biodegradation of xenobiotic compounds
TRANSCRIPT
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Biodegradation of Xenobiotic compound, PAH, PCB, Nitrobenzene & DDT
Gunjan Mehta,
VSC,
Rajkot
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Biodegradation of Xenobiotic Compounds
• Xenobiotics : compound have been
produced artificially by chemical synthesis for
industrial or agricultural purposes e.g.
halogenated H.C., aromatics, pesticides, PCB,
PAH, lignin, humic substances
• Recalcitrant : compound totally resistant to
biodegradation e.g. unusual substitute (Cl- or
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H-), unusual bond sequences (3΄ & 4΄), highly
condensed aromatic rings, and excessive
molecular size (polyethylene)
• Co-metabolism : an organic compound is
converted to metabolic products but does
not serve as a source of energy or nutrients
to microorganisms
Ex. insecticides, aliphatic & aromatic H.C.
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Source of xenobiotic compounds
1. Petrochemical industry : oil/gas industry, refineries,
and the production of basic chemicals e.g. vinyl
chloride and benzene
2. Plastic industry :
- closely related to the petrochemical industry
- uses a number of complex organic compounds
such as anti-oxidants, plasticizers, cross-linking
agents
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3. Pesticide industry : most commonly found central
structures are benzene and benzene derivatives,
often chlorinated and often heterocyclic
4. Paint industry : major ingredient are solvents,
xylene, toluene, methyl ethyl ketone, methyl
isobutyl ketone and preservatives
5. Others : Electronic industry, Textile industry, Pulp
and Paper industry, Cosmetics and Pharmaceutical
industry, Wood preservation
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1. Failure of the compound to induce the
synthesis of degrading enzyme.
2. Failure of the compound to enter the m.o. cell
for lack of suitable permease.
3. Unavailability of the compound due to
insolubility or adsorption.
Why compounds are recalcitrant?
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4. Excessive toxicity of the parent compound
or its metabolic products.
5. Unavailability of the proper electron acceptor.
6. Unfavorable environmental factors e.g. temp.,
light, pH, O2 , moisture.
7. Unavailability of the other nutrients (N, P)
and growth factors.
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Factors effect the xenobiotic biodegradation by m.o.
1. Substrate specificity: such as for the type of
aromatic cpd., for the ring position (o-, m-, or p- ),
and for the atom or group removed. Specificities
could reside at the level of enzymes, organisms, or
broad physiological groups
2. Electron acceptors : oxygen, nitrate, and sulfate
most often inhibit dehalogenation by anaerobic
communities
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3. Other nutrients : addition of various nutrients as
electron donors, C-source, N-source, P-source, or
micronutrients can stimulate the reaction or support
the growth of the microorganisms
4. Temperature : affected both the acclimation period
and the rate of biodegradation activity
5. Substrate availability : the hydrophobicity of many
xenobiotic cpd. affects their biodegradation through
its effect on their availability to microorganisms
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Biodegradation of Petroleum compounds
Petroleum compounds are categorized into 2 groups Aliphatic hydrocarbon e.g. alkane, alcohol,
aldehyde Aromatic hydrocarbon e.g. benzene, phenol,
toluene, catechol
H.C. (substrate) + O2 H.C.-OH + H2O
H.C. (substrate) + O2 H.C.OH
OH
monooxygenase
dioxygenase
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Straight chain
aliphatic H.C.
