The TLV-TWA of a chemical is the average airborne concentration to which

nearly all workers may be exposed for an 8-hour day and a 40-hour week without adverse effects

workers may be exposed for 15 minutes but never longer

half of the exposed test animals die within 96 hours

nearly all workers may be exposed for an 8-hour day and a 40-hour week without adverse effects

the general public may be exposed continuously for a lifetime

the protozoan Entamoeba histolytica

Which of the following are phase II biotransformation reactions? Select all that apply.

Conjugation with glucuronic acid

Hydroxylation by cytochrome P450

Which plant is a well-known hyperaccumulator used in the phytoextraction of arsenic?

Water hyacinth, Eichhornia crassipes

Assertion (A): Outbreaks of cryptosporidiosis can occur even from properly chlorinated drinking water. Reason (R): Cryptosporidium oocysts are highly resistant to chlorine at the doses used in water treatment. Choose the correct option.

Both A and R are true, and R is the correct explanation of A

Both A and R are true, but R is not the correct explanation of A

In toxicology, the margin of safety of a substance is defined as

the dose lethal to 1% of the population divided by the dose effective in 99% (LD₁ ÷ ED₉₉)

the difference between the NOAEL and the LOAEL

the median lethal dose divided by the median effective dose (LD₅₀ ÷ ED₅₀)

the median effective dose divided by the median lethal dose

Environmental Biology IV: Toxicology and Microbiology — Absorption, Distribution, Biotransformation and Excretion, Acute and Chronic Toxicity, Bioassay, TLV, Margin of Safety and Therapeutic Index, Water-Borne Diseases and Airborne Microbes — and Environmental Biotechnology: Bioremediation, Bioindicators, Biofertilisers, Biofuels and Biosensors

Unit III ends with two applied items. The first is toxicology and microbiology: how a toxic agent is absorbed, distributed, transformed and excreted; the difference between acute and chronic toxicity and the dose-response curve; bioassays; the threshold limit value, margin of safety and therapeutic index, with the arithmetic worked; and the major water-borne diseases and airborne microbes. The second is environmental biotechnology: bioremediation, in situ and ex situ, by microbes and plants; bioindicators; biofertilisers; biofuels; and biosensors. The chapter is written at textbook level: effects on health are stated factually, as the examiner asks for them.

1. Absorption, distribution, biotransformation and excretion of toxic agents

Toxicokinetics follows what the body does to a toxicant. Absorption occurs through the lungs (gases, vapours and fine particles, the fastest route to the blood), the gastrointestinal tract (contaminated food and water) and the skin (fat-soluble solvents and many pesticides). Lipid-soluble, non-ionised molecules cross membranes most easily. Distribution by the blood carries the toxicant to its sites of action and storage: fat-soluble organochlorines and PCBs are stored in adipose tissue, lead, fluoride and strontium in bone, cadmium in the kidney and liver; the blood-brain barrier excludes many substances but not methylmercury, and the placenta passes many toxicants to the fetus. Storage depots can release their contents later, as when fat is mobilised during weight loss or lactation.

Biotransformation, mainly in the liver, converts fat-soluble chemicals into water-soluble ones that can be excreted. Phase I reactions (oxidation, reduction and hydrolysis, above all by the cytochrome P450 monooxygenases) add or expose a reactive group; phase II reactions conjugate the product with glucuronic acid, sulphate, glutathione or amino acids. Usually this detoxifies, but sometimes it bioactivates: benzo[a]pyrene is harmless until P450 enzymes convert it to a diol epoxide that binds DNA, which is why it is carcinogenic. Excretion is mainly by the kidneys in urine and by the liver in bile and faeces; volatile compounds leave through the lungs, and some toxicants are excreted in milk, sweat, hair and nails, which is why hair is used to monitor mercury and arsenic exposure. The bioconcentration factor, the ratio of the concentration in an organism to that in water, measures how strongly a chemical accumulates; a fish holding 5 mg/kg of a pesticide present at 0.002 mg/L in the water has a BCF of 2,500 L/kg.

