P382-225Q77
Toxicology of Pesticides
Yugoslav Academy of Sciences and Arts, Zagreb
Prepared for
Health Effects Research Lab
Research Triangle Park, NC
>b 32
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
MTIS
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PB82-226077
EPA-600/1-82-OOL
February 1982
TOXICOLOGY OF PESTICIDES
by
Elaa Reiner
Institute for Medical Research
and Occupational Health
Zagreb, Yugoslavia
Grant No. R 804539010
Project Officer
William JVv .'Durham
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO. ' 2.
EPA-600/1-82-001 ORD Report
4. TITLE AND SUBTITLE
Toxicology of Pesticides
7. AUTHOR(S)
Elsa Reiner
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Institute for Medical Research and Occupational
Health Zagreb, Yugoslavia
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Research, and Development
Health Effects Research. Laboratory
U.S.. Environmental Protection Agency ..
Rccoirch Trisncrlsi Par!' N^1 27"'ll '
3. RECIPIENT'S ACCESSION NO.
pe>%
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NOTICE
THIS DOCUMENT HAS BEEN-.REPRODUCED
FROM THE BEST COPY FURNISHED US BY
THE SPONSORING AGENCY. ALTHOUGH IT
IS-- RECOGNIZED THAT CERTAIN PORTIONS
ARE. ILLEGIBLE, IT IS BEING RELEASED
IN THE INTEREST OF MAKING AVAILABLE
AS MUCH INFORMATION AS POSSIBLE.
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DISCLAIMER
This report has been reviewed by the Health.Effects Research Laboratory,
UoS. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation for use.
ii
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FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk of
existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to enhance
the quality of our environment in order to promote the public health and
welfare and the productive capacity of our Nation's population. ;
The Health Effects Research Laboratory, Research Triangle Park, conducts
a coordinated environmental health research program in toxicology, epidemi-
ology, and clinical studies using human volunteer subjects. These studies
address problems in air pollution, non-ionizing radiation, environmental
carcinogenesis and the toxicology .of pesticides as well as other chemical
pollutants. The Laboratory participates in the development and revision of
air. quality criteria documents on pollutants for which national ambient air
quality standards exist or are proposed, provides the data for registration of
new pesticides or proposed suspension of those already in use, conducts
research on hazardous and toxic materials, and is primarily responsible for
providing the health basis for non-ionizing radiation standards. Direct
support to the regulatory function of the Agency is provided in the form of
expert testimony adn preparation of affidavits as well as expert advice to the
Administrator to assure the adequacy of health care and surveillance of
persons having suffered imminent and substantial endangerment of their health.
Of all chemical pollutants, pesticides pose one of the most direct
threats to human health.' Thus, to assure the safety of pesticides before
their commercial marketing, researchers must investigate their effects through
extensive toxicological testing. In addition to laboratory testing and
analysis of pesticide compounds, these researchers must also develop effective
monitoring techniques for determining the safety of workers handling
pesticides.
F. G. Hueter, Ph.D.
Director
Health Effects Research Laboratory
iii
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ABSTRACT
Documented in this report are the results of five toxicological studies
of pesticide compounds conducted by the Institute for Medical Research and
Occupational Health, Zagreb, Yugoslavia, for the U.S. Environmental Protection
Agency*
In the first study, the reactions of two groups of esterases (cholin-
esterases and arylesterases) with substrates and inhibitors were investigated.
Procedures for monitoring the absorption of phosalone and malathion in
occupationally exposed workers by determination of pesticide residues in the
urine were developed in the second study. This detection technique was
compared to the traditional blood cholinesterase inhibition method to
determine which was a more rapid detector of poisoning.
The third study reported surveyed the residues of chlorinated hydro-
carbons in human milk and blood samples taken from the general population, and
compared the levels with those found in the serum of workers exposed to
pesticides. In the fourth study, cholinesterase activity was used to assess
the effects of recent changes made in the protective procedures for
occupationally exposed workers. Finally, in the last study, the alleged
effect of pesticides on the eye and on vision were studied and the results
discussed.
In addition, the report also includes summaries of seven students' theses
related to the work conducted by the Institute.
iv
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CONTENTS
Disclaimer ii
Foreword iii
Abstract. iv
Tables vi
Acknowledgments . . vii
1 . Introduction 1
2.. Esterases and organophosphorous compounds 2
3. Residues of organophosphorus pesticides in human urine 16
4. Residues of chlorinated hydrocarbons in human milk and blood ... 28
5. Assessment of biochemical and clinical effects of pesticides
in humans 32
6. Effect of pesticides on eye vision ....... 37
References. . 41
Appendices 49
A. Tables 49
B. Summaries of Theses. . 55
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TABLES
Number Page
1 Reactivation of AChE by 16 Pyridium Compounds. . . . . ..... 3
2 Reactivation of Methylethaoxyphosphonylated AChe by
Bispyridinium Aldoximes .............. ...... 4
3 Reactivation of Inhibited AChE by Oximes ....... ..... 5
4 Efficiency of Oximes and Atropine Against VX and DDVP Poisoning. 6
5 Effect of Temperature on Inhibition of AChE by Maloxon ..... 7
6 Michaelis Constants and Substrate Inhibition Constants for
AChE and ChE .......... ............... 8
7 Competition Between Two Substrates for AChE and ChE. .... . . . . . 8
8 Brain and Plasma ChE Activities of Rats Injected I.V. with
Netrifonate or DDVP ............... . ...... 9
9 Erythrocyte and Plasma ChE Activities in Schoolchildren ..... 10
10 Kinetic Constants for the Reaction of ChE with ATCh and BTCh . . 11
11 Inhibition of ChE by DDVP in Parasite Species .......... 11
1 2 ChE Activities in Parahphistonum Microbothrium and Ascaris suum
for 10 mM ATCh and 10 mM BTCh. . ...... ......... 12
1 3 Hydrolysis of DDVP and PA in Homogenates of Parasitic Helminths 1 3
14 Hydrolysis of DDVP and E600 in Plasma and Erythrocytes ..... 13
15 Heat Inactivation of the Hydrolytic Activities in Plasma .... 14
1 7 Distribution of Hydrolytic Activities Between Stroma and
Hemolysate in Human Erythrocytes ...... ......... 14
18 GC Working Conditions ..... ................. 17
19 Absorption of Malathion and ChE Activity ..... ....... 21
20 Absorption of Phosalone in TJwo Volunteers ............ 22
21 Excretion of Metabolites dnd ChE Activity in a Worker Exposed
to Phosalone for Six Hours ......... . . ....... 23
22 Excretion of Metabolites and ChE Activity in A Worker Exposed
to Phosalone for Three Days .................. 24
23 Metabolites in Urine of a Worker Exposed to Phosalone for
Three Days ............. ....... ..... 25
24 Concentrations of Chlorinated Hydrocarbons ........... 30
25 Blood ChE Activity and Evidence of Poisoning .......... 33
26 Vitamin A in Serum of Workers Exposed to Phosalone ....... 34
27 ChE Activity and DDT in Serum of Workers Exposed to Phosalone. . 36
28 Exposure to Pesticides for Two Groups Tested for Eye Defects . . 37
29 External Eye Examination in Workers Exposed to Pesticides. ... 38
30 Kerathometric Measurements of Workers Exposed to Pesticides. . . 39
31 Intraocular Pressure in Workers Exposed to Pesticides ...... 40
vi
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ACKNOWLEDGMENTS
The authors wish to acknowledge the assistance provided for this work by
consultants S. Horvat, I. Ledie, and M. Piukovie-Plestina (Department of
Ophthamology, University Hospital, Zagreb), N. Marion (Chromos Chemical Works,
Zagreb), and Z. Stefanac (Faculty of Science, Zagreb).