compounds
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Cyclic aliphatic
H.C. compounds
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Aerobic degradation
of aromatic compounds
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- are metabolized by a variety of bacteria, with ring fission
accomplished by mono- and dioxygenases
- catechol and protocatechuate are the intermediates
mostly found in aromatic cpd. degradation pathway
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Some m.o. involved in the biodegradation of xenobiotics
Organic Pollutants Organisms
Phenolic - Achromobacter, Alcaligenes,
compound Acinetobacter, Arthrobacter,
Azotobacter, Flavobacterium,
Pseudomonas putida
- Candida tropicalis
Trichosporon cutaneoum
- Aspergillus, Penicillium
Benzoate & related Arthrobacter, Bacillus spp.,
compound Micrococcus, P. putida
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Organic Pollutants Organisms
Hydrocarbon E. coli, P. putida, P. Aeruginosa, Candida
Surfactants Alcaligenes, Achromobacter,
Bacillus, Flavobacterium,
Pseudomonas, Candida
Pesticides P. Aeruginosa DDT
B. sphaericus Linurin
2,4-D Arthrobacter, P. cepacia
P. cepacia 2,4,5-T
Parathion Pseudomonas spp., E. coli,
P. aeruginosa
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Genetic Regulation of Xenobiotic Degradation
plasmid-borne
mostly in the genus Pseudomonas
PLASMID SUBSTRATE
TOL Toluene, m-xylene, p-xylene
CAM Camphor
OCT Octane, hexane, decane
NAH Napthalene
pJP1 2,4-Dichlorophenoxy acetic acid
pAC25 3-Chlorobenzoate
SAL Salicylate
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Anaerobic degradation
1) Photometabolism : in bacteria this light-induced
“bound oxygen” (OH•) was used to oxidized substrates
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2) under nitrate-reducing condition : Nitrate-reducing bacteria couple the oxidation of org. cpd. with water to the exergonic reduction of nitrate via nitrite to N2
OH OH O
Metabolicpool
3H2 H2O
H2
3) dissimilation through sulfate respiration: Sulfate- reducing bacteria couple the oxidation of org. cpd. with water to the exergonic reduction of sulfate via
sulfite to sulfide
CH3
COOH
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4) The anaerobic fermentation of many polyphenolic substances : m.o. derive their energy from substrate-level phosphorylation while org. cpd. serve as e-donors and acceptors.OH O
Intermediarymetabolism
NADPH H2O
5) Methanogenic fermentation :
OH OH
O
OH OH OH OH
OH C
HOOC CH2
HOOC CHOH
CH2
O
OH
OH
H
H
OH 3H2
OHOH
H2
O
OH O
H2
H2
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1) reductive dehalogenation : two-electron transfer reaction which involves the release of the halogen as a halogenide ion and its replacement by hydrogen
Cl Cl H Cl
C C + XH- C C + Cl - + X
Cl Cl Cl Cl
Mechanisms of Dehalogenation
COOHCl
+ O2 + NADPH + H+
OHOH
+ NAD+ + CO2 + HCl
2) oxygenolytic dehalogenation : catalyzed by mono- or dioxygenases, which incorporate atom of molecular oxygen into the substrate
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3) hydrolytic dehalogenation : catalyzed by halido-
hydrolases, the halogen is replaced by a OH group
which is derived from water.
H2O Cl-
COOH Cl COOH OH
4) thiolytic dehalogenation : in dichloromethane-utilizing
bacteria, a dehalogenating glutathione S-transferase
catalyzes the formation of a S-chloromethyl glutathione
conjugate, with a concomitant declorination taking place.
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5) intramolecular substitution: involved in the
dehalogenation of vicinal haloalcohols
CH2OH-CHOH-CH2Cl CH2OH-CH-CH2 + HCl
O
CH2Cl2 + GSH [GS-CH2Cl] + HCl
[GS-CH2Cl] + H2O GS-CH2OH + HCl
GS-CH2OH CH2O + GSH
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7) hydratation : a hydratase-catalyzed addition of water
molecule to an unsaturated bond can yield dehalogenation
of vinylic compounds
HOOC-CH=CHCl + H2O [ HOOC-CH2-CHOHCl]
HOOC-CH2-CHO + HCl
Cl
Cl
Cl
Cl
ClCl
Cl
Cl
Cl
Cl
Cl
+ HCl
6) dehydrodehalogenation : HX is eliminated from the
molecule, leading to the formation of a double bond
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Metabolism of monochlorophenols by Pseudomonas sp. B13
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Cl
Cl
Cl
OH
Cl
Cl
Cl
Cl
OH
OH
Cl
Cl
Cl
OH
OH
Cl
Cl
Cl
OH
OH
Cl
HCl HCl HCl
Proposed degradation pathway for pentachlorophenol in Flavobacterium sp. and coryneform-like strain KC-3
Proposed degradation pathway for 2,4,5-trichlorophenol in P. cepacia
Cl
Cl
Cl
OH
Cl
Cl
OH
OH
OH
Cl
OH
OH
HCl HCl
hydroquinone
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2,4-dichlorophenol
reductive
dechlorination
4-chlorophenolphenol
reductive
dechlorination
carboxylation in p -position4-hydroxybenzoate
Cl
Cl
OH
Cl
OH OH
COOH
benzoate
3 Acetate + CO2 + 3 H2
CH4 + CO2
COOH
OH
ring fission
methanogenesis
decarboxylation
Sequential degradation of 2,4-dichlorophenol under anaerobic (methanogenic)conditions in a lake sediment
Organism 1 Organism 2
Organism 3
Organism 4
Organism 5 & 6
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• Bacteria, fungi, yeasts, and algae have the ability to metabolize both lower and higher M.W. PAHs found in the natural environment most bacteria have been found to oxygenate the PAH initially to form dihydrodiol with a cis-configuration, which can be further oxidized to catechols.