2. Acute and chronic toxicity, bioassay, TLV, margin of safety and therapeutic index

Acute toxicity results from a single dose or a short exposure, usually within 24 to 96 hours, and is measured by the median lethal dose (LD₅₀, the dose in mg per kg of body weight that kills half the test animals) or, for exposure through water or air, the median lethal concentration (LC₅₀). The lower the LD₅₀, the more toxic the substance. Chronic toxicity results from repeated exposure to lower doses over a long period, a large part of the lifespan, and shows as cancer, organ damage, reproductive and developmental effects or neurotoxicity; it is characterised by the no-observed-adverse-effect level (NOAEL) and the lowest-observed-adverse-effect level (LOAEL). The dose-response curve for most toxic effects is S-shaped with a threshold below which no effect is seen; for genotoxic carcinogens regulators generally assume no threshold, so that any dose carries some risk. Two toxicants together may act additively, synergistically (asbestos and smoking multiply the risk of lung cancer) or antagonistically.

A bioassay measures the effect of a substance or effluent on living organisms under standard conditions, and it captures the combined toxicity of a mixture that chemical analysis of single substances cannot. Standard aquatic bioassays include the 96-hour fish LC₅₀, the 48-hour Daphnia immobilisation test and algal growth inhibition; the luminescence of the bacterium Vibrio fischeri gives a rapid screening test. A "safe" concentration for continuous exposure is often estimated by multiplying the 96-hour LC₅₀ by an application factor, commonly between 0.01 and 0.1.

Measure	Definition	Interpretation
Threshold limit value, time-weighted average (TLV-TWA)	The average airborne concentration to which nearly all workers may be exposed for an 8-hour day and 40-hour week, day after day, without adverse effect (set by the ACGIH)	An occupational, not an ambient, standard
TLV-STEL and TLV-C	Short-term exposure limit, a 15-minute average not to be exceeded; ceiling, a concentration never to be exceeded even briefly	Protect against acute effects of peaks
Therapeutic index	TD₅₀ ÷ ED₅₀ (or LD₅₀ ÷ ED₅₀): median toxic or lethal dose divided by median effective dose	The larger it is, the safer the substance
Margin of safety	LD₁ ÷ ED₉₉: the dose lethal to 1% divided by the dose effective in 99%	More conservative than the therapeutic index because it compares the tails of the two curves

A worked case: if a pesticide used as an antiparasitic in livestock has an LD₅₀ of 400 mg/kg and an ED₅₀ of 20 mg/kg, its therapeutic index is 400 ÷ 20 = 20. Two substances with the same therapeutic index can differ in margin of safety if one has a much steeper dose-response curve. In environmental risk assessment the term margin of safety (or margin of exposure) is also used for the NOAEL divided by the estimated human exposure, where a large ratio means the exposure is well below the level at which effects begin.

⚠️ TLVs are for workers, not for the public

A TLV assumes healthy adults exposed for 8 hours a day, 5 days a week, with time to recover in between. It cannot be used as an ambient air-quality standard for children, the elderly or the sick breathing the air continuously; that is what the National Ambient Air Quality Standards are for.

3. Major water-borne diseases and airborne microbes

Pathogen group	Disease and agent	Note
Bacteria	Cholera (Vibrio cholerae), typhoid (Salmonella Typhi), bacillary dysentery (Shigella), diarrhoea from pathogenic Escherichia coli	Transmitted by the faecal-oral route; controlled by safe water, sanitation and chlorination
Viruses	Hepatitis A and E, poliomyelitis, rotavirus diarrhoea	Hepatitis E causes large water-borne outbreaks in India and is especially dangerous in pregnancy
Protozoa	Amoebic dysentery (Entamoeba histolytica), giardiasis (Giardia), cryptosporidiosis (Cryptosporidium)	Cysts and oocysts resist chlorine; Cryptosporidium is removed mainly by filtration
Helminths	Guinea-worm disease (Dracunculus medinensis), ascariasis	India was certified free of guinea-worm disease by the WHO in 2000

