1 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 2 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 3 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Organophosphates and Germany
• 1820s: investigations into organophosphate (OP) chemistry began
• Early 1900s: several OP compounds synthesized
• 1930s: toxicity of OPs becoming recognized
• 1940s: insecticidal action observed by Germany during WWII
4 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Organophosphates and Germany
• Group led by Gerhard Schrader searching for substitutes to nicotine as an insecticide – Nicotine in short supply during WWII – Developed a number of incredibly toxic nerve agents
5 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Organophosphates and Germany
• Schrader’s group also created some of the first commercial OP insecticides
Dimefox TEPP Schradan
Parathion
6 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 After WWII
• Schrader’s research records were captured by Allied forces – Led to massive increase in interest in OP insecticides • Early OPs – very effective against insects – Much more toxic to vertebrates than organochlorine insecticides – Nonpersistent and chemically unstable
7 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Malathion
• First produced by American Cyanamid in 1950 • Very safe due to its low vertebrate toxicity • Used on most fruits, vegetables and forage crops • Works on a wide range of insect pests
8 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 9 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Diethyl 2-[(dimethoxyphosphorothioyl)sulfanyl]butanedioate
10 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 11 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 O Diethyl Maleate C CH O CH CH 2 3 S + CH O CH CH 2 3 CH O P O CH C O 3 3 S C S O CH CH O CH CH O P S 2 3 H O 3 O CH O CH C 2 2 CH + CH C 3 CH O CH 2 CH 3 Malathion 3 O CH 3 CH 2 O CH C O Diethyl Fumarate
12 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Very Simple Synthetic Reaction
Michael Addition during Condensation Reaction Nucleophilic addition of an SH-group to the double bond of diethyl maleate or diethyl fumarate.
13 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Maleic Acid
Fumaric Acid
14 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Carboxylic Acid, Hydrogen Bonds The carbonyl group is an electron withdrawing group when conjugated with a double bond. This decreases the electron density and activates the double bond to nucleophilic addition.
H O 2 C C C C C C C C + RSH O O O O RS H S CH O OCH -d 3 P 3 -d
.. O
O ..
S H H C OC 2 H 5 C OC 2 H 5 H +d C +d C C C H C 2 H 5 O C H C OC 2 H 5 O O Diethyl Fumarate O Diethyl Maleate S C CH O P S CH O C 2 H 5 3 O CH 2 O C 2 H 5 CH C 3 O Malathion CH 3 O CH 3 O P SH CH 3 O CH 3 S CH 3 O CH 3 O P SH S P SH O O
Diethyl Maleate or Diethyl Fumarate
Malaoxon Malathion Isomalathion
18 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 1. Malathion is considered to be one of the least toxic (i.e., safest) insecticides to mammals. 2. The above statement is conditional to the absence of any highly toxic and or strong potentiating impurities. 3. Malathion is not protected by any patent and can, therefore, be synthesized by any manufacturer.
19 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 4. Most malathion impurities are more potent than malathion itself and some of them strongly potentiate its mammalian toxicity. 5. Furthermore, some impurities act similarly and dissimilarly to malthion mode of action. 6. The above statement tremendously complicates the risk of formulated malathion.
20 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 7. Malathion safety will be in jeopardy if manufacturing companies irresponsibly produce it without assuring the absence of any detrminntal levels of its impurities. 8. Regulatory authorities should make sure that registered malathion in their regions be analyzed to ensure the absence of malathion risk-induced impurities. Yehia A. Ibrahim (June 2012)
21 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Test Material % a.i. LD (mg/kg) 50 Malathion TC 94.6 1650
Malathion TC, containing 0.44% Isomalathion 96.2 1850
Malathion TC, containing 0.20% Isomalathion 96.2 2400
Malathion TC 96.8 5100
Malathion TC 96-98 5550
22 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Safety Margin
Above 97.5%
23 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 24 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • Cholinergic Neurotoxicity (AChE Inhibition)
• Organophosphorus Ester-Induced Delayed Neurotoxicity (OPIDN)
• Organophosphorus Ester-Induced Chronic Neurotoxicity (OPICN)
25 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • Acetylcholinesterase (AChE) is an enzyme that degrades the neurotransmitter acetylcholine, producing choline and an acetate group. It is mainly found at neuromuscular junctions and cholinergic synapses in the central nervous system, also in red blood cell membranes.