• Most fungi oxidize PAHs via a cytochrome P450 catalyzed mono- oxygenase reaction to form reactive arene oxides that can isomerize to phenols
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• White-rot fungi oxidize PAHs via ligninases (lignin peroxidases and laccase) to form highly reactive quinones.
• little is known about the potential of PAHs for anaerobic metabolism
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Compound Organisms Metabolite
Naphthalene Acinetobacter calcoaceticus , Alcaligenes denitrificans,Mycobacterium sp. , Pseudomonas sp., Pseudomonas putida , Pseudomonas fluorescens , Pseudomonas paucimobilis , Pseudomonas vesicularis , Pseudomonas cepacia , Pseudomonas testosteroni , Rhodococcus sp. , Corynebacterium renale , Moraxella sp., Bacillus cereus , Streptomyces sp.
Naphthalene cis -1,2 – dihydrodiol,1,2 – dihydroxynaphthalene, 2 - hydroxychromene - 2 – carboxylic acid, trans – o – hydroxybenzylidene pyruvic acid, salicylaldehyde, salicylic acid, catechol, gentisic acid, naphthalene trans – 1,2 – dihydrodiol .
Acenaphthene Beijerinckia sp., Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas cepacia
1- Acenaphthenol, 1- acenaphthenone, acenaphthene – cis – 1,2 – dihydrodiol,1,2 – acenaphthenedione,1,2 – dihydroxyacenaphthylene,7,8 – diketonaphthyl – l – acetic acid,1,8 – naphthalenedicarboxylic acid, 3 – hydroxyphthalic acid .
Bacterial strain degrading PAHs
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Compound Organisms Metabolite
Anthracene Beijerinckia sp., Mycobacterium sp., Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas paucimobilis, Pseudomonas cepacia, Rhodococcus sp., Flavobacterium sp, Arthrobacter sp.
Anthracene cis – 1,2 – dihydrodiol,1,2 – dihydroxyanthracene, cis – 4 – ( 2 – hydroxynaphth – 3 – yl ) – 2 – oxobut – 3 – enoic acid, 2 – hydroxy – 3 – naphthaldehyde, 2 - hydroxy – 3 – naphthoic acid, catechol, 2,3 –dihydroxynaphthalene, salicylic acid
Phenanthrene Aeromonas sp., Alcaligenes denitrificans , Arthrobacter polychromogenes , Beijerinckia sp. , Mycobacterium sp. , Micrococcus sp., Vibrio sp., Pseudomonas putida, Pseudomonas paucimobilis, Rhodococcus sp., Nocardia sp., Flavobacterium sp., Streptomyces griseus, Acinetobacter sp.
3,4 – dihydroxyphenanthrene,cis – 4 – ( 1- hydroxynaphth – 2 – yl ) – 2- oxobut – 3 – enoic acid,1 – hydroxy – 2 – naphthaldehyde, 1 – hydroxy – 2 – naphthoic acid,1,2 – dihydroxynaphthalene, o – phthalic acid , 2 – carboxybenz- aldehyde, protocatechuic acid .
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Compound Organisms Metabolite
Fluoranthene Alcaligenes denitrificans , Mycobacterium sp. , Pseudomonas putida , Pseudomonas paucimobilis, Pseudomonas cepacia , Rhodococcus sp.
7- Acenaphthenone, 1- acenaphthenone, 7- hydroxyacenaphthylene, benzoic acid, phenylacetic acid, adipic acid, 3- hydroxymethyl – 4,5- benzocoumarin,9- fluorenone – 1 – carboxylic acid,8- hydroxy – 7- methoxyfluoranthene,9- hydroxyfluorene , 9- fluorenone,phthalic acid, 2- carboxybenzaldehyde
Pyrene Alcaligenes denitrificans , Mycobacterium sp. , Rhodococcus sp.
