Lethal= causing death

Sub-Lethal=below a level that directly causes death

1)Population: density, productivity, mating, etc 2)Individual: Mortality, reproduction, necrosis (cancer), teratogensis (tumour), etc

3) Biochemical: Enzyme inhibition, DNA, endrocine, stress proteins

1. Acute: A stimulus (contaminant) including a rapid response. Brief intense exposure. Short test (4 days)

2. Chronic: Effects after prolonged use. Stimulus that lingers/continues for a long period of time. Approximatley 10% of life span.

3. Subacute: Stimulus is less servere than acute stimulus. Response in longer period of time.

4. Cummulative: Toxicity brought about by successive additions (could be different times/ different ways)

A test for either lethality, growth, reproduction, development over the entire life cycle of an organism

How does Cirtical life stage testing differ from early life stage testing?

Critical : Toxicity at an important life stage

Early : Subset of critical which focuses on the early stages and it is the most sensitve phase

What is weakest link incongruity?

1. Time- Time required to get a response

2. Toxicant dose/concentration- The amount required for average orangism to respond in a lifetime

3. Test type (Static toxicity=same medium, Static renewal-change it, flow through=constant concentration)

Biological Assay

Experiment for estimating the potency of a material by the reaction of a biological organism.

Smallest dose required to kill an individual

- Same individual will response different if conducting the same test

Conc: the amount of toxicant on a volume basis given externally

Dose: the amounf of toxicant applied internally

No Observed Effects Concentration

-Before inflection

Lowest observed effect concentration

-inflection point

Minimum Threshold Concentration

-Based on dose

Maximum Allowable Toxicant Concentration

-Set up by the government

-Usually :

NOEC<MATC<LOEC

-increases confidence of the EC50

-But more error as you move away from it

-At low concentrations there is a positive sitmulation (positive response)

- a stimulatory effect exhibited with exposure to low subinhibitory levels of some toxicants or physical agents

-a biphasic dose-response phenomenon characterized by a low-dose stimulation and a high-dose inhibition 

Threshold:

- Common conc. response (0-100%)

-used in most cases except cancer risk and the recommendations of the safe drinking water act

-assumptions don't always hold (at low conc and chronic exposure)

-lacks the ability to provide accurate low-dose predictions, thus failing to deliever societial need

Hormesis:

-biphasic conc. response (-30 - 100%)

-Occurs about 40% of the time

-"to excite"

-A biphasic dose response characterized by a low-dose stimuation and a high-dose inhibition

A pratcial means to assess many doses in the below toxic threshold zone

-Lower concentration and more concentrations are readily measured.

When testing whole organism what is the expected outcome of:

1) Acute

2)Chronic

3)Multi-species

1) Endpoint is survival

2)  Growth/Reproduction

3)Survival/Growth

1. Needs to be widely availalbe

2. Readily cultured in labs

3. Genetic history known

4. Relative toxicity of random toxicant should be known

5. Should respresent sensitivity of the env.

6. For multi-species test, interaction should be understood

7. Meets regulatory madates

NO phenomenom/Expected results

Null effect

Describe the Daphia magna Test.

Advantages+Disadvantages

- Fresh water micorcrutacean, cladocreran, water flea

Test (For 48 HOURS): 

-10 neonate (<24 hour old) in a stravation test

- 100 mL (5 conc. + negative control X3)

- Room light ( 16 hr light+8 hr dark)

- Static renewal

- Endpoint = death / immobility 

- measure every 24 hr

-Positive control: Pentachlorophenol, copper

Advantages: Fast, small volumes, small waste, very sensitive, either chronic or life cycle test

Disadvantages: Very sensitive, no food (stress), dose response very steep

When water is churrened it adds oxygen which oxidizes the luminescence menchanism 

-it stimulates the electron transport by turing on the luminescence mechanism

Biotic:

-Test organism (variation between each species)

-Life stage& size (development stages more susceptible, while large mammals are less sensitive)

-Health & Nutrition ( Potential for stress)

-Acclimation/Acclimatization (modification in biological function, can increase tolerance)

-Sex differences ( different endocrine disruptors impact reproduction differently) 

-Genetics (different populations of the same species have different sensitivity)

Abiotic:

-Temperature (more stress)

-Dissolved O2 (Low O2 adds stress, but high o2 can create oxygen radicals)

-pH ( ammonia toxicity inceases as ph increases, while low metal count decrease pH)

-water hardness (more toxic in soft water- less competition with Ca)

-Binding& sorption ( suspended metals complex/chelate out)

-Light (increase toxicity)

-Salinity (no impacts?)