26 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • first neurotransmitter identified (Otto Loewi, 1921)
• important role in movement: causes muscle contraction
• also found in brain: important role in attention, learning & memory
6/26/2012 Malathion Impurities Dr. Yehia Ibrahim 27 28 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 6/26/2012 Malathion Impurities Dr. Yehia Ibrahim 29 ACh is broken down in the synaptic cleft by the enzyme acetylcholinesterase (AChE)
30 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • An esteratic site – The amino acid serine • An anionic site – The amino acids Tyrosine (3 of them), Aspartic Acid, and Tryptophan
Serine Tyrosine Aspartic Acid Tryptophan 31 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012
O H
Reaction Mechanism H
Acetylcholine – CH H C 3 O 3 O CH H C 3 + CH 3 N 3 + CH H C O N 3 3 H C O O 3
R2 - R1 N – - O H O - R2 CH H C 3 R1 Anionic Site 3 N + CH H O N 3 HO
Serine - – Choline O O - O – H C O HH O H H C OH 3 H 3 O H C Acetate 3 OH O
– R2 - O H R1 R1 R2 R2 N - R1 - N H O H - N H Serine O Anionic Site O 32 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • Inhibition of Acetylcholinesterase (AChE), an enzyme essential for life.
33 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • AChE inhibitors — block the function of AChE and thus cause excessive acetylcholine to accumulate. The excess acetylcholine causes neuromuscular paralysis (i.e. interminable muscle contractions) throughout the entire body, leading to death by asphyxiation.
34 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • Phosphates are biologically active as acetylcholinesterase (AChE) inhibitors, whereas phosphorothioates need bioactivation to their phosphate analogues (oxons) to become biologically active.
35 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 S O +d CH O +d P S X CH O P S X 3 3 O O
Malathion CH Malathion-Oxon CH 3 3
36 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 (b) acyl pocket Peripheral binding site (d)
Trp 286
phe 297 Asp 74
phe 295
O N
O- Glu 334 H Trp 86 Choline N d+ N (c) binding H d- O site His 447 (a) Catalytic triad of AChE Ser 203
Active site gorge of AChE
37 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Malathion
Malathion-Oxon (Malaoxon)
AChE Inhibition
ACh Accumulation
Cholinergic Neurotoxicity
38 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Peripheral binding site acyl pocket (d) (b) Trp 286
phe 297
Asp 74 phe 295
N
O Trp 86 O Choline
d+ (c) binding Glu 334 O- P O H site N Nd+ H d- O (a) His 447 Catalytic triad of AChE Ser 203
MalaoxonSARIN phosphonyl-AChE Complexenzyme
39 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Peripheral binding site acyl pocket (d) (b) Trp 286
phe 297
Asp 74 phe 295
N
O Trp 86 O Choline
d+ (c) binding Glu 334 O- P O H site N Nd+ H d- O (a) His 447 Catalytic triad of AChE Ser 203
DimethylSARIN phosphonyl Phsphorylenzyme AChE
40 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Peripheral binding site acyl pocket (d) (b) Trp 286
phe 297
Asp 74 phe 295
N
O Trp 86 O Choline
d+ (c) binding Glu 334 O- P O H site N Nd+ H d- O (a) His 447 Catalytic triad of AChE Ser 203
AgedSARIN Phosphoryl phosphonyl enzymeAChE
41 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 42 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 O CH 3 CH 2 O C S O CH CH 3 2 P CH S O CH 3 C CH 3 CH 2 O O Malathion
43 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Oxidase
O CH 3 CH 2 O C S O CH CH 2 3 Carboxylesterase P Methyl Transferase CH S O CH 3 C CH 3 CH 2 O O
Organophosphorus Hydrolase
44 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 O O CH CH 3 C 2 CH O S 3 CH P 2 CH 3 O S CH C Malathion O O CH 2 CH 3
O O H O O CH CH 3 C C 2 CH O S CH O O 3 CH 2 3 CH P P 2 CH 3 O S CH CH 3 O S CH C C O O H O O CH 2 CH 3 Malathion Diacid Malaoxon
45 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 46 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 H
47 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Optic System Brain Pupil Constriction Headache Blurred Vision Dizziness Lacrimation Vertigo Anxiety Apathy Confusion Respiratory System Anorexia Bronchospasm Insomnia Bronchial Secretion Lethargy Pulmonary Edema Fatigue Tightness of Chest Inability to Concentrate Wheezing Memory Impairment Cough Convulsion Difficulty Breathing Coma
Gastrointestinal Tract Salivation Cardiovascular System Nausea Tachycardia Cramps Increased Blood Pressure Abdominal Pain Vomiting Diarrhea Fecal Incontinence Musculature Weakness Tremor Fasciculations Urinary - Genital Twitching Urinary Incontinence Cramps Impotence Increased Sweating Uterus Contraction