vii
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SECTION 1
INTRODUCTION
This Is the ffnal report of our four year study of the toxicology of
pesticides. It consists of six sections, each written as an Independent
entity. The report also Includes summaries of seven students's these related
to the work. The theses were supervised by senior members of our Institute,
which Is also a teaching Institution of the University of Zagreb. The
references Include our publications since 1976.
Most studies described In this report are a continuation of work started
before 1976. Studies were Initiated by EPA-and are now Included In the
Institute's research program. Studies described under section 4 are part of
the World Heallth Organization/United Nations Environmental Planning (WHO/UNEP)
"Pilot project on assessment of human exposure to pollutants through biological
monitoring". The Institute Is a "Collaboratong Center for Pesticides
Toxicology" of WHO..
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SECTION 2
ESTERASES AND ORGANOPHOSPHORUS (OP) COMPOUNDS
Reactions of two groups of esterases, the cholInesterases (ChEs) (EC
3.1.1.7 and EC 3.1.1.8) and the aryIesterases (EC 3.1, 1.2), with substrates
and Inhibitors were studied.
In addition to this report, we have published these results In nine
papers. (Reiner 1980, Reiner et al. 1977b, 1978a,b, 1979a, 1980, Simeon et al.
1977, 1979, Skrtngove -Spoljar et al. 1980), seven communications (Reiner-
1977a, Reiner et al. 1976, 1979b, Simeon et al. 1978a, b, Skrenjarlc-SpolJar
et al. 1978, 1979) and one thesis (Radlc 1979). Details of experimental
procedures and references to the IIterature^ may be found In these pub 11catIons.
Material and Methods
The following enzyme preparations were used: purified bovine erythrocyte
acethylchol Inesterase (AChE), purified horse serum ChE, native plasma and
erythrocytes from man,.rabbit, rat, hamster and chicken, liver and brain
homogenates from rat and hamster, and homogenates from different parasitic
helminths.
The enryme activities were determined either by spectrophotometrlc-or
manometrlc methods. (El Iman et al. 1961, Krupka 1966, Reiner et al. 1978).
Partial purification of arylesterases from human erythrocytes was done by gel
filtration.
RESULTS AND DISCUSSION
Reactivating and Protective Effects of Pyrldlnlum Compounds In AChE
Inhibition by OP
It Is well known that pyrldlnlum oxlmes are effective reactlvators of ChEs
Inhibited by OP compounds; these oxlmes are also effective In protection
against and therapy for OP poisoning (SIPRI 1976). More recently, pyrldlnlum
compounds with no oxlme group have also shown some activity In vivo.
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The reactivation of human erythrocvte AChE (EC 3.1.1.7) Inhibited by
O-ethyI-S-2-dllsopropylamJnoethyl methyIphosphonoth!oate (VX) and the protec-
tion against AChE Inhibition by 0-1,2,2-trlmethylpropyl
methylphosphonofluorldate (Soman) were studied with sixteen quartern I zed
pyrldlnlum compounds. Table 1 gives the structural formulae of these
compounds, the dissociation constants of the AChE - reacttvator complex In the
presence of 1.0 mM acetylthlolIne (Kl(app)) and rate constants for 1.0 mM
acetyIthIocholine hydrolysis In the presence of pyrldlum compounds (k1).
TABLE 1. REACTIVATION OF AChE* BY 16 PYRIOINIUM COMPOUNDS
'* - CRj - Y - CHj - *
(a)R
Abbreviation
I
II
III
17
7
71
711
Till
BG6-12
BOG -42
HI-6
TMB-4
R
CB
CH
CB
CH
CH3
CB3
CB
CR
CB
CB
CB
CB
ROB
NOB
NOB
NOB
NOB
NOB
NOB
NOB
NOB
NOB
,.,
4.
4
4
4
2
2
4
4
2
2'
2
4.
Y
CH2
CHj
CH2
CH2
CO
CO
CO
co
0
0
0
CHj
R1
*
CONH(CR2)3OB
CONB(CH2)3OH
-
CONH2
CH3
CH3
CH3
COCgHs
oocgHii
CONH2
CR - NOB
(b)
4
3
4
-
3
2
2
4
3
3
' 4
4
Z
Br
Br
Br
Br
Br
Br
Br
Br
a
y
d
Br
*i(app
(OH)
0.06
0.08
0.27
0.23
0.04
0.26
0.02
0.06
0.11
0.18
0.16
1.0
.. V1
<*-' mla-1)
31
38
35
10
7
31
26
37
30
22
69
+B - CH2 - R
Xbbnriatioa
IX
Z
CB NOB
CB - NOH
()
(CH2)2BS
(CB2>2-(I
Br
(OH)
0.11
0.07
k1
(M Bin-')
34
26
ZI
III
CDCTjBR
COCBjBR
OL04 0.20
Br 0.05
*Inhlbit«d by OX and Soman
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TMB-4, which Js known as a good reacttvator of AChF Inhibited by OPs proved to
be the most effective reactlvator. Of the newly synthetlzed compounds tested,
three were fairly good reactlvators of mathylethoxyphosphonylated AchE. These
compounds have two pyrldlnlum rings connected by a dtmethylether link and a
hydroxylmtnomethyl group on position 2 of one pyrldlnlum ring; while the
radicals of the other pyrldlnlum ring are benzoyI carbonyI (HG6-12),
cyclohexylcarbonyl (HGG-42) or amtdoearbonyl (HI-6) residue.
The rate of reactivation with these compounds followed a two-phase
pattern, being fast at the beginning and then slowing down to an equilibrium.
Kinetic treatment of the first-phase reaction course yielded the second-order
rate constants of reactivation (kr) (Table 2). Air three compounds had
TABLE 2. REACTIVATION OF METHYLETHOXYPHOSPHONYLATED AChE BY
BISPYRIDIN|UM ALDOXIMES
Evaluated Constants*
Compound
HGG-42
HGG-12
HI-6
TMB-4
k+2 ,
(mln-1)
0.04
0.06
0.09
0.03
Kr
(uM)
50
45
26
2
kP 10-3
(M-1 mln-1)
0.8
1.4
3.6
19.4
Protective
Index
2.51
3.27
3.99
Nil
The above constants refer to the following reaction:
k k
Phosphorylated + Oxlme + 1 .» Comp I ex +2 > Free* Phosphorylated
AChE *_ AChE oxlme
k
-1
k k
where Kr equals: +2 + -I
similar reactivating efficiency (kr values range from 0.8 x 103 to 3.6 x 103
M-1min-') but In effective concentrations (1 to 100 uM) they also Inhibited
AChE (Kj (app) values range from 0.11 to 0.19 mM). Their reactivating proper-
ties were not better than those revealed by TMB-4 (kp = 19.4 x 10-* M~') which
was tested as a reference compound.