Avian Toxicity Testing?

What are the 3 types of tests?

- Main factors: DDT and the thinning of egg shells due to pesticides

-acute, sub-acute, reproduction

Acute testing

- Oral dose based on mg active ingredi/kg body

-dosing 24 hr after fasting (clean gut)

-5 dose (3 mortality level)

-4 day test

Subacut testing

-younger birds

-chemcial in diet

-longer duration 

Reproduction test:

-endpoint: fertility, viable eggs

-chemical in diet with light conditions that does not promote breeding (7 hr light) then (16 hour light) 

-most sensitivite 

Acclimation = lab

Acclimatization = field

Both - the modificatin of biological functions, especially those physiological, or structures to maintain or minimize deviations from homeostasis despite changes to the environmetal qualiites 

Which is more toxic brown lakes or clear lakes?

brown less toxic which can be seen through the biological life that exists

clear lakes more toxic more harsher chemicals less biological life

1) Narcosis: membrane damage

(high levels of chemcials = attack lipids= leaky)

2)biochemical toxicity

-enzyme inhibition

-chemicals bind&block enzyme making

-can attack ribosomes (no DNA/RNA)

3)Reactive oxygen species

-oxidiative damage

4)Endocrin toxicity (hormones mimic& reproduction)

5) Genotoxicity (mutations, stops replication)

Random no threshold 

-mutations in genotoxicity can divide out of control since there is no threshold

-finite probability since no end

1) Detoxificiation : Degradation and excretion

2) Acclimation: Defense response by adding sugars

Metabolic: Iron, Calcium, Zinc, Sodium, Potassium

Non-Metabolic: Lead, Cadmium, Tin, Nickel, Mercury, Silver

Class A metal - Binds to O stronger than S

-Attacks sugars, proteins and DNA

Class B - Binds to S stronger than O

-Attacks proteins, and active sites of enzymes (sulphide bridges)

-Since there are alot of O sites more competetion so not as much of a threat

Enzymes

Fe: Electron transport, Super oxide Dismutase, CytoP450

Zn: Super Oxide dismutase

Mg&Mn:Photosynethesis

Cu: Electron transport

Ca: Cell signalling

Ca&Na: Substrate in ion pumps

Structural 

Fe: hemoglobin, cell structure

Zn:Zinc fingers

Ca: Bone structure, cell structure

-Biological Ligand Model

- An approach that integrates chemcial equilibrium predictions and potential metal binding with ligand associated with external phases and biological surfaces to model the potential for metal effects

More toxic in soft

soft ( 10-20 for Cu and 50-110 Zn)

Hard (30-100 for Cu and 1000-1500 Zn)

Hard has higher amount of Ca in the water - competetion for Cu and zinc

-Reactive Oxygen Catalytic Mechanism

-Creates a super oxide (O2 -)

ROS : Reactive oxygen species

1. Hydroxide radicial (OH')

2. Superoxide (O2 - )

3. H2O2 (long lifespan, least reactive and most stable)

-Metabolic

-Evidence for acute toxity through the inhibition of ion pumps

-in Plants: inhibits photosynethesis (PSII at high conc.- BLM)

-Cu inhibits respiration at high conc. (BLM)

-Cu binds to DNA distrubing base pairs (BLM)

-at low concentrations (RCM) damaging lipids, protein and DNA

BLM - BIOLOGICAL LIGAND MODEL

- Direct binding to biological receptor

-Damage to membrane lipids, membrane of proteins, ion pumps, enzymes,

-Dominant at high concentration or at long times

-Acute effects

RCM - Reactive Oxygen Catalytic Mechanism 

- Catalytic production of ROS

-More than just 1:1 effect: Amplification

-Dominates at lower concentrations

-Chronic effects

-Damages lipids, proteins, DNA& Rna

-non metabolic

-Creates the superoxide

-Binds to the SH group on proteins-imparing the function(BLM)