Pyrene cis - and trans - 4,5 – dihydrodiol,4 – hydroxyperinaphthenone, phthalic acid, 4- phenanthroic acid, 1,2 - and 4,5 – dihydroxypyrene, cinnamic acid, cis – 2 –hydroxy – 3 – ( perinaphthenone -9-yl ) propenic acid
Chrysene Rhodococcus sp. None determined
Benz [a] anthracene
Alcaligenes denitrificans , Beijerinckia sp. , Pseudomonas putida
Benz [a] anthracene cis – 1,2, cis- 8,9-, and cis – 10,11- dihydrodiols, 1- hydroxy – 2 – anthranoic acid, 2- hydroxy – 3 – phenanthroic acid, 3- hydroxy – 2 – phenanthroic acid .
Benz [a] pyrene
Beijerinckia sp., Mycobacterium sp.
Benz [a] pyrene cis -7,8 - and cis -9,10 – dihydrodiols .
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Biodegradation of toluene
Anaerobicpathway
Aerobic pathway
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p-Xylene
The metabolic enzymes in this pathway have been shown to have similar specificty for toluene, para-xylene, and meta-xylene
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Biodegradation of ethyl benzene
• Under aerobic conditions
ethylbenzene degradation involves
oxygenase reactions that can
proceed in either of two primary
pathways
• A Pseudomonas sp. (strain NCIB
10643) has been shown to utilize a
wide range of n-alkylbenzenes
(C2-C7), of which ethylbenzene is
a single example.
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Bacterial co-metabolism of halogenated organic compounds
• TCE (trichloroethylene), widely used industrial solvent,
is unreactive halocarbon compound. • Anaerobic bacteria can reductively dehalogenate
TCE to form vinylchloride (strongly carcinogenic).• No one has succeeded in obtaining a TCE-degrading
bacteria able to use TCE as sole carbon and energy
source, that driven the studies on the co-metabolism
of TCE
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• An oxygenase
• monooxygenase might yield TCE- epoxide
• dioxygenase yield 1,2 – dihydroxy TCE
e.g. Toluene dioxygenase from
Pseudomonas putida F1
• Methane monooxygenase (MMO) from
Methanotrophs (Methylococcus, Methylosinus)
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40Theoretical pathways of TCE oxidation by monooxygenases and dioxygenases
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TCE oxidation by soluble methane monooxygenase (MMO) oxidizes methane to methanol in the first step of C1 oxidative metabolism by methanotrophs
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Oxygenases and organisms implicated in TCE oxidation Organism enzyme
Methylosinus trichosporium OB 3 b
Pseudomonas cepacia G 4
P. mendocina
P. putida
Nitrosomonas europaea
Methylocystis parvus OBBP
Mycobacterium sp.
Alcaligenes eutrophus JMP 134
Rhodococcus erythropolis
soluble methane monooxygenase
Toluene 2- monooxygenase
Toluene 4- monooxygenase
Toluene dioxygenase
Ammonia monooxygenase
Particulate methane monooxygenase
Propane monooxygenasePhenol hydroxylase
Isoprene oxygenase
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• TCE 1. TCE - epoxide (major product)
2. Chloral (minor product)
• TCE 1. D - formate (major product)
2. Glyoxylate (minor product)
MMO
Conclusion
Toluene dioxygenase
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e.g. heme, vitamin B12, coenzyme F430
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Bacterial transition-metal coenzyme
Coenzyme F430
Vitamine B12
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Cl
n
Cln
General structure(there are 210 theoretically possible PCB molecules)
Microorganism-specific nature of PCB degradation (Unterman et al., 1988)
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Overview
What are PCBs? Why are they a problem? What can we do with them? How do the microbial methods work?
