommends
diluting 1 part from the bottle with 64 parts of water (= 0.0084%) before
application to pets. Major effects on Lumbriculus rapidly occur at 0.000084%,
or less. This is ≤1/100th of the
recommended concentration for pets.
SAFETY
Obtain and study “Material Safety Data Sheets” (MSDS)
and/or International Chemical Safety Cards for all solvents and test chemicals
that you will use in testing. MSDS
sheets and International Chemical Safety Cards are readily available from the
University of Vermont which maintains a huge electronic data base relating to
chemical safety. The address is: http://hazard.com/msds/
Learn and follow all safety measures for
the laboratory facility in which you will be carrying out your study. Learn and follow all written safety
precautions for the chemicals you are using.
Handle all volatile or toxic materials in a fume hood.
Wear a lab coat, protective vinyl (or latex) gloves and use protective
eyewear when opening or handling any chemical storage containers, stock
solutions, pipettes, or exposure containers.
Clearly
label the contents and concentrations
of all chemical solutions in containers.
Properly dispose of all used
solutions, surplus solutions, or chemically-exposed materials such as pipette
tips or filter paper. Use absorbent
towel to thoroughly remove any drips or spills of solutions to which humans may
come in contact. Thoroughly and
carefully scrub and clean all
glassware or plasticware that was exposed to chemicals. Use ethanol and then water rinses to clean
containers that held water-insoluble chemicals.
It is very important not to leave any chemical residues on glassware,
thus the emphasis on careful cleaning.
Finally, it is important to begin experiments
without any traces of soap residue on glassware. Soap residues are especially toxic to many
aquatic invertebrates.
EXPOSURE
METHODS
A
simple way to expose worms to water-soluble chemicals is by immersion. Worms are placed in individual containers
along with a small volume (about 20-30 ml) of
test solution of known chemical concentration. The chemical is thus absorbed through the
skin (termed contact exposure). Always
use just one worm per container, since a dead, decaying worm may be toxic to
others.
For
water-insoluble (and non-volatile) chemicals, there is a simple and reliable
alternative to exposure by immersion.
This involves placing the worm in direct contact with wet filter paper
that has been uniformly pre-treated with the insoluble test chemical. Pre-treatment is done by placing a dry filter
paper disk in the bottom of the glass exposure container. The disk should fit snugly and flatly at the
bottom of the container. Then, prepare
stock solutions as described in section “E” below. Each solution should contain a known amount
of the water-insoluble chemical dissolved in a known volume of suitable
solvent, such as ethanol or isopropyl alcohol.
Using
a calibrated, hand-held pipette, transfer just enough of the desired stock
solution to completely saturate the filter paper disk. Allow the solvent to evaporate completely in
a fume hood. This leaves behind a known
and nearly uniform residue of the test chemical on the paper (assuming that the
test chemical is not volatile). Next,
add a known volume of spring water into the container so that the paper is
immersed in shallow water. Use enough
water volume so that the worm could be easily drawn up into a disposable pipet
if later transfer is needed. For
example, 5 ml of water is adequate for a 6 cm diameter plastic petri dish. Next, add a worm.
All
toxicity tests should include a control group in which the paper in test
containers is initially wetted with an identical volume of solvent (but no
chemical in it). Once the solvent
evaporates, water and a worm are added, just as in treated groups.
PRELIMINARY
EXPERIMENTS AND CONCENTRATION RANGE-FINDING
Sublethal
effects of some chemicals may occur within a narrow range of
concentrations. High concentrations may
rapidly kill organisms while lower ones may cause no effect. Since concentration ranges for sublethal
effects differ among chemicals, an important step in toxicity testing for a
chemical is to determine its threshold
concentration, NOEL, and dose-response relationship (see
Glossary). This requires preliminary
range-finding experiments which are time-consuming but lead to more meaningful
results during final stages of toxicity testing.
To
make a stock solution, dissolve a known amount of pure chemical (liquid or
solid) in a small, known volume of water or other suitable solvent (such as,
ethanol or isopropyl alcohol for non-water soluble chemical). Typically, a few milligrams or milliliters of
the chemical are dissolved in 100-1000 ml of solvent. The concentration should be expressed as: mg
of chemical per liter of solvent if the chemical is a solid. This is the same as “parts per million” (see Glossary).
If the chemical is a liquid, then concentrations will be in milliliters of chemical per liter of
solvent. This concentrated stock
solution is used to make a series of weaker stock solutions by serial dilution (see Glossary). Each concentration step may be several times
weaker than the preceding one, such as 25, 5, and 1 ppm.