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Protective effIclency was evaluated by comparing AChf- Inhibition bv Soman;
with and without the tested compound (0.1 mM final). . The time course of
Inhibition.by Soman was measured', and log percent activity was plotted as a
function of time. The first-order rate constants of AChE Inhibition by Soman
were evaluated from the linear part of the Inhibition curves with and without
the addition of the pyrldlntum compound* As a measure of protective
efficiency, quotients of the rate constant without and the rate constant with
0.1 mM pyrtdlnlum compound present were calculated (protective Index). HGG-12,
HGG-42 and HI-6 were also found to exert a good protective effect against AChE
Inhibition by Soman; no protection was obtained with TMB-4 (Table 2).
In addition, nine other blspyrldlnlum oxlmes were synthesized and tested
for reactivating potency and therapeutic effect on two OPs: 0,0-d!methyl-2,
2-dIchIorovlnyI phosphate (DOVP), and VX. The reactivation was measured on.
human erythrocyte AChE and the therapeutic effect was evaluated on male albino
rats. Tested compounds contained two pyrldlnlum rings linked by dimethyl ether;
each compound had a hydroxyimJnomethyl group on position 2 or 4, on one of the
pyrldinlum rings, while the other ring was unusubstltuted or had a methyl or a
dydroxytminomethyl group In position 2 or 4 (Table 3).- The oxlmes with a
TABLE 3. REACTIVATION OF OP INHIBITED AChE BY OX1MES
Ca)HON = CH
N - CH2 - 0 - CH2 - N
2C1~
o
R 00
Abbreviation (a)
kr10-3/M-1 mlm-1
VX DDVP
HI4
4.2-MEDP
4.4-MEDP
Toxogon 1 n
HS-3
HS-14
2.2-MEDP
2.4-MEDP
HS-4
PAM-2
(4)
(4)
(4)
(4)
(4)
(2)
(2)
(2)
(2)
CsH4N-C
-H
-CH3
-CH3
-CH =
-CH *
-H
-CH3
-CH3
-CH =
W3
NOH
NOH
NOH
(-)
(2)
(4)
(4)
(2)
(-)
(2)
(4)
(2)
7.7
7.9
7.8
7.5 8.6
7.8 8.4
7.4
-
7.6 -
6.7 7.7
7.8
4.1
6.3
4.3
6.6
8.6
0.34
,.
*' "' '
*
1.2 '"
13
6.2
*
14
4.8
*
#
' *' '
0.07
2.5
(2CH = NOH1CI
No reactivation was observed.
hydroxyimino group in position 4 in the pyrldiniurn ring were good reactivators
of both phosphorylated and phosphonylated AChE. The same oximes were also very
effective given with atroplne against VX and OOVP poisoning (Table 2). The
compounds are almost as effective as PANfc-2, but PAM-2 Is less toxic.
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TABLE 4. EFFICIENCY OF OXIMES AND ATROPINE AGAINST VX AND-DDVP POISONING
Toxlclty
Antidotes
Atroplne (alone)
Oxlme + atroplne
HI-4
4,2-WEDP
4,4-MEDP
Toxogon I n
HS-3
HS-14
2,2-MEDP
2,4-MEDP
HS-4
PAM-2
LDSO/mg
l.v.
«
47
59
100
100
168
66
54
170
67
101
kg'1
1 .p.
^
56
59
119
200
149
-
-
178
168
212
Relative efficiency
Aqalnst
DOVP
VX and
OOVP
(p.o.) (s.c.)
7.7
8.8
7.0
8.4
8.4
7.0
9.9
6.5
-
6.5
8.4
12.
-
37
34
34
34
43
-
-
24
DDVP
VX
(s.c.)
4.6
43
47
47
40
89
26
-
28
-
47
(10 mg/kg)
l.v. = Intravenous
I.p. = Intraperltoneal
p.o. = by mouth
s.c. = subcutaneous
Binding Sites for Substrates and Inhibitors In AChE and ChoHnesterase
It Is known that certain substrates act also as Inhibitors of the
cholInesterases. The mechanism of this reaction Is complex and not fully
understood (Aldrldge et al. 1972, Reiner 1975). Studies of the substrate-
Inhlbltlon-slte are described In this section.
AChE (EC 3.1.1.7) from bovine erythrocytes Is Inhibited by haloxon
(dI-(2-chloroethyl) 3-chloro-4-methylcoumarfn-7-yl phosphate), both Irreversibly
and reverslbly (temperature range 5 'C-40 °C).
AChE
Reversible complex
Ha Ioxon
Phosphorylated enzyme
The second-order rate- constants of Irreversible Inhibition (ka) Increase with
temperature, and the activation energy Is 84 kJ mot"' (Table 2). The dis-
sociation constants of the enzyme-Inhibitor reversible complex (Kj) range from
-------
3.6 uM (at 5*C) to 6.5 uM (at 40°C). The dissociation constants of the
enzyme-acetythlocholIne complex, K(s), derived from the reversible Inhibition
experiments range from 2.5 mM (at 5*C) to 4.1 mM (at 40°C) (Table 5). These
TABLE 5. EFFECT OF TEMPERATURE ON INHIBITION OF AChE BY HALOXON
Temp.
-c
3
11
18
25
32
40
10-3 ka
M-1 s-1
0.165
0.377
0.656
2.08
3.97
9.08
Kl
WM
3.6
4.2
5.5
6.4
9.1
6.5"
N*
28
12
3
87
3
7
K(S)
roM
2.5
1.9
2. 1
2.3
7.7
4.1
N a Number of experiments
constants are considerably larger than the Michael Is constant (Km) for AChE and
acetylthiocholIne, and agree more closely with the substrate Inhibition
constant (Kss) for acetylthiocholIne, Indicating that the competition between
haloxon and acetylthiocholIne occurs close to the substrate-Inhibition site.
The kinetics of competition of pairs of two substrates for bovine
erythrocyte AChE (EC 3.1.1.7) and horse serum ChE (EC 3.1.1.8) were studied In
such way that the hydrolysis of only one substrate was measured at a time. The
substrates were acetylthiochotIne, phenylacetate and benzoylcholine; the same
compounds, and also acetylcholIne, were used as competing substrates i.e.
Inhibitors. The substrate inhibition constants (Kss) and Michael is constants
for the reaction of a single substrate were also determined.
In Table 6, the Km and Kss values are given, the dissociation constants
for the enryme-substrate (K(S)T and enzyme-inhibitor (K(l) complexes are
listed in Table 7. Comparing these sets of data, it was concluded that the
substrate-Inhlbltion-slte In the enzyme does not show up In the competition
between two substrates.