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PCBs
Synthesized chemicals from petro-chemical industry used as lubricants and insulators in heavy industry
Used because Low reactivity Non-flammable High electrical resistance Stable when exposed to heat and pressure
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Uses
Hydraulic fluid Casting wax Carbonless carbon
paper Compressors Heat transfer systems
Plasticizers Pigments Adhesives Liquid cooled electric
motors Fluorescent light
ballasts
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Manufacturing
First manufactured in 1929 by Montesano Manufactured around the world Production ended in 1977 in the US Manufacture and unauthorized use banned in
1978 by USEPA Made in countries world wide – Europe
(France), Japan, former USSR
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Where found
They are ubiquitous: Water: rain and groundwater Soil: through direct disposal and leaching from
disposal sites Animals: bioaccumulation Food: bioaccumulation and production methods
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Basic Aromatic Carbon structure
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Interesting Facts
Between 1929 and 1970, 4*10^5 tons produced in US
Around 4,000 tons per year get into waterways through dumping and leakage
As of 1975, 4.5 million kg lost to environment through vaporization, leaks, spills, and landfills
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Risks
PCBs are toxic Soluble in fats, oils, solvents Some more than others: more Cl = more toxic Position of Cl affects toxicity
Ortho position less toxic than meta or para Para + meta = “dioxin like”; flat plane molecule,
particularly toxic
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Food Chain EffectsBioaccumulation
Primary producers and lower trophic level organisms take up PCBs, accumulates in the food chain
Higher organisms eating primary producers get more concentrated amounts of toxin
Often people consuming higher organisms are exposed to more toxic forms than factory workers
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Human Health carcinogenous:
Liver cancer melanoma
immune system studies done on rhesus
monkeys which have similar systems
effects noted in people exposed to PCB contaminated rice oil
suppressed swollen thymus gland in
infants
reproductive system humans and animals reduced birth weight reduced conception
weight decrease in gestational
ages still births and abortions
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Human Health Cont.
nervous system infant neurological
functions Recognition short-term memory Learning effects seen at levels
present in breast milk
endocrine system thyroid health
other health effects
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Marine and Animal Health
Inhibits plankton growth and photosynthesis affecting the food chain reduce trophic pathways
Reduce plankton size Reduce size of higher
feeders Divert carbon flow to non-
harvestable species less plankton = less bigger
food fish
Toxic to crustaceans, mollusks, and fish at concentrations of only a few ppb
Human health concerns apply to animals as well
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Mitigation Extraction or separation of contaminants from
environmental media
Immobilization of contaminants
Destruction or alteration of contaminants Chemical Thermal Biological
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Bioremediation
The process by which organisms use toxic chemicals as a food source in an environment which is favorable to that process
Generally enhanced by addition of nutrients, oxygen, moisture or adjusting pH levels
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Pathways of PCB Degradation
Anaerobic/Aerobic removal Photochemical Degradation Thermal Degradation Fungal Degradation
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Methods for PCB removal
Natural Attenuation: Microbes already in the soil are allowed to degrade as they can naturally and the site is closely monitored.
Biostimulation: Microbes present in the soil are stimulated with nutrients such as oxygen, carbon sources like fertilizer to increase degradation.
Bioaugmentation: Microbes that can naturally degrade PCB’s are transplanted to the site and fed nutrients if necessary
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Microbial Methods
Microbes either: Use PCBs as a carbon source Microbes initiate reductive de-chlorination
Problems Generally slow Use other carbon sources in natural systems first Microbes prefer lower chlorinated biphenyl Prefer para and meta positions
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Ortho, Meta and Para Chlorination of Biphenyl
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Pathways of Aerobic Degradation
dihydroxybiphenyl
Oxygenase attack
Bugs attack benezoate
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Pathways of Aerobic Degradation cont.
metaclevage
Most bugs degrade this
M
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Degradation of PCB Products
Dioxygenase attack on
2,3 catechol
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Degradation of PCB Products cont.
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Take Home Message of Aerobic Degradation
Works at 10% oxygen content or at 4ppm minimum. Anaerobic degradation needs to occur first if more
that 4 chlorines exist per ring. Degradation means chlorine removal from the ortho,
meta or para positions. Degradation happens most often through 2,3-
dioxygenase on the 2,3 carbons with a metacleavage, or a metacleavage of unchlorinated 2,3-carbons.
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Fungal Degradation
Aspergillus niger: fillamentous with cytochrome p450 that attacks lower chlorinated PCB’s
Phanerochaete chrysosporium: White rot fungi can attack even highly chlorinated PCB’s at low conc. (less than 500ppb) while aerobic degradation is occuring at a level of 10ppm.