48 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 49 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 O CH 3 CH 2 O C S O CH CH 3 2 P CH S O CH 3 C CH 3 CH 2 O O Malathion
O O CH 3 CH 2 O CH 3 CH 2 O C C O O CH O S CH CH 3 CH 3 2 P 2 P CH S O CH 3 CH S O CH 3 C C CH 3 CH 2 O O Malaoxon CH 3 CH 2 O O Isomalathion
50 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 O CH 3 CH 2 O C S O CH CH 3 2 P CH S O CH 3 C CH 3 CH 2 O O Malathion O S CH O O CH 3 3 P P CH 3 S O CH 3 CH 3 S O CH 3 MeOSSPO-triester MeOOSPO-triester
S O CH S O CH 3 3 P P CH CH 3 O O CH 3 CH 3 S O 3 MeOOOPS-triester MeOOSPS-triester 51 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Malathion Impurity Rat Oral LD 50 (mg/kg bw) − Isomalathion, CAS 2244-12-5 113 − Malaoxon, CAS 1634-78-2 215 − MeOSSPO-triester, CAS 22608-53-3 26* − MeOOSPO-triester, CAS 152-20-5 47* − MeOOOPS-triester, CAS 152-18-1 562 − MeOOSPS-triester, CAS 2953-29-9 628
*,* Impurities that were not detected in the technical malathion manufactured by Cheminova.
52 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Several organophosphate impurities present in technical malathion are known to synergize malathion toxicity probably through blocking malathion detoxification.
53 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 The toxicity of several malathion impurities alone is also very high (eg the LD50 of O,O,S-trimethyl phosphorothioate in rats is 15 mg/kg, or 833 times more toxic than pure malathion) and cause delayed toxicity suggesting a mode of action other than AChE inhibition.
54 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Malathion degradation to products of higher toxicity under improper storage conditions is well documented, however effects due to impurities and degradates during normal use are not (with the exception of the mass poisoning of 2,800 spray men in Pakistan in 1976 resulting in 5 deaths, Aldridge et al 1979).
Aldridge, W.N., Miles, J.W., Mount, D.L., and Verschoyle, R.D. (1979). The toxicological properties of impurities in malathion. Arch Toxicol 42. 95-106.
55 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012
58 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 59 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 The new assessment by the rapporteur Member State and the new conclusion by the EFSA concentrated on the concerns that lead to the non-inclusion. Those concerns were in particular the presence in the technical material of varying levels of isomalathion, an impurity that contributes significantly to the toxicity profile of malathion and the genotoxicity of which cannot be excluded. Because of this fact it was impossible to assess the risk to operators, workers and bystanders. Moreover, information on the effects of certain toxicologically relevant metabolites was insufficient, and it had, hence, not been demonstrated that the estimated exposure of consumers resulting from the acute and chronic intake of edible crops is acceptable. New data and information were submitted by the notifier in the new dossier and a new assessment was performed, as included in the additional report and in the EFSA conclusions. Those new data show that the genotoxicity of malathion containing not more than 2 g/kg isomalathion can be ruled out. It has therefore been possible to determine acceptable levels of exposure of operators, workers and bystanders. Use by non-professionals could, however, raise concerns, as it cannot be assumed that these have recourse to the adequate personal protective equipment. As a consequence, such use should not be authorised. 62 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Relevant impurities of Malathion Malaoxon (CAS No. 1634-78-2; butanedioic acid, (dimethoxyphosphinothioyl)-, diethyl ester) Maximum: 1 g/kg
Isomalathion (CAS No. 3344-12-5; succinic acid, mercaptodiethylester, S-ester with O,S-dimethyl phosphorodithioate) Maximum: 4 g/kg
MeOOSPS-triester (CAS No. 2953-29-9; phosphorodithioic acid, O,O,S-trimethyl ester). Maximum: 15 g/kg
MeOOOPS-triester (CAS No. 152-18-1; phosphorothioic acid, O,O,O- trimethylester) Maximum: 5 g/kg
Acidity (CIPAC MT 31) Maximum: 2 g/kg, calculated as H2SO4
63 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012
Impurity Content % Recommendations Malaoxon Maximum: 1 g/kg 0.1 1. Malathion that is less than 97% should not Isomalathion Maximum: 4 g/kg 0.4 be accepted. 2. Analyze Malathion MeOOSPS-triester Maximum: 15 g/kg 1.5 First, then impurities. 3. Analyze impurities in MeOOOPS-triester Maximum: 5 g/kg 0.5 an order of analytical easiness. Acidity Maximum: 2 g/kg, 0.2 4. Ismalathion should calculated as H2SO4 not exceed 0.2% instead of 0.4% due to Total 27 g/kg 2.7 its genotoxicity.