Reaction of DDVP and 0,0-OImethyl-2t2,2-Trichloro-l-Hvdroxyethyl-Phosohonate
and (Metrifonate) with Cho11nesterases and AryIesterases of Various Species
Metrifonate (also called trlchlorfon) and DOVP are widely studied
compounds because metrifonate Is a drug used against schIstosomIas Is and DOVP
is a well known pesticide (Holmstedt et at. 1978, Nordgren et al. 1978, Wright
et al. 1979). Furthermore, metrifonate rearranges spontaneously Into DOVP and
all reactions of metrifonate are therefore accompanied by simultaneously'
proceeding reactions of CDVP. It is known that mammalian cho11nesterases (EC
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TABLE 6. MICHAELIS CONSTANTS AND SUBSTRATE INHIBITION CONSTANTS FOR
AChE AND ChE .
Substrate (mM)
(Range of Cone.)
KSs/mM
AcetylcholInesterase
Acetylchollne (0.01-1.0)
Acetylchollne (1.0-100)
Acetylthlocholfne (0.1-50)
PhenyI acetate (0.1-10)
ChoiInesterase
BenzoylcholIne (0.01-10)
Benzoylchollne (10-100)
Acetylchollne (0.1-10)
Acetylthlochollne (0.1-10)
Phenylacetate (0.1-10)
0..15
0.11
2.6.
0.4(nH» a 0.7)
1.2-3.2
0.6(nM
3.B
0.8)
9
14
56
nH = Hill coefficient
TABLE 7. COMPETITION BETWEEN TWO SUBSTRATES FOR AChE AND ChE
Substrate
K(S)mM
Inhibitor (nW)(Range)
K(l)irM
Acetylcholtnesterase
AcetylthtocholIne
AcetylthlocholI he
Phenylacetate
Phenylacetate
ChoiInesterase
AcetyIthlocho11ne
Acetylthlochollne
AcatyithiocholIne
Phenylacetate
Phenylacetate
BenzoylcholIne
0..17 ± 0.17 Acetylchollne (0.5-10) 0..22 ± 0.22
0.22 ±0.14 Phenylacetate (2.6-15) 4.9 ± 6.1
3.8 ± 0.8 Acetylthlochollne (0.2-5) 0.25 ± 0.03
6.2 ± 1.2 Acetylchollne (0.5-5) 0.54 ± 0.05
0.53 ± 0.17 Acetylchollne (5-20) 2.6 ±0.77
1.0 Phenylacetate (1-10) -4.0
± 0.55 Benzoylchollne (0.05-1) 0.03 ± 0.04
± 0.4 AcetyIthlocholine (0.2-4) 0.69 ± 0.08
±0.4 Acetylchollne (5-15) 3.1 ±0.7
±0.45 Acetylthlochollne (10-50) 1.3 ± 3.3
0
0.37
2.5
1.6
0.17
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3.1.1.7 and 3.1.1.8) are Inhibited by DOVP. The rates of the In vitro .
Inhibition, spontaneous reactivation, and aging of the inhIblteS" enzymes are
also known. A calculation Is presented here In which rate constants determined
In vitro are applied to enzyme activities measured In vlvo« The purpose was to
discover whether regeneration In vivo Is due to spontaneous reactivation of the
Inhibited enzyme or to enzyme synthesis. Two groups of data are used, one for
human blood ChE's and the other for rat brain and plasma ChE's. Two OP
compounds were studied, DDVP, and metrlfonate. The latter Is not a ChE
Inhibitor, but, In aqueous solutions It rearranges spontaneously Into DDVP,
which Is an Inhibitor of the enzyme.
The data for the In vivo regeneration of rat brain and plasma chollnes-
terases are given |n Table 8. The kinetic analysis of the data has proved
TABLE 8. BRAIN AND PLASMA ChE ACTIVITIES OF RATS INJECTED I.V. WITH
METRIFONATE OR ODVP*
Choi Inesterase activity (#)
Metr I f onate
Time-
after dosing
30 mln
60 mln
90 mln
2. h
3 h
6 h
12 h
24 h
2 days
6 days
8 days
300
Brain
2
«.
13
14
: '-35-. , .
68
68
84
78
83
77
mg/kq
P 1 asma
7
7. - ' "
14
.19
..33' -. :' .
73
74-
95
93
95
102
Brain
15
30
36
50
56
85
83
87
90
82
82
DDVP
2.5 mq/ka
P 1 asma
41
44
59
74
_^ 93
94
100
103
-
-
The activities are expressed as percentage activity of untreated animals.
Each value Is the mean value obtained In 5 to 12 animals. The number of
untreated rats was 40.
that regeneration of the enzyme activities after treatment with DDVP can be
attributed entirely to spontaneous reactivation of the Inhibited enzymes,wlth
half-times of 2 and 2.5 h for the brain and plasma ChEs, respectively. Table 9
gives data for the In vivo regeneration of human plasma and erythrocyte ChEs
after treatment with metrlfonate. The first determination of ChE activities
was done 6 h after dosing. It follows from the In vitro results that after
that time almost all enzyme is present in a non-reactable (aged) form.
Regeneration of the ChE activities (which was much slower than predicted from
-------
TABLE 9. ERYTHRCCYTE AND PLASMA ChE ACTIVITIES* IN SCHOOLCHILDREN
TREATED ORALLY WITH METRIFONATE
___=_--__. _ -. \ '.
. ' '**.
Choi Inesterasa activity (55)
Time
Group A (21)
7.5 mq/lq Metrlfonate
Group B (19)
Group C (19)
after dosing Erythrocyte Plasma Erythrocyte Plasma Erythrocyte Plasma
6 hr
24 hr
3 days
7 days
14 days
78
87
72
80
92
5
19
28
64
76
43
51
63
65
72
0
17
35
47
71
59
60
76
62
80
0
14
31
56
72
The activities are expressed as percentage activity before treatment, and
each number Is the mean value obtained for the group.
spontaneous reactivation) was therefore attributed to enzyme synthesis.
calculated half-times for the synthesis of human erythrocyte and plasma
ChEs are 15 and 6.7 days, respectively.
The
The reaction of DDVP with ChEs In parasitic helminths was
studied, because DDVP formed from metrlfonate might be the active compound of
the antI paras I tic drug.
The chol Insterase of the nematode parasite Metastronqy I us aprl (M_. aorl)
was studied at 25°C In 0.1 M phosphate buffer pH 7.4. The activity vs
substrate concentration curves for acetylthfocho!tne (ATCh) and
butyrylthiochol Ine (BTCh) are both be 11-shaped, and each has an optimum at 10mM
substrate. The Michael Is constants for ATCh and BTCh are 0.1 and 0.8 mM,
respectively. The substrate Inhibition constants for ATCti and BTCh are MOO
and »30 mN, respectively. At optimum substrate concentration, ATCh Is
hyrolyzed 15 times faster than BTCh. (Table 10). The enzyme Is soluble
(97$) In 0.15 M NaCI. DOVP and haloxon are progressive Inhibitors with rate
constant of Inhibition 7.2 x 10* and 2.5 x 105 M~1mtn~1, respectively.