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Anaerobic Reductive Dechlorination of PCBs
Not as well-characterized a process as aerobic degradation
Anaerobic bacteria responsible were not identified until more recently
Anaerobic PCB degradation first observed in Hudson river sediments (a site of historic contamination)
Since then, it has been noted in many other places
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• For the uncontaminated (PCB-free) sites, this was determined by introducing PCBs to sediments from these areas in the lab
• Indicates that dechlorinating activity may be due to a common, widespread reductive pathway
(Abramowicz,1995)
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Anaerobic dechlorination is complementary to aerobic degradation
The less chlorinated products of anaerobic pathways are better substrates for aerobic pathways than more chlorinated congeners
A combination of the two could result in complete PCB breakdown
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A reduction pathway, with Cl as the terminal electron acceptor
At least one species (o-17) likely uses acetate as the electron donor
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Anaerobic congener selectivity
Most (but not all) observed microbial degradation of PCBs removes Cl only in the meta or para positions (primarily ortho products)
Even highly chlorinated congeners can be mostly dechlorinated
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Aerobic PCB Degraders
Numerous soil bacteria break down PCBs via dioxygenase pathways
Most identified seem to be Pseudomonas species Others: Achromobacter, Acinetobacter, Alcaligenes,
Arthrobacter, Corynebacterium, Rhodococcus, Burkholderia (fairly diverse)
In general, the more highly chlorinated the PCB is, the fewer species that are able to degrade it aerobically.
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(Abramowicz, 1990)
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Some aerobic bacteria are capable of degrading a broader range of PCB congeners, notably: Burkholderia xenovorans LB400 (Gram -)
Widest range of congener substrates ~9.5 Mb genome - one of the largest sequenced
Rhodococcus erythropolis RHA (Gram +) These possess different enzymatic pathways, and
the genes for them(“ohb” and “rod/cat+” respectively) are often used in the genetic construction of PCB degraders
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Anaerobic PCB degraders
Although PCB dechlorination in anaerobic sediments was noted fairly early on, first responsible bacteria was not identified until 2001 “o-17”, from Baltimore Harbor sediments Was discovered by monitoring 16s rRNA of an enriched
ortho-PCB degrading culture. Growth of o-17 was dependant on the growth and
dechlorination of 2,3,5,6-tetrachlorobiphenyl
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(Cutter et. al. 2001)
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Biodegradation of Nitrobenzene
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Nitrobenzene degradation pathways.
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• in about 1868 a chemical compound used as a commercial pesticide was Paris green [Copper acetate
meta-arsenate, Cu(CH3COO)2 • 3Cu(AsO2)2]• DDT (dichloro-diphenyl-trichloroethane) is the first of a number of chlorinated H.C. to be developed as pesticides in 1939• Pesticides can be classified in a number of different ways for example; by their chemical nature ( natural organic cpd., inorganic cpd., chlorinated hydrocarbon, organophosphates, carbamates, and others)
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Proposed pathways for the microbial degradation of DDT
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85
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Summary of Biodegradation of Pesticides
There are many mechanisms involved on the biodegradation of pesticides and other contaminants. These may be summarised as follows: CATEGORY/REACTION EXAMPLE
Dehalogenation
RCH2C1 Õ RCH2OH Propachlor (C,S)
ArC1 Õ ArCH Nitrofen (S)
ArF Õ ArCHFlam-prop methyl
(S,C)
ARC1 Õ ArH Pentachlorophenol
(S,C)
Ar2CHCH2C1 Õ Ar2C=CH2 DDT (C)
ArCHCHC12 Õ Ar2C=CHC1 DDT (C,S,W)
Ar2CHCC13 Õ Ar2CHCHC12 DDT (C,S)
Ar2CHCC13 Õ Ar2C=CC12 DDT (S,C)
RCC13 Õ RCOOH N-Serve (S), DDT (C)
HetC1 Õ HetOH Cyanazine (S)
CATEGORY/REACTION EXAMPLE
Deamination
ArNH2 Õ ArOH Fluchloralin (S)
Decarboxylation
ArCOOH Õ Ar4 Biofenox (S)
ArCHCOOH Õ Ar2CH DDT (C)
RCH(CH3)COOH Õ RCH2CH2 Dichlorfop-methyl (S)
ArN(R)COOH Õ ArN(R)H DDD(S)
Methyl oxidation
RCH3 Õ RCH2OH Õ RCHO Õ RCOOH
Bromacil (S)
Hydroxylation and ketone formation
ArH Õ ArOH Benthiocarb (S)
R(R’)CHR" Õ, (R’)CHOH(R") Bux insecticide (S)
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1. Measurement of oxygen consumption by manometric and electrolytic system
2. Measurement of CO2 evolution by infrared or
chemical methods
3. Use of radio-labeled substrates
4. Measurement of the disappearance of the chemicals
by GC
5. Determination of the reduction of DOC
6. Chemical biodegradability under anaerobic conditions
(measuring gas production, CH4+ CO2)
The assay systems for biodegradability test