Because pathogens are many and scarce, water is tested instead for indicator organisms that are always present in faeces in large numbers and survive about as long as pathogens: total coliforms, faecal (thermotolerant) coliforms and above all Escherichia coli, whose presence proves faecal contamination. Airborne microbes, or bioaerosols, include bacteria such as Mycobacterium tuberculosis and Legionella pneumophila, which grows in cooling towers and hot-water systems and causes Legionnaires' disease; viruses such as influenza, measles and SARS-CoV-2, spread by droplets and fine aerosols; and fungal spores of Aspergillus, Penicillium, Alternaria and Cladosporium, which cause allergies and asthma and, in people with weakened immunity, infection. Crowded, poorly ventilated and damp buildings concentrate them. They are sampled with impactors such as the Andersen sampler, which sorts particles by size onto culture plates.

4. Bioremediation: in situ and ex situ, by microbes and plants

Bioremediation is the use of living organisms, chiefly microbes and plants, to degrade, transform, remove or immobilise pollutants in soil, water or sediment. Microbes mineralise organic pollutants to carbon dioxide and water or transform metals to less toxic or less mobile forms. Among the best-known agents are Pseudomonas species, including the oil-degrading strain for which Ananda Chakrabarty obtained the first patent on a genetically engineered organism after Diamond v. Chakrabarty (United States Supreme Court, 1980); the white-rot fungus Phanerochaete chrysosporium, whose lignin-degrading peroxidases also break down PAHs, dyes and chlorinated compounds; and Deinococcus radiodurans, which tolerates intense radiation. In India a bacterial consortium developed by TERI, marketed as Oilzapper, has been used to clean oil-contaminated soils and sludges.

Approach	Techniques	Principle
In situ (treated in place)	Biostimulation, bioaugmentation, bioventing, biosparging, monitored natural attenuation	Supply oxygen, nutrients or microbes to the contaminated zone without excavation; cheaper and less disruptive but slower and harder to control
Ex situ (excavated or pumped out)	Landfarming, biopiles, composting, slurry-phase bioreactors, pump-and-treat with bioreactors	Treat the material under controlled conditions; faster and more certain but costly

Phytoremediation uses plants. Phytoextraction takes metals up into the shoots, which are harvested; hyperaccumulators such as Noccaea (formerly Thlaspi) caerulescens for zinc and cadmium, Indian mustard (Brassica juncea) for lead and the Chinese brake fern Pteris vittata for arsenic are the models. Phytostabilisation immobilises metals in the root zone to stop erosion and leaching; phytodegradation and rhizodegradation break down organics within plants or through microbes in the rhizosphere; rhizofiltration absorbs contaminants from water onto roots, as sunflowers did for radionuclides in ponds near Chernobyl; and phytovolatilisation releases selenium or mercury to the air in volatile form, which moves rather than removes the problem.

5. Bioindicators, biofertilisers, biofuels and biosensors

Tool	Examples	Environmental use
Bioindicators	Lichens, which disappear near sources of SO₂ (the "lichen desert" of cities); mayfly, stonefly and caddisfly larvae in clean streams; Tubifex worms and red chironomid larvae in organically polluted water; E. coli for faecal contamination	Integrate pollution over time; biotic indices score a stream by which groups survive
Biofertilisers	Rhizobium in legume nodules; free-living Azotobacter and associative Azospirillum; cyanobacteria and the Azolla-Anabaena symbiosis in rice fields; phosphate-solubilising bacteria such as Bacillus megaterium; arbuscular mycorrhizal fungi such as Glomus	Supply nitrogen and phosphorus biologically, reducing chemical fertiliser use and its runoff
Biofuels	First generation: ethanol from sugarcane molasses and grain, biodiesel from vegetable oils; second generation: cellulosic ethanol from crop residues; third generation: algal oils; biogas	Replace fossil fuels; first-generation fuels compete with food crops for land, which later generations avoid
Biosensors	A biological recognition element (enzyme, antibody, nucleic acid or whole cell) coupled to a transducer (electrochemical, optical or piezoelectric)	Rapid field detection: acetylcholinesterase sensors for organophosphate and carbamate pesticides, microbial BOD sensors, luminescent bacterial sensors for toxicity