-Drinking water Decreases lifespan of rats 10% (Chronic RCM)

-Western canada has high levels of Cd and on the rise everywhere due to batteries

-liver failure in mammals

-40mg inhaled = fatal in humans (RCM)

-inhibits growth in plants

-impars reproduction in Daphnia and Rainbow trout (low conc., chronic=RCM)

- toxic form is inorganic Pb

-But exists as organo-metalics (Tetraethyl lead, and tetramethyl lead)

-once in organism looses the organic and the Pb is in it toxic form

-non-metabolic 

-toxic as inorganic Pb, but exists as organo-lead

-loses the organic once its in the organism

Mammals:

- initally accumulates in liver and kidneys then enters the blood stream attacking red blood cells (anemia) 

-small amount crosses the blood-brain barrier (CNS problems, Placental barrier)

-Accumulates in bone and hair

-cummulative poison 6 year half life(BLM) 

-Renal failure (protein in urine) + CNS collapse

- Inhibits Aminolaevulinci acid dehydratase (ALAD)

-enzyme in heme biosythnesis (BLM)

-Specifically and rapidley inhibited by Pb

-slow reversal

- land plant typically dont take up Pb since it is not mobilize 

- slows cell division at 1-5 ug/L

-can impair germination, growth, photosynethesis at 5-10 ug/L (BLM)

-LC50 - 1000-2000 ug/L

- non-metabolic

-Toxic as inorganic Hg but exist as organometalic

-methyl-mercury (very toxic once methyl removed)

-Microbes convert Hg-> methyl mercury

-can crosslink DNA strands causing single strands to break (cancer)

Animals: effects on CNS+ brain (methylmercury accumulates in the brain) 

-CNS affects the transfer of charge/neurons (BLM) 

-High doses = tremors- loss of consciousness- death (BLM)

-reproduction - fetus 2X more methylmercury than mom

-Metals which cannot be metabolised are sequestered - Metallothioneins (phytochelation in plants) (Cu, Zn, Cd, Ni)

- superoxide dismutase (reduces superoxide radical)

-Catalase (removes h2o2)

-glutathione peroxidase (remove hydrogen peroxide)

- polypeptides resembling equine

-Small proteins, including phytochelation, which are polypeptide

-function in the uptake, internal compartmentalization, sequestration, and excretion of essential and nonessential metals

- can uptake 2 metals/proteins (metals bind cooperatively to the sulfhyrul group of the cysteine)

-high cysteine 

-stress related protein

- High Metallothioneins mean tolerance

-indicator of gene probes 

-superoxide dismutase (SOD) 

-decreases the amount of the superoxide radicals in the cell through this equation

2O2^- + 2H⁺ -> H₂O₂ + O₂

-Catalase or Glutathione peroxidase

-decreases the levels of hydrogen peroxide

-2H₂O₂-> 2H₂O + O₂

-2GR + H2O2 -> GRox + H2O

Metal transcription factors

MTF-1 :

 A zinc responsive transcription factors responsible for regulating the expression of major metallothionenin genes in varied species 

- produce antioxidants (Vitamine E, C, Beta carotene, catecholamines, etc) 

-Metallothionein - because of the high number of sulfhydryl groups

Hypersensitivity 

- Anything will trigger death?

Energy that can be transferred from one body to another through a vacuum

Nonionizing radiation: Light, radio, etc (harmless)

Ionizing Radiation: High energy photons from radioactive nuclides

- its the excitation or ionization that causes disruption in molecular bonds & biological damage

beta particle: electron or positron ejected from nuclear decay

Alpha particle: Pieces of nucleus ejected from a radioactive atom to reduce excess energy and gain stability (charge of He +2= 2 neutron and 2 protons)

Ionization: 

Gamma ray hits a molecule= Electron ejected + unstable molecule

-it can either try to recapture the electron (unlikely) or react with other molecule to get an electron

Excitation:

Gamma ray hits a molecule= Excited molecule

-returns to ground stat by luminescening

-or dissociates (breaks more bonds causing damage)

-transfers energy to another molecule (damage or heat)

Direct: hits molecules and breaks bonds

Indirect: Creates a free radical or excited molecule which does damage

no defence mechanism for radiation

OR

Somatic

- damage to enzymes(proteins-most damage), cell membrane, cell organelle

-rapid and acute cytotoxicity(damage to protein)