Sometimes
the exact amount of chemical may be unknown because it is present in an
unpurified, crude form. In this case,
the volume or weight of crude material should still be measured and recorded in
making a stock solution. Then, dilutions
of stock solution are used for range-finding experiments, with concentrations
expressed as percentages of the original stock solution.
FINAL STAGES
OF TOXICITY TESTING
Preliminary
experiments should provide an indication of concentration range and duration of
exposure for final stages of
testing. The following are essential
considerations in this testing.
Concentrations in treated
groups. Try to use at least 2-4
concentrations which, based on preliminary testing, will likely cause sublethal
effects. Also, try to use at least one
slightly lower concentration that causes no effects. A minimum of 5-6 worms should be used for
each concentration, although 8-10 provide even more statistical power. Select worms of similar size for all
groups. Use a separate container for
each worm.
Controls. In addition to groups of treated worms, it is essential to have a control group
(see Glossary). The purpose of the
control group is to verify that effects in exposed groups are, in fact, due to
the chemical itself rather than to some other aspect of the procedure. Therefore, the number of organisms, handling procedures, temperature, lighting,
testing methods, use of solvents to distribute chemicals, exposure times, etc.
should all be identical to those used in treated groups. If control conditions cause effects, then
these must be subtracted from effects in treated groups in order to obtain true
measure of the chemical’s effects.
Effects. Results from preliminary experiments often
provide clues regarding expected types of sublethal effects and expected timing
for appearance and disappearance of effects.
Exposure duration and frequency of
testing. One strategy for toxicity testing is to make a
single set of short-term observations or tests of organisms after exposure to
the chemical for a fixed time period,
such as 24 or 48 hours. This minimizes
handling of organisms and provides a standardized basis for comparing results
between different researchers and laboratories.
Another
testing strategy is to perform a series of repeated tests and measurements that
better describe the sequence and
time-table of symptoms and effects caused by a chemical. This may be especially important for
chemicals that rapidly cause neurotoxicity effects that, in turn, lead to other
effects. So, if worms are not observed
or tested frequently, important effects may be missed.. There are no standard
procedures for doing this and the experimenter should exercise his/her own
judgment based on results from preliminary experiments.
Ideally,
effects should be determined while worms are still in their original exposure
container. However, this may not always
be practical or desirable, especially if filter paper is used in the container,
because it may obscure viewing or interfere with testing. In such instances, a worm may be very
carefully removed from the test solution with a disposable pipet so that it may
be briefly examined or tested while in another container without the
chemical. Before doing this, however,
worms should be briefly and quickly rinsed in spring water and then transferred
to the new container for viewing and/or testing.
After
testing, the worm should be replaced into the original test solution if further
exposure is desired. Use a disposable
plastic pipette for transferring worms.
Special care should be taken to avoid cross-contamination of containers
or implements that are used to handle treated worms or fluids. Repeated observations and testing may be done
at any desired interval, but the frequency of testing should be the same in all
groups, including a control.
Reversibility and rescue. If chemical effects on an organism are truly
“sublethal,” then organisms should survive if exposure is promptly
stopped. But survival does not always
mean full or immediate recovery from effects.
Study the persistency or reversibility of toxicity effects (recovery) by
simply placing organisms into chemical-free conditions and continuing
observations and testing. Effects may
disappear in minutes, hours, or days.
TYPICAL EQUIPMENT AND
SUPPLIES
Here is a list of materials and equipment that may
be used for toxicity testing in freshwater or terrestrial oligochaetes. Check with instructor/mentor for approval or
modification of the list.
Materials List
1) Calibrated pipetter (e.g., Pipetman) for
measuring milliliter or microliter quantities of chemicals that are fluids
2) Balance for measuring milligram or sub-milligram quantities
of chemicals that are solids
3) Uncontaminated glassware for making serial
dilutions and storing chemical stock solutions
4) Large supply of identical covered dishes or jars (such as clean, dry baby food jars with covers; jars should thoroughly cleaned but must not contain any soap residues since these will kill many aquatic invertebrates); containers must not be prone to spills and should be easy to handle; one organism recommended per container (such as, 6 organisms/group x 5 groups = 30 containers)
5) Filter paper discs that easily fit into the
bottom of the exposure dish or jar (some trimming may be necessary)
6) Sturdy box or tray for storage and transfer of
treatment dishes or jars
7) Counter space covered with clean, absorbent,
disposable material, such as paper towel
8) Safe storage location for all stock solutions,
solvents, and all experimental containers (fume hood, if possible)
9) Disposable vinyl (or latex) gloves
10) Protective eyewear and lab coat (as advised and
needed)
11) Spring water (Note: Freshwater organisms
survive and perform best in spring water.