64 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 65 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 66 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 67 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 68 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 69 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 70 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 71 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 O CH 3 CH 2 O C S O CH CH 3 2 P CH S O CH 3 C CH 3 CH 2 O O Malathion Malathion is determined by gas chromatography (GC) using a non-polar capillary column and FID, with quantification by internal standardization (Sørensen, 2002a). It is the same as Method VAM 001-02 (Knold, 2002a).
The method was collaboratively tested, shown to be suitable for malathion TC, EC, EW and DP (Sørensen, 2002b) and adopted by CIPAC in 2002 (CIPAC, 2002).
72 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 O CH 3 CH 2 O C O O CH CH 3 2 P CH S O CH 3 C CH 3 CH 2 O O Malaoxon
Malaoxon in the TC is determined by reversed phase liquid chromatography (HPLC), using a C18 column and UV detection with quantification by external standard (Method VAM 008-02, Hinz, 2001a). The absorption at 215 nm is used to determine malaoxon. In Method VAM 208-01 for DP formulations, the sample portion is sonicated with acetonitrile prior to HPLC analysis (Hinz, 2002c). The signal at 230 nm is used to determine malaoxon.
73 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 74 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 O CH 3 CH 2 O C O S CH CH 3 2 P CH S O CH 3 C CH 3 CH 2 O O Isomalathion
Isomalathion is determined by reversed phase liquid chromatography (HPLC), using an ODS2 column and UV detection with quantification by external standard (Method FAO VAM 005- 03, Petersen 2001a). The absorption at 200 nm is used for measurement of the isomalathion concentration, while the absorption at 225 nm is used to check for the presence of interfering compounds.
75 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 76 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 S O CH 3 P CH 3 S O CH 3 MeOOSPS-triester
MeOOSPS-triester in technical malathion is determined by gas chromatography (GC), using a non-polar capillary column and FID with quantification by an external standard (Method VAM 006-02: Sørensen, 2002c, Knold, 2002c). A portion of test material is weighed into a sample bottle and diluted with acetonitrile, ready for GC analysis. Method VAM 206-01 is essentially the same method which was applied to technical malathion and EW and DP formulations (Knold, 2002f).
77 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 S O CH 3 P CH 3 O O CH 3 MeOOOPS-triester
MeOOOPS-triester is determined by gas chromatography (GC), using a non-polar capillary column and FID with quantification by an external standard (Method VAM 006-02: Sørensen, 2002c, Knold, 2002c). A portion of test material is weighed into a sample bottle and diluted with acetonitrile, ready for GC analysis. MeOOOPS-triester in technical malathion and in EW and DP formulations is determined by gas chromatography (GC), using a non-polar capillary column and FID with quantification by an external standard (Method VAM 206-01: Knold, 2002f).
78 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 79 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 What’s Safe and What Isn’t
80 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 81 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 82 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • In mammals, the relative safety of malathion, compared with many other cholinesterase inhibitors, has been attributed to its rapid enzymatic hydrolytic degradation.
83 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • Impurities that inhibit hydrolytic activity have the ability to potentiate the toxicity of malathion and therefore it is important to control the impurity profile of malathion products.
84 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 • Some impurities are toxic in their own right and at least one of them strongly potentiates the toxicity of malathion.
85 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Jensen (2001) listed six impurities which, because of their toxicological properties, should be considered in this context.
Jensen M. K., 2001 Malathion FAO specification - potential relevant manufacturing impurities. Cheminova report No. 398 FYF. Unpublished.
86 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 The main formulation types available are: − Dustable powders, DP, − Ultra-low volume liquids, UL, − Emulsifiable concentrates, EC, − Emulsions, oil in water, EW.
These formulations are registered and sold in many countries throughout the world.
87 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 88 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 Extracted from CIPAC Cumulative Index
89 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 90 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 91 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 92 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 93 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012
And you think your day was tough!! 94 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012 THE END, THANK YOU!
95 Malathion Impurities Dr. Yehia Ibrahim 6/26/2012