Metrifonate Is not an Inhibitor. The results Indicate that M. aprl has only
one ChE, but It could not be established whether It Is the E.G. 3.1.1.7 or
E.C. 3.1.1.8 enzyme.
DDVP was used to establish the number of ChEs In certain
species. The protein fractions, soluble In 0.15 M NaCI, of Paramphlstomum
mlcrobothrium. Ascarls suum and Neoascarls vltulorum each contain at least two
ChEs. This is deduced from the degree of Inhibition by DDVP which
is different when measured with ATCh than with BTCh as substrate (Table 11).
As Table 12 shows, the ChEs of these species are only partially soluble In
saline.
10
-------
TABLE 10. KINETIC CONSTANTS .FOR THE REACTION OF:ChE WITH ATOh AND RTCh
Enzyme
*Metastronqy 1 us
aprl ChE
ATCh
BTCh
Erythrocyte ChE
ATCh
BTCh
Serum ChE
ATCh
BTCh
.VmM '
0.1
0.8
0.11
0.42
0.60
0.41
Kss/mM
>100
» 30
14
30
-
v for ATCh
v for RTCh
15
107
0.46
The activities for the £J. aprl enzyme were measured at nine different concen-
trations of ATCh and sev"en different concerttrations of BTCh, In enzyme pre-
parations from five and eight batches of parasites respectively.
TABLE 11. INHIBITION OF'ChE* BY DDVP IN PARAS.!TE SPECIES
Parasite
species
*At 25'C and pH 7.4
N * number of assays
ATCh
Inhibition CO
determined with
(N) . BTCh.
(N)
Paramohistomum
microbothrium
Ascaris suum
Ascaris suum
anterior part
Neoascaris
v i tu I orum
10 uM DDVP
5 min
10 min
2.0 uM DDVP
3 min
5 min
2.0 uM DDVP
1 min
2 min
3 min
4 min
2.0 uM OOVP
1 min
3 min
5 m!n '
44
64
. 74
82
51
57
58
67
25
45
57
(4) .
(19)
(8)
(8)
(6)
(6)
(5)
(4)
(5)
(4)
(3)
6
' 31
25
28
17
23
36
48
Zero
27
19
(2)
(18)
(3)
(7)
(5)
(5)
(5)
(5)
(4)
(3)
(4)
11
-------
TABLE 12. ChE ACTIVITIES*
ASCARIS SUUM FOR
IN PARAMPHISTOMUM MICROROTHRIUM AND
10 mM ATCh AND 10 mM BTCh
P.
mlcroUothrlum
A. suum
Activity In homogenates
against ATCh
Mean (n)
Range
Activity ratio
ATCh/BTCh In homogenates
Mean (n)
Range
umol frc
47 (6)"
35-62
1.0 (6)
0.73-U5
Ratio
A3 (7)
34-58
2.1 (7)
1.2-3.1
Percent activity In
soluble protein fractions*
Mean against ATCh (n)
Range
Mean against BTCh (n)
Range
82 (fir)
72-93
90 (5)
77-100
Activity
43 (7)
29-58
66 (7)
44-77
At 37°C and pH 7.7..
The activity of the total homogenate was taken as
--n - number of parasite batches.
The hydrolysis of DDVP, E600 (0,0-dIethyl-4-nltrophenyl phosphate) and
MeE600 (0,0-dImethyl-4-nItro-phenyl phosphate) was studied (37°C; bicarbonate
buffer, pH 7.4) In homogenates of Ascarls suum, Fasctola hepatlca, JM. aorl,
Neoascarls vltulorum, Paramphlstomum mlcrobothrlum and Schlstosoma manson I,
In plasma and erythrocytes of man, rat, rabbit, hamster and chicken, and In
liver homogenates of rat and hamster. AM preparations hydrolyzed DOVP (Tables
13 and 14), and, for 10 mM DDVP, the activities ranged from 2 to 60 pmol/h/g
wet weight tissue. Mammalian plasma hydrolyzed E600 at about the same rate as
ODVP; (Table 14) little or no hydrolysis of E600 (5 mM) and MeE600 (5 mM) was
found In vertebrate erythrocytes In chicken plasma and In parasitic helminths.
The Michael Is constants for DDVP were all In the ml 11Imolar ranqe. The
hyrolysts of DDVP In parasitic helminths and In vertebrate plasma was Inhibited
by MgCl2 and AgN03, while the hydrolysis In the erythrocytes was unaffected;
the non-competitive Kj for rabbit plasma was 5.1 mM and 0.5 uM for MgCl2 and
AgN03, respectively.
12
-------
TABLE 13. HYDROLYSIS OF DOVP AND PA IN HOMOGENATES OF PARASITIC HELMINTHS*
Hydrolysts/ymol h-1 q-1 wet weIqht
Species
A. suum
F. hepatlca
M. aprl
N. vltulorum
P. microbothrlum
S. mansonl
DDVP
10 mM
6.9
57
33
8.6
21
6.6
n
12
2
1
2
6
1
PA
10 mH
138
67
260
28
n .
5
1
2
1
n = number of parasite batches
*AI I activities determined manometrIca11y
TABLE 14. HYDROLYSIS OF DDVP AND E600 IN PLASMA AND ERYTHROCYTES'
Species
DDVP
Hydro lysis/vino I h~1 ml~l
E600
10 mH
Plasma Erythrocytes
5 mH
Plasma Erythrocytes n
Rabbit
Rat
Man
Hamster
Ch Icken
61
12
11
3.7
1.9
5.8
14
5.6
7.9
7.5
8
13
20
1
17
93 2.3
18 1.9
9.1 0.94
- -'
Not hydro lyzed
6
9
14
-
7
All activities determined manometricatly.
n = number of parasite batches
Heat Inactlvation (at 80°C) of the hydrolytlc activities In erythrocytes
(against DDVP and MeE600) revealed two enzymes, (Table 15). In plasma (at
53*C) there was no evidence of more than one enzyme (rate constants are given
In Table 16). Both enzymes In the erythrocytes hydrolyzed the OPs and PA;
their activity was fully abolished by proteolytlc digestion. The enzymes In
erythrocytes were fully soluble (Table 17), In human erythrocytes, a partial
separation from hemoglobin was achieved by gel filtration on Sephodex G-1CC.
The proteins which hydrolyzed DDVP In all studied preparations can be
classified under arylesterases (EC 3.1.1.2).
13
-------
TABLE 15. HEAT INACTIVATION* OF THE HYDROLYTIC ACTIVITIES IN ERYTHRCCYTES.
Species
Man
Man
Man
Man
Rat
Rabbit
Chicken
Substrate
DDVP '
MeE600
PA
ACh
DOVP
DDVP
DDVP
Remain Inq
activity/?!
54
39
Zero
Zero
23
51
29
n*
13
5
4
5
2
2
3
1 hr at 80"C. All activities determined manometrlcally.
^n = number of experiments with erythrocytes of different Individuals.