Examples of brand names of “worm-friendly” spring water are Evian, Poland Spring, Naya, and
many others). Aqueous stock solutions of
chemicals and serial dilutions of stock solutions all should be made with this
water and stored in separate, clearly labeled containers. Chlorinated water, directly from the tap, is
highly toxic to freshwater invertebrates.
However, tap water that has been aged in a open container for at least a
week is often just as safe to worms as spring water.)
12) Ethanol, or other solvent (needed only if
chemical is not water-soluble)
13) Capped or covered containers for storage of
stock solutions (approximately 100-500 ml volume)
14) Scientific calculator
15) Paper towels
16) Adhesive labels or colored labeling tape for
labeling test dishes and containers with stock solutions
17) Permanent marking pen
18) Experimental organisms with appropriate maintenance
or culture containers (large dishes, aquaria. etc.)
19) Supplies for handling, feeding, and care of
organisms (such as air pump for aquatic species)
20) Thermometer (to document temperature of all
experiments)
21) Test chemical, along with MSDS sheets and/or
International Chemical Safety Card with pertinent technical information about
density, solubility, formula weight, handling instructions, hazards, safety,
storage, etc.
22) Bound notebook and pen for record keeping
23) Dissecting or compound microscope with light
source
24) Recording/monitoring devices (e.g., camera,
camcorder, video camera, etc.), if desired for documentation of behavioral
effects (optional)
25) Blackworms (Lumbriculus
variegatus); commercial sources may be found at:
http://www.eeob.iastate.edu/faculty/DrewesC/WORMSO5.htm
OTHER ORGANISMS, OTHER IDEAS
In environmental toxicology the selection of
an invertebrate test organism and test chemical are often closely
inter-related. Chemicals that are
relevant to terrestrial/soil ecosystems, for example, might be tested using
commonly available invertebrates such as
earthworms, pillbugs, insect larvae, or nematodes. Tests with chemicals that are relevant to
freshwater ecosystems might utilize aquatic invertebrates such as water fleas, ostracods, copepods, hydra,
planaria, snails, or amphipods (scud).
The effect that is tested might have special ecological relevance to
predator avoidance, food acquisition, ability to react to stimuli, or ability
to locomote. Behavioral effects could be
quantified using some defined scoring system, or effects could be analyzed using
videotape playback.
Another
approach to environmental toxicity is collection and testing of soil or water samples from real-world
sites where contamination is suspected.
Water samples from a site may be used in laboratory toxicity tests and
effects may be compared to those in control groups as well as to groups treated
with concentrations of a pure chemical which is the suspected contaminant in
the water samples. Such experiments
utilize invertebrates as a true “bioassay”
organisms (see Glossary). In cases of
soil samples, organisms could be exposed to water extractions (leachates)
derived from soil samples.
For
toxicity testing relatING to human health concerns, attempts should be made to
match the kinds of effects that might be expected in humans (say, neurotoxicity
effects or developmental effects) with organisms in which similar effects might
be present and readily testable.
OBTAINING
BACKGROUND INFORMATION
If possible, locate general texts in
toxicology which may contain more helpful or specialized information. Recommended reference books include: Kamrin
(1988), National Research Council (1991), Viccellio (1993), Ware (1996),
Hodgson and Levi (1997), Ottoboni (1997).
Additional reference books that are likely to have key technical information
are:
“CRC Handbook of Chemistry and Physics” and “Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals.”
The
most reliable and up-to-date information about toxicity effects of chemicals on
invertebrates and other organisms is found in primary references -- namely,
original articles that are published in scientific journals. References to such journal articles can be
located in many college, university, or medical libraries using several
different electronic data bases for scientific literature. Three of the most useful for toxicology
purposes are: AGRICOLA, MEDLINE, AND BIOLOGICAL AND AGRICULTURAL INDEX
(BIAG).
A
very limited amount of credible and relevant information about effects of
specific toxicants may be available on the world wide web. Considerable caution should be exercised in
evaluating any web-derived information relating to chemicals or chemical
effects.
[
NOTE: Upon request, I will gladly send
copies of detailed background information about Lumbriculus biology, or reprints of any research papers below, to
students or teachers. ]
ACKNOWLEDGMENTS
I
thank G. Atchison, F. Bahls, K. Cain, and J. Coats for suggestions and
comments. Supported in part by Howard
Hughes Medical Institute Funding to Iowa State University for educational
initiatives in biology.
REFERENCES
Drewes,
C.D. 1996a. Head or tails? Patterns of segmental regeneration in a freshwater
oligochaete, Tested Studies for
Laboratory Teaching, Volume 17, Association for Biology Laboratory
Education (ABLE), New Haven, CT, pp. 23-34.