ℹ️ The first biosensor

Leland Clark described the enzyme electrode in 1962: glucose oxidase trapped over an oxygen electrode, so that the fall in oxygen measured glucose. The same design principle, an enzyme whose activity is converted to an electrical signal, underlies most environmental biosensors, including those that detect pesticides by the inhibition of acetylcholinesterase.

Key takeaways

Toxicants are absorbed through the lungs, gut and skin, stored in fat, bone or kidney, biotransformed by phase I (P450) and phase II (conjugation) reactions, sometimes into more toxic forms, and excreted mainly in urine and bile.
Acute toxicity is measured by LD₅₀ or LC₅₀ and chronic toxicity by NOAEL and LOAEL; bioassays capture the toxicity of whole effluents.
The TLV-TWA is an occupational 8-hour, 40-hour-week limit; the therapeutic index is TD₅₀ (or LD₅₀) ÷ ED₅₀ and the margin of safety LD₁ ÷ ED₉₉.
Cholera, typhoid, hepatitis A and E, amoebiasis, giardiasis and cryptosporidiosis are water-borne and traced by E. coli as the indicator; Legionella, tuberculosis, influenza and fungal spores are airborne.
Bioremediation is in situ (biostimulation, bioaugmentation, bioventing, biosparging) or ex situ (landfarming, biopiles, bioreactors); phytoremediation uses hyperaccumulators such as Pteris vittata for arsenic; bioindicators, biofertilisers, biofuels and biosensors complete the toolkit.

Practice questions (10)

Attempt each one before opening the answer. Every explanation names the tempting wrong option as well as the right one, because that is where marks are lost.