TABLE 16. HEAT INACTIVATION OF HYDROLYTIC ACTIVITIES IN PLASMA
Species
Man
Rat
Rabbit
Chicken
*
k/mln-1
0.026
0.097
0.017
0.002*
nf
4
2
2
5
_ Is the first order rate constant of Inactlvatlon obtained with DOVP and £600
as substrates. AM activities determined manometrlcalIly.
1"n Is number of experiments with plasma from different Individuals.
^Obtained with DDVP only.
TABLE 17. DISTRIBUTION OF HYDROLYTIC ACTIVITIES BETWEEN STROMA AND
HEMOLYSATE IN HUMAN ERYTHROCYTES*
Substrate
Activity
umol h-1 m
Whole erythrocytes
Percent
activity
Stroriia
Haemolysate
DDVP (10 nW)
MeE600
PA (10 rrW)
ACh (13.8 mM)
ATCh (1 mM)
6.7
3.2
79
140
229
Zero
Zero
49
73
61
100
100
51
27
39
5
2
5
5
7
"All activities were determined manometrtcally except towards ATCh which was
determined spectrophotometrlcally.
n = number of experiments.
14
-------
SUMMARY
Twenty five pyrtdtnfum oxlmes were synthetlzed and tested In vitro for
reactivation of phosphorylated and phosphonylated AChE, and ?n vtvo against
poisoning by OP compounds. Some of the compounds were about equally effective
as PAM-2, TMB4 and toxogonln, which are the best known antidotes so far.
The binding sites In AChE for several Inhibitors were studied, In order to
establish whether the substrate-lnhlbltton-slte takes part In the reaction.
It was concluded that the substrate-lnhlbltton-slte Is not Involved In the
competition between two carboxyl esters, but when the OP compound haloxon Is
the Inhibitor, the reaction occurs close to that site.
Comparative studies of chollnesterases and aryIesterases In different
species were conducted using DOVP as Inhibitor (of ChE) and substrate (of
aryIesterases). No difference was observed In the kinetics of the reaction In
different species, and the activity of the aryIesterases (expressed per wet
weight) was of the same order In mammalian and In non-vertebrate tissues. The
kinetics of the reaction were also used as a tool for determining the number of
ChEs In a given tissue. Further, the return of ChE activity In vivo after
Inhibition by DDVP was used to evaluate the rate of synthesis of human plasma
and erythrocyte ChEs.
15
-------
SECTION 3
RESIDUES OF ORGANOPHOSPHORUS PESTICIDES IN HUMAN URINE
Alkali metal salts of the dialkyl esters of phosphoric, thtophosphorteand
dlthlophosphorlc acid are produced In the course of hydrolytlc and metabolic
degradation of OP pesticides. Therefore, a sensitive and specific determina-
tion of amounts of these salts In urine and blood samples Is of great value In
protecting against the bad effects of absorption of OP pesticides. Several
procedures have been described for monitoring the absorption of pesticides by
humans and animals based on acidification of a urine sample and conversion of
the acidic form of the excreted OP species Into trlalkyl derivatives suitable
for gas chromatographlc (GC) analysis (Shaftk et al. 1973, Blair and Roderick
1.976, Lores and Bradway 1977,. Brad way and Sbaflk 1977). An analogous procedure
for monitoring these pollutants In surface waters Is based on collection of OP
species on Amberllte XAD-4, with simultaneous separation of the greater part of
the Inorganic phosphates (Daughton et al. 1976).
Until recently, In Yugoslavia,, a.depress Ion of either plasma or blood ChE
activity has served as the only Indicator for absorption of OP pesticides
(Svetltclc and'W!I helm 1973). A significant depression of ChE activity In
persons manipulating pesticides was taken as a signal for job-change to avoid
further contact with toxicants until the normal cholInesterase activity was
restored. Urinary metabolite analysis was Introduced to detect absorption of
OP pesticides at a phase preceding a depression of ChE activity, so protective
measures could be taken sooner (Drevenkar et al. 1979a).
The high concentrations of OP pesticide residues found In the morning
urine samples of workers occupationally exposed to phosalone led us to
investigate the rates of excretion of metabolites In the urine of a volunteer
experimentally exposed to phosalone during one working day and then again
during three subsequent days. On the basis of the results of these tests, we
established the period necessary for the complete excretion of residues and the
appropriate time for urine sampling. (Drevenkar et al. 1979b).
We compared two methods, measuring blood ChE Inhibition and measuring OP
pesticide residues In urine of occupational Iy exposed persons,, to establish
which provides the more reliable Indication of absorbed OP pesticide.
The results of our work are published In five papers (Drevenkar et al.
1976a, c, 1979b, c, Stefanac et al. 1976) seven communications Drevenkar et al.
1976b, 1977, 1979a, 1978, Frobe and Stipcevic 1977, Vasll!c et al. 1978, 1979)
and five theses (Frobe 1977, Meczner 1979, Stipcevic 1978).
16
-------
MATERIALS AND METHODS
Standards
0,0-Oimethyl phosphorodtthfoate potassium salt (DMDTPK) Lot. No. 5057;
0,0-dIethyl phosphorodlthfoate potassium salt (DEDTPK) Lot. No. 4224;
0,0-dIethyl phosphorothlonate potassium salt (DETPK) Lot. N o. 6803;
0,0-dTethy! phosphate (DEP) Lot. No. 7302, purity 98%, were all obtained from
the United States Environmental Protection Agency Repository, Research Triangle
Park, NC, U.S.A.
Dtazomethane ethereal solution (10±5 mg CH2N2/ml) was prepared from
N-methyl-N-nltroso-p-toluensulphonamlde (Merck, Germany) following the usual
procedure (Vogel 1956), or, according to the usual d 1st! I lat I on procedure,' from
N-methyl-N-nltroso urea. N-methyl-N-nltroso urea was a gift of the Laboratory
for Organic Chemistry and Biochemistry, Faculty of Science, University of
Zagreb. -
Ion-exchanger Amber lite IR-120, H+-form, 28-35 mesh, was obtained from
Fluka AG, Switzerland..
Instruments
Varlan Aerographs, Series 1400 (columns 11 andd III) and Series 2800
(columns la and Ib), equipped with Alkali Flame lonlzatlon Detectors with a
rubidium sulphate-tip and a Pye Unlearn GC 204 GC (column IV) with an Flame
Photometric Detector were used.
The GC columns used were: glass, 1.8m x 2 mm I.d., packed with 4$ SE-30 +
6% OV-210 on 100-120 mesh Gas Chrom Q. (columns la and Ib); stainless steel, Im
x 2 mm I.d., packed with 25% Triton X-305 on 80-100 mesh Chromosorb W-AW/DMCS
(column II); glass, 1.5 m x 2 mm I.d., packed with 25% Triton X-305 on 80-100
mesh Chromosorb W-AW/DMCS (column 111); glass, 2m x 2 mm I.d., packed with 4%
SE-30 + 6% OV-210 on 100/120 mesh Gas Chrom Q.
The working conditions are listed In Table 18.