Drewes,
C.D. 1996b. Those wonderful worms. Carolina
Tips,Vol. 59, pp. 17-20.
Drewes,
C.D. 1997. Sublethal effects of environmental toxicants on oligochaete escape
reflexes, American Zoologist. Vol. 37,
pp. 346-353.
Drewes,
C.D. 1999. Helical swimming and body reversal
behaviors in Lumbriculus variegatus
(Family Lumbriculidae), Hydrobiologia
(in press).
Drewes,
C.D. & Cain, K. 1999. As the worm
turns, American Biology Teacher, Vol.
61, (In press).
Hodgson,
E. & Levi, P.E. 1997. A Textbook of
Modern Toxicology, 2nd Ed., Appleton and Lange, Stamford, CT, 496 pp.
Kamrin,
M.A. 1988. Toxicology: A Primer of
Toxicology Principles and Applications, Lewis Publishers, Chelsea, MI., 145
pp.
Karr,
L.L., C.D. Drewes & Coats, J.R. 1990. Toxic effects of d-limonene in the
earthworm, Eisenia foetida (Savigny),
Pesticide Biochemistry and Physiology, Vol.
36, pp. 175-186.
Lesiuk,
N. & Drewes, C.D. 1999. Blackworms, blood pulsations, and drug effects. American Biology Teacher, Vol. 61, pp.
48-53.
National
Research Council. 1991. Animals as
Sentinels of Environmental Health Hazards, National Academy Press,
Washington, DC, 160 pp.
Ottoboni,
M.A. 1997. The Dose Makes the Poison: A
Plain Language Guide to Toxicology, 2nd Ed., Van Nostrand Reinhold, New
York, NY, 244 pp.
Rogge,
R. & C.D. Drewes. 1993. Assessing sublethal neurotoxicity effects in the
freshwater oligochaete, Lumbriculus
variegatus, Aquatic Toxicology, Vol. 26,
pp. 73-90.
Viccellio,
P. (Ed.) 1993. Handbook of Medical
Toxicology, Little, Brown and Company, Boston, MA, 812 pp.
Ware,
G.W. 1996. Complete Guide to Pest Control
- With and Without Chemicals, Thomson Publications, Fresno, CA., 388 pp.
GLOSSARY OF TOXICOLOGICAL TERMS
absorption. Entry of a
chemical into the body through a surface such as the skin, digestive tract, or
respiratory tract.
acute toxicity. Adverse effects
of a chemical on an organism after brief exposure
to a relatively large amount of the chemical.
Often, acute effects occur a few minutes or hours after exposure begins.
[compare to chronic toxicity]
ataxia. Inability to produce coordinated movements or
locomotion due to neurotoxicity effects or neurological disorder.
behavioral toxicology. Study of the disruptive effects of chemicals
on the behavior of organisms.
bioassay. Strict definition: Use of a living organism to estimate the amount
of a chemical in a test sample. In
toxicology, this is done by comparing the toxicity effects produced by a test sample,
which contains an unknown amount of a chemical, to the toxicity effects
produced by known amounts of the chemical.
Loose definition: The use of an organism to investigate or test
for toxicity effects of chemicals.
chronic toxicity. Adverse effects
of a chemical on an organism as a result of long-term exposure to a relatively
small amount of the chemical. Often,
chronic effects become evident only many days or weeks of repeated or
continuous exposure. [compare to acute
toxicity]
contaminant. A chemical that taints or corrupts soil,
water, food, or air, thus making it impure. [compare to toxicant and toxin and pesticide.
control group. A group of
organisms that has not been exposed to the test chemical but which has, in
every other way, been subjected to conditions and procedures that are identical
to those in groups exposed to the test chemical.
contact exposure. Exposure of an
organism to a chemical by direct contact with a surface of the body, such as
skin.
dose. The total amount of a chemical
given to an organism at one specific time. [compare to dosage]
dosage. The rate of administration of a chemical or
drug to an organism. A stated dosage
includes the dose, dose frequency, and total period of time that a chemical is
administered to the organism. [compare to dose].
dose-response relationship. A quantitative relationship between the
amount of chemical given to (or taken in) by organisms in a group and the
measured effect of the chemical in the organisms, as determined by some type of
toxicity test. In a dose-response graph,
the amount of chemical is shown on the x-axis.