A veterinary antiparasitic compound has an LD₅₀ of 400 mg/kg and an ED₅₀ of 20 mg/kg in the target animal. What is its therapeutic index?
Numerical answer — type the value.
Show answer
Answer: 20
Therapeutic index = LD₅₀ ÷ ED₅₀ = 400 ÷ 20 = 20; the larger the index, the wider the gap between the effective and the lethal dose. Dividing the ED₅₀ by the LD₅₀ (0.05) inverts the ratio.
The TLV-TWA of a chemical is the average airborne concentration to which
1. workers may be exposed for 15 minutes but never longer
2. half of the exposed test animals die within 96 hours
3. nearly all workers may be exposed for an 8-hour day and a 40-hour week without adverse effects
4. the general public may be exposed continuously for a lifetime
Show answer
Answer: C — nearly all workers may be exposed for an 8-hour day and a 40-hour week without adverse effects
The TWA is an occupational limit for a normal working day and week. A lifetime limit for the public is an ambient standard, a concentration killing half the animals is the LC₅₀, and the 15-minute limit is the short-term exposure limit (STEL).
Cholera is caused by
1. the hepatitis E virus
2. the bacterium Vibrio cholerae
3. the bacterium Salmonella Typhi
4. the protozoan Entamoeba histolytica
Show answer
Answer: B — the bacterium Vibrio cholerae
Cholera is a bacterial disease of Vibrio cholerae spread through faecally contaminated water, causing profuse watery diarrhoea. Entamoeba causes amoebic dysentery, hepatitis E virus causes jaundice, and Salmonella Typhi causes typhoid.
Which of the following are phase II biotransformation reactions? Select all that apply.
1. Conjugation with sulphate
2. Conjugation with glucuronic acid
3. Conjugation with glutathione
4. Hydroxylation by cytochrome P450
Show answer
Answer: A — Conjugation with sulphate; B — Conjugation with glucuronic acid; C — Conjugation with glutathione
Phase II reactions attach a polar group to the molecule, making it water-soluble for excretion: glucuronide, sulphate and glutathione conjugation are the main ones. Hydroxylation by cytochrome P450 is a phase I oxidation that usually prepares the molecule for conjugation.
Which plant is a well-known hyperaccumulator used in the phytoextraction of arsenic?
1. Lantana camara
2. Water hyacinth, Eichhornia crassipes
3. The Chinese brake fern, Pteris vittata
4. Sunflower, Helianthus annuus
Show answer
Answer: C — The Chinese brake fern, Pteris vittata
Pteris vittata can accumulate arsenic in its fronds at concentrations thousands of times those in soil and is the standard arsenic hyperaccumulator. Sunflowers were used in rhizofiltration of radionuclides, and water hyacinth absorbs many pollutants from water but is not the arsenic model.
Assertion (A): Outbreaks of cryptosporidiosis can occur even from properly chlorinated drinking water. Reason (R): Cryptosporidium oocysts are highly resistant to chlorine at the doses used in water treatment. Choose the correct option.
1. Both A and R are true, and R is the correct explanation of A
2. Both A and R are true, but R is not the correct explanation of A
3. A is true, but R is false
4. A is false, but R is true
Show answer
Answer: A — Both A and R are true, and R is the correct explanation of A
Both are true and R explains A. Because chlorine at normal doses does not inactivate the oocysts, removal depends on effective coagulation and filtration or on ultraviolet disinfection; the Milwaukee outbreak of 1993, which sickened hundreds of thousands, followed a failure of filtration.
In toxicology, the margin of safety of a substance is defined as
1. the dose lethal to 1% of the population divided by the dose effective in 99% (LD₁ ÷ ED₉₉)
2. the difference between the NOAEL and the LOAEL
3. the median lethal dose divided by the median effective dose (LD₅₀ ÷ ED₅₀)
4. the median effective dose divided by the median lethal dose
Show answer
Answer: A — the dose lethal to 1% of the population divided by the dose effective in 99% (LD₁ ÷ ED₉₉)
The margin of safety compares the lowest lethal tail with the highest effective tail of the two dose-response curves, LD₁ ÷ ED₉₉, which makes it stricter than the therapeutic index, LD₅₀ ÷ ED₅₀. The inverted ratio and the NOAEL-LOAEL gap are not the definition.
Which of the following are in situ bioremediation techniques? Select all that apply.
1. Biosparging
2. Landfarming of excavated soil
3. Bioventing
4. Biopiles
Show answer
Answer: A — Biosparging; C — Bioventing
Bioventing supplies air to unsaturated soil and biosparging injects air below the water table, both treating contamination where it lies. Landfarming and biopiles first excavate the soil and treat it elsewhere, which makes them ex situ methods.
Which organisms are most widely used as bioindicators of sulphur dioxide pollution in air?
1. Coliform bacteria
2. Water hyacinth
3. Lichens
4. Tubifex worms
Show answer
Answer: C — Lichens
Lichens absorb water and gases over their whole surface and have no protective cuticle, so they are highly sensitive to SO₂ and disappear near sources, leaving "lichen deserts" in polluted cities. Tubifex indicates organic pollution of water and coliforms faecal contamination.
A pesticide is present in lake water at 0.002 mg/L, and fish from the lake contain 5 mg/kg of it. What is the bioconcentration factor in L/kg?
Numerical answer — type the value.
Show answer
Answer: 2500
BCF = concentration in organism ÷ concentration in water = 5 mg/kg ÷ 0.002 mg/L = 2,500 L/kg. A high BCF marks a fat-soluble, persistent chemical; dividing the water concentration by the fish concentration gives 0.0004, the inverse.