TABLE. 18. GC WORKING CONDITIONS
Condition
Columns
la Ib II III IV
Column temp., 8C 190
Injector temp., °C 235
Detector temp., °C 255
Nitrogen carrier
flow, ml/min 3.5
Air flow, ml/min 235
Hydrogen flow, ml/min 35
115
160
175
30
235
35
175
230
235
35
235
35
140
190
235
30
235
35
110
250
250
30
30
.30
17
-------
Methyl derivatives were'Identified with a GC-mass spectrometer (MS) system
by courtesy of Professor D, Jeremle (Faculty of Science, University of
Belgrade). A Varlan Aerograph Series 1200 GC, equipped with a thermal
Conductivity Detector (TDC), was coupled with Varlan MAT CH 5 MS by means of a
separator, lonlzatlon source temperature was 190°C, with electron energy of 70
eV. The column was heated to 80'C and 110°C for the determination of the
products obtained by methylatlon of CMDTPK and DEPTPK respectively, and helium
was used as a carrier gas.
Methods
Simulated samples, prepared by adding a definite amount of standard salt
to urine of nonexposed persons, were treated by three different procedures.
Procedure I
A dilution series of the standard aqueous DEDTPK solution (4 ug/ml) was
prepared. AlIquots of 0.5 ml were added to 2.0 ml of water or urine containing
no DEDTPK. Four 2-ml portions of each urine sample were measured Into separate
10-ml centrifuge tubes. A known amount of DEDTPK was added to two of them as
Internal standard. Successively, 2 g of sodium chloride, 4 ml of dlethyl ether
and 1 ml of 6 N hydrochloric acid were added. The solution was shaken for 1
mln on a Vortex mixer, then centrlfuged at 425 G. An aliquot of 2 ml was
transferred from the organic layer Into a test-tube fitted with a ground-glass
stopper. One ml of ethereal dlazomethane solution was added and mixed In
thoroughly, and the tube was left for 10 mln. The solution was evaporated to 1
ml under a stream of nitrogen and made up with water to 5 ml. Then, 4 g of
sodium chloride was added, and the mixture was extracted with 2-ml portions of
hexane. The combined extracts were evaporated to 1 ml In a stream of nitrogen,
and subjected to GC analysis.
Procedure 11
To a 5 ml aliquot of a urine sample, 0.5 ml of standard salt dissolved In
bldlstllled water (or bldlstllled water alone In the case of a blank) was
added. Inorganic phosphates were removed by adding O.lg Ca(OH)2, shaking for 1
min on a Vortex mixer, and centrlfuglng at 425 g for 3 mln. One ml of the
supernatant was transferred Into a test tube with a ground stopper; and 4 ml of
di ethyl ether and 1 ml of 6 N HCI was added. This mixture was shaken again on
the Vortex mixer and 2 ml of the ethereal layer was separated, and then
alkylated by the addition of 1 ml of dlazomethane solution. The sample was
left to stand for 10 mln, and then evaporated under a stream of nitrogen to 1
ml, which also removed the excess of diazomethane.
Procedure 11 I-
The samples were treated according to the method of Blair and Roderick
(1976). For the analysis of urine samples collected from the persons
occupattonally exposed to qulnalphos, 9 ml of the final sample obtained by this
procedure was transferred Into a test tube with a ground stopper and evaporated
under a stream of nitrogen to 1 ml.
Recovery rates for.Procedures II and III were determined using solutions
prepared In the following way: 3 - 5 mg of standard salt was dissolved in
2-3 drops of 6 N HCI and 5 ml of benzene was added. After being mixed In a
18
-------
Vortex mixer for 1 mfn, 3 ml of the benzene layer was separated and alkylated
with dlazomethane. The excess of CH2N2 was removed and a dilution series of
reference solutions prepared. '
Sample solutions for GC-MS characterization of DMDTPK andd DEDTPK methyl
derivatives were prepared In the same way.
Standard solutions for quantitative evaluation of malathlon (Procedure
III) and phosalone (Procedure II) residues were prepared by adding amounts
increasing from 0.1 to 0.5 ml of water solutions of DMDTPK (0.230 mg/ml) and of
DEDTPK (0.11 mg/ml), respectively, to 5 ml urine samples of non-exposed persons.
Control samples for quantitative evaluation of qulnalphos andd phosalone
residues (Procedure III) were prepared by adding 0.1 to 0.5 ml of DEDTPK
(0.0134 mg/ml), DETPK (0.01123 mg/ml) and DEP (0.0107 mg/ml) to 5 ml of urine
of a non-exposed person.
Blood and plasma ChE activities were measured as described by ElIman et
al. 1961.
RESULTS AND DISCUSSION
Gas Chromatoqraphlc Determination of Alkali'Metal 0,0-OIethyl Phosphorodlthlo-
ate"Present In Trace Amounts
Pesticide formulations containing phosalone are widely produced In
Yugoslavia for agricultural and domestic use so there Is a demand for an
analytical method for assessing exposure and pollution with respect to this
pesticide. To meet this demand, a determination of 0,0-dtethyl
phosphorodithloate alkali metal salts was worked out as being the best
indicator of phosalone (Drevenkar et al. 1979c). MetabolIc degradation of
phosalone causes the urinary excretion of alkali metal salts of the dlethyl
esters of dithiophosphorlc, thlophosphorlc and phosphoric acid. The last two
of these species might also result from derivatives of thlophosphorlc acid; and
the last one from derivatives of phosphoric acid. Therefore, the determination
of alkali metal salts of dlethyl phosphorodithloate (Procedure I) was chosen
because these can only come from dlethyl esters of dithiophosphorlc acid, which
Include phosalone.
Various dlazoalkanes were applied for conversion of the salts into the
more volatile trlalkyl derivatives. Diazopentane Is repeatedly recommended
(Shafik et al. 1073, Lores and Bradway 1977) as promoting the GC separation of
the amyl derivatives obtained from all OP moieties possibly present. A
drawback reported in the literature was that the reagent for the preparation of
diazopentane is not easily accessible (Blair andd Roderick 1976). We encoun-
tered additional problems related to the purity, of prepared diazopentane.
The GC obtained with blanks showed an abundance of peaks, one of them having
the retention,time expected for 0,0-diethyl^S-amylphosphorodIthio-ate. All
attempts to separate the Impurities efficiently enough to remove the
interference in the GC analysis were unsuccessful. These draw-backs were
eliminated when diazomethane, purified by distillation was used as
- . -... . -..-. 19,' . .. ; ;.. ,...-. "" ' '. :
-------
alkylatlng reagent. The.clean up procedure on a silica gel column; unavoidable
after the alkyiatlon with dlazopentane, could be omitted'., interferences In the
GC analysis arose only when the dtazomethane concentration exceeded 15 mg/ml,
but such concentrations are easily avoided by dilution, without affecting the
results.
The alkyiatlon product of the DEDTPK with dlazomethane was characterized
by GC-MS.
The retention times of the methylated 0,0-dIethyl phosphorodtthloate are
75 sec on column la and 190 sec on column II, with a detection limit slgnal-
to-nolse ratio 4:1 of 40 pg.