EC50. In a dose-response relationship showing
sublethal effects, the EC50 is the concentration that produces a level of
effect = 50% of the maximum effect. For
example, the EC50 may be the concentration that causes a particular behavioral
effect in 50% of the organisms that are tested. [compare to LC50]
effect. Any observable or measurable biological
response of an organism to chemical exposure. The measured effect in a toxicity test may be
lethality -- that is, death caused by chemical exposure -- or the measured
effect may be sublethal, such as a change in an organism’s behavior,
physiology, and/or biochemistry.
environmental toxicology. A subdivision of toxicology that deals with
the presence, movement, chemical fate, and biological effects of chemical
contaminants within air, land, or water environments, especially in relation to
individual organisms, populations of organisms, food chains, or habitats.
exposure. Contact of an organism with a chemical. [see chronic toxicity and acute toxicity]
hazard. A danger or threat that a chemical poses in
terms of some toxicity effect(s)
[compare to risk]
LC50. In a dose-response relationship, the LC50 is
the concentration of chemical that is expected to produce death in 50% of the
organisms that are exposed to that concentration. [compare to EC50]
lethality. Death of an organism caused by chemical
effects.
lipid soluble/lipophilic. Refers
to chemicals that tend to be soluble in lipids but not water. Lipophilic substances tend to easily cross
cell membranes and enter the body. [compare to hydrophilic]
mode of action. Refers to the biological/biochemical
mechanism (or mechanisms) by which a toxicant is known to (or is believed to)
exert its effects on an organism.
mortality. The frequency of deaths in a group of
organisms exposed to a chemical. [compare to moribund]
moribund. Describing a state in which an organism is
beginning to die or is near death. [Compare to mortality]
MSDS. Material Safety Data Sheet. [Source for
MSDS: http://hazard.com/msds/ ]
neurotoxicology. Study of the
adverse effects of chemicals on the structure or function of the nervous
system; neurotoxicity effects often cause behavioral effects. [see behavioral toxicology]
neurotransmitter. A chemical (such as acetylcholine) that is
released by a nerve cell at a chemically transmitting synapse.
no observed effect level (NOEL).
In a dose-response relationship, the NOEL is the highest concentration
of a chemical that causes no observable effect in a group of organisms.
[compare to threshold concentration/dose]
non-target organism. An organism that is exposed to, but is not
the intended target for, an applied pesticide.
paralysis. Inability to move the body or body parts due
to effects of disease or toxicity.
parts per million (ppm). A unit
of chemical concentration. The
concentration of a chemical is 1 ppm if one weight unit of chemical (for
example, 1 milligram) is dissolved in one million weight units of water
(1,000,000 milligrams of water = 1 liter).
Very low concentrations of chemicals may be expressed in parts per
billion or high concentrations in parts per thousand.
pesticide. A chemical used to kill organisms that are
considered pests. [see non-target
organism]
poison. Synonym = toxicant. Any chemical that causes harmful biological
effects.
recovery. The disappearance of toxicity effects in an
organism and return to normal function and behavior. If this occurs, it often occurs at some point
in time after sublethal exposure to a chemical has ended.
risk. The probability that
adverse effects will occur if an organism is exposed to a chemical under a
specific conditions.
serial dilution. Creation of a series of separate solutions
with concentrations that differ in a regular, step-wise fashion, such as a
series of concentrations that decrease by a factor of five: 50, 10, 2, 0.4 ppm. Serial dilutions may be used for both
range-finding and final stages of toxicity testing.
solvent. A liquid that is
capable of dissolving other chemicals.
sublethal concentration. A
concentration of chemical that does not kill an organism.
sublethal effect. A biological
effect caused by chemical exposure at a concentration below that which causes
death.
threshold concentration/dose. In
reference to a dose-response relationship, the threshold dose/threshold
concentration is the minimum amount of a chemical that just causes an
observable effect in a group of organisms. [compare to no observed effect level]
toxic. Synonym = harmful or
poisonous.
toxicant. Synonym = poison.
Any chemical that causes harmful biological effects.
toxicity. The capacity of a
chemical to produce harmful effects.
toxicity test. A controlled test in which the effects of a
toxicant are studied on living cells, tissues, or organisms.
toxin. A toxicant produced by a
living organism. [compare to toxicant
and contaminant]
toxicology. The study of the
adverse effects of chemicals on living organisms.
voltage-gated channel: A
membrane channel protein, usually in nerve and muscle, that opens (or closes)
in response to membrane depolarization.
Voltage-gated channels generate electrical impulses (= action
potentials).
water soluble/hydrophilic.
Refers to chemicals that are soluble in water but not in lipids.
[compare to lipid soluble]
xenobiotic. Any chemical that
does not occur in the normal biochemical pathways of an organism; a xenobiotic
compound is a compound that is “foreign” to the organism.