-impacts to bone marrow, gastrointestinal, and CNS

Genetic

-Damage to DNA with mutation that can lead to cancer

-Chronic effect

-Low water soluble

-lipophilic

-Hydrophobic

-unreactive (unless activated)

Photochemical Activation 

- PAH absorbed solar UV radiation ( ↑ Light ↑ PAH toxicity)

- Photosensitization: Simulation exposure of the organism and PAH to UVs (inside the organism)

-Photomodification: Pre-exposure of PAH to UV is Pah photooxidation (oxyPAH) 

Biochemical Activation

- Enzymatic transformation of PAH (hydroxylation) where the organism excretes a soluble PAH, but oxyPAH more reactive

Endocrine Disruption

- some oxyPAH mimic hormones

-Induces cytochrome P450

-Can be outside of the organism 

-Oxidizes the PAH to a triplet state

-UV modification of a compound that involve photooxidatin or photolysis

- has to be within the organism

-impacts greatest on the organism transparent to UV

-Tissue damage

-Forms a singlet state oxygen which can do damage

-can inhibit photosynethisis and respiratioon 

-1,2 dhATQ mimics UQ

-creates a hydroxyl radical instead of water 

- heme containing enzyme that catalyzes the oxidative (sometimes reductive) biotransformation of organic compounds 

-detoxication and toxication (activation) process

Cytochrome P450

epoxide hydroxylase 

- Induces Cytochrome P450 Expression 

-AH receptors induce live p450IA1 at the transcriptional level

-metabolism and elimination 

To Eliminate: 

Hydroxylation(Phase 1)->Conjugation(Phase II)-> Elimination 

- narcosis at high concentrations

- usually metabolised ( no biomagnification) unless activated 

- higher MW of PAH bigger problems

- do not magnify in fish = Cytochrome p450 + glutathione S-transferase

-Oxidized in liver than excreted

- more cases of cancer in industrialized regions 

Examinationof tissues of plants/animals

- Common endpoint

Polychlorinated biphenols

- highly lipophilic

-persistent

- More toxic when eclipsed

- 2 benzene rings with at least 2 Cl

-typically from paper mills

- uptake -> blood-> liver metabolism -> excreted

- more Cl the harder it is to metabolise

- induces cytochrome p450

-to metabolise need 2 adjacent carbons without chlorines

-if they cannot be metabolised then they remain persistent in the organism( bioaccumulation)

-Narcosis after long time ( membrane damage)

- mutation - animals

Endocrine disruption: alter the p450 activity increassing hormone level, or can also mimic hormones

Pophyria 

-Increased activity of aminolevulenic acid synethetase

- increases porphyrin levels in blood, liver and bile

-photosensitization results

- highly lipophilic 

- 100% assimilation 

-most toxic and potent

-used as a herbicide

- once used for agent orange

- liver necrois, liver hemorrhaging

- immo-suppressant (endocrine)

- teratogenic (tumor) (mutagenic and carcinogenic)

-induce cytochrome p450 (need adjacent hydrogens)

- too much for CP450 to control

-Potency of different dioxins related to AH association and activation 

Cytochrome P450 

even tho it wont do anything since it can metabolise

-Insecticides, herbicides, rodenticides

-use mechanisms well know designed to kill pest

Integrated Pest management 

- pests to control pest

-insecticide

-inexpensive but chronic toxicity and persistance 

- genetic resistence is a growing problem

- chlorinated ethanes

 -attacks Central Nervous system by closing the Na+ channels = immobilization of specie

- creates inoluntary nerve impulses in non target species

dichloro-dibenzo-trichloroethane

-insecticide

-egg shell thinning

-high persistance 

-insecticide

-inexpensive but more acutely toxic to non target species

-more water soluble

- more degradable

-less persistance

- attacks the target species nerve system

-inhibits ACETYLCHLOLINE ESTERASE

-for it to be degradable needs NO2 or P no Cl

Nicotine:

-Targets Aphids

-Nerve poision at acetylchloine esterase receptor

-Degrades in light

-Toxic for mammalian - addiction

Rotenoid

-Targets aphids

-electron transport inhibitor

Pyrethrums

- highly toxic to insects, with little effect on humans

- nerve poison - ion channels

Permanent Removal: Diuron or Atrazine

-  high dose & non selective

Temporary Removal: Paraquat or glyphosate

-non persistent & non selective

- 2,4-D selectively removes dicots (dandilions not grass)

- inhibits photosystem II

       - competition to inhibit Quinone binding site

       - toxicity to algae and any other plants down stream

-Binds to QUinone B preventing it from binding which in turn inhibits photosynethesis

- Takes oxygen from triplet state to singlet state = damage

- Corn is tolerant of atrazine (enzyme hydroxylates at Cl)

- Roundup

- Inhibitor of metabolism 

- Inhibits synthesis of aromatic amino acids

- inhibits 5-enolpyruvylskikimate-3-phosphate synthetase

-rapidely degrades in soil (1 year)

- non toxic to non plant

-Paraquat

- Free radical generator

- accepts electrons from Photosystem I - Creating superoxides 

- fast

- attacks everything (non specific) 

-can attack mitochrodia 

-LC50 mammals = 100 mg/kg

-it requires photosysthesis to occur

-mixtures(you'll never get just one chemcial) 

        -metals+oxyPAH=synergistic effects making it worse

-sunlight

         -uv increases toxicity

-temperature (oxidative stress)

-drought

-pests

-partioning of chemicals

         -can increase or decrease availablity

-blocks respiration 

-produces ROS

- Cu redoxs creating hydroxy radical

- increase toxicity 

- when its just 1,2-dhATQ - inhibits respiration by 50%

-1,2-dhATQ +Copper = inhibits by 100% (even at low conc.)

Biological response at higher organizational levels 

(populations, community, ecosystems)

Demonstrates adverse effects on organism 

-measure of real toxicity effect

Indicates exposure of the organism to a contaminant 

BUT does not give information of the degree of effect

Biomarker: Inhibition of ALAD

- Mechanism should be understood 

- Need to connect it to the whole organism's toxicity 

- Does not require know of history of contamination 

- response in test often occurs at low conc levels while the whole organism may respond differently

- Should be easy to measure and see the same effect on the whole organism 

-Aminolevulinic acid dehydratase (creates heme)

- Biomarker for lead

Advantages: More sensitve, accurate than chemcial analysis, measures impacts to organism

Disadvantages: Only useful for lead, since nothing else inhibits ALAD

- Ethoxy-Resorufin-O-Deethylase 

-measures levels of CP450

-P450IA1 induced by PAH, PCB, Dioxins, Furans for detoxificiation 

- need to measure with a substrate that induces the change

- ↑fluorescence = ↑ P450 

Advantages: rapid, sensitivity, and logical response

Disadvantage: Measure exposure, and only the compounds that induces measurable AH receptor change

- Expose an organism to control & contaminant for 1-4 days

       -Vertebrates: isolate liver

       - Cell system : take all cells

       - Invertebrates/ plants: take whole tissue 

- Plants biomarker for contaminant exposure/impact

           - CO2 fixation : inhibits Calvin cycle

           - O2 evolution: Electron transport

           - Chlorophyll A fluroescene: Photosystem II & electron transport

Advantages: measures impact, directly relates the effects to whole plant, can detect many toxicants

Disadvantage: complex response, equipment, given in terms of growth usually

- Northern hydridization 

- Polymerase chain reaction (PCR) 

-Micro Array techniques

Advantages: Sensitive to low conc, reveals mechanism, molecular fingerprint

Disadvantages: hard to connect to the mechanism, labour intensive, open to artifacts

- need to ensure a correlation of concentration response of indicator to the whole organism

- Assumes molecular response (molecular connect whole organism)

- Kinetics of biomarker should mirror kinetics of whole organism

- should detect biomarker in field trial under same conditions that cause whole organims response

- uses plants to remove contaminant from water, soil and air

- cost effective, easy, works 24hour/day

- can be used in conjuction to other treatment

- Phytotoxicity can be an issue

- takes a long time (2-5 yr)

Advantages: Improves natural structure & texture of soil, uses solar energy, cost effective, plants can provide sufficeint biomass for rapid remediation, increases rhizosphere activity, remote areas

What process affect photoremediation?