Systematic errors arising from Incomplete methyl atIon and recovery, as
well as from changes caused by variations In working conditions during the GC
analyses were corrected by running standards and samples alternately. Peak-
height measurement was used for quantitative evaluation..,
The calibration graph obtained with a series of aqueous samples covering
the range 0.04 - 1 ng of DEDTPK was a straight line (slope b=6.80; standard
error of the slope Sb=0.55; variance about ±he regression s2* 0.86). The
detection limit clearly shows the necessity of Including an efficient
collection procedure (Daughton et al. 1976) In the pollution control of surface
waters. A complete separation of Inorganic phosphates Is not critical, because
of the good differentiation of trlmethyl phosphate In GCs.
For urine samples In the same concentration range, the calibration graph
was non-1Inear. .
The results of a series of analyses performed on urine samples of
occupationally exposed persons sre listed In Table A-1 (Appendix). In the urine
samples of all 12 persons working In the production of pesticide formulations
containing phosalone DEDTPK was found. Only the two highest values were
accompanied by a depression of the AChE activity In the blood, thus affirming
the usefulness and reliability of our method for early detection of exposure.
For definite proof of the relation of concentration of metabolites to
depression of AChE activity, a GS/MS system with a capillary column Is
necessary.
Comparable results have been obtained using the procedure described and
the particularly simple one of Blair and Roderick (1976) to analyze exposure
monitoring. However, a common drawback has to be pointed out. The procedures
based on alkyiatlon with dlazomathane are not feasible for simultaneous
determination of all OP pesticide residues. The necessity of removing
Inorganic phosphates seriously complicates the sampler-treatment procedure when
0,0-dimethyl phosphate has to be determined. This drawback does not apply when
the alkylation Is done with dlazopentane, but then there Is the problems of
obtaining a sufficiently pure reagent for preparation of the dlazopentane.
20
-------
The Rate of Urinary Excretion of Phosalone Residues In Occupationally Exposed
Persons
The absorption of OP pesticides by two groups of occupational Iy exposed
workers was evaluated by determining the residues In urine samples (Drevenker
et al. 1979b). One group of persons was exposed to malathlon and the other to
phosalone. To assess the extent of exposure, urine samples were analyzed for
DMDTPK and DEDTPK..
The Identity of 0,0-dlmethyl-S-methyl phosphorodlthtoate, the only product
obtained by methylatlon of DMDTPK, was confirmed by SI-MS analysis.
Procedure III was chosen to test for malathlon because It yielded 73 to
Q9% recovery of DMDTPK from spiked urine samples compared to only 37 to 57%
recovery by Procedure II. For phosalone, Procedure II was preferable, giving
slightly better recovery'(89 to 107J& as opposed to 87 to 995?) of DEDTPK than
Procedure III.
Urine samples of workers exposed to malathlon were collected at the end of
work hours, whereas blood ChE activities of some exposed persons were measured
a day later. The results obtained are listed In Table 19. DMDTPK concentra-
tions found In ten out of a total fourteen urine samples were In the range from
TABLE 19. ABSORPTION OF MALATHION AND ChE ACTIVITY
Exposed
workers
Initials
P.M.
L.P.
G.A.
Z.A.
K.D.
S.I.
H .M.
K.B.
F.A.
B.J.
Cone.
ng DMDTPK/ml of
urine
1072 ± 76
760 t 65
690 t 62
641 ± 85
641 ± 91
608 ± 49
545 ± 149
449 t 80
474 ±- 80
471 ± 24
ChE activity
% of pre-exposure value
_
'
-
-
65
85
95
-
449 to 1072 ng/ml of urine. In remaining urine samples, as well as in urine
samples of nine non-exposed persons, the malathlon residue was not detected.
Detection limit was 20 pg.
As only three workers had their ChE activities measured before exposure,
the relevant data are for the most part missing In the Table 19 but the highest
concentration of malathlon residue In urine is accompanied by the lowest blood
ChE activity and vice verse. Two workers (noti,'fn*Table 19} from the exposed
21
-------
group with no metabolite detected In urine had blood ChE activities 96 and 100*
of the pre-exposure values.
To check If urinary metabolite excretion Is completed within a 24-h
period, urine samples of two workers exposed to phosalone collected before and
after work were analyzed. Table 20 shows that the concentrations of the
residue of phosalone, DEDTPK, In urine samples col Iected be>ore work were,
TABLE 20. ABSORPTION OF PHOSALONE IN TWO VOLUNTEERS*
Cone.
Workers
Initials
O.M. Day 1
O.M. Day 2
O.M. Day 3
B.M. Day 1
B.M. Day 2
B.M. Day 3
nq DEDTPK /ml
Beginning of Day
314 ± 15
405 ± 5
347 ± 4
1274 ± 42 .
064 ± 10
913 t: 43
of urine
End of Day
311 ± 71
317 t 34
739 ± 122
1084 ± 94
476 ± 30
474 ± 63
All results are the mean vslues of two determinations.
within the range of experimental error, equal to, of higher than, those found
In urine samples collected after work. The results Indicate that the excretion
of phosalone metabolites Is still going on at the beginning of the next working
day.
To verify this conclusion, the rate of DEDTPK, DETPK and DEP excretion In
the urine of a volunteer experimentally exposed to phosalone was Investigated
(Drevenkar et al. 1979b). The experimentally exposed person, who had no
previous professional contact with pesticides, worked In the same locations,
and performed exactly the same Jobs, as other workers.
The results, showing concentrations of metabolites fn urine after an
exposure lasting 6 h, are presented In Table 21. Urine sampling was carried
out at 2 - 4 hour Intervals,
In the case of exposure during three succeeding working days (Table 22),
urine samples were taken at the beginning and at the end of the work, and
4 -5 hours after termination of exposure. The 24-h urine was also collected,
and blood ChE activity before work was measured.
The concentrations of DEDTPK shown In Tables 21 and 22 are expressed In
nanograms per mil I Miter of urine. The concentrations of DETPK and DEP are
expressed in peak heights relative to the peak helaht of 0,0-dlethyl ohosoho-
rodlthioate methyl derivative, which was used as internal standard. Due TO
this difference In expressing concentrations, only the elutlon patterns, not
the absolute amounts, of excreted compound can be compared.
22
-------
TABLE 21. EXCRETION OF METABOLITES AND ChE ACTIVITY IN A WORKER EXPOSED TO PHOSALOME
FOR SIX HOURS
Hours Attar Exposure
0£P
Amount of petabolIta*
OETPK
DEOTFK
ChE Activity In Blood
(t of preexposure value
is)
Exposure O.t)
0.2
4.1
6.6
B.O
II
U
16
21
25
26
28
32
36
41
46
49
53
"Peak halqht of DEW3.
Nat detected
Hot datected
30
36
Not measured
195
92
60
50
44
Not measured
36
55
51
46
13
Not datected
Not detected
DEMTP, or OEMHP
Not detected
Not detected
84
166
Not measured
1246
250
124
as
70
Not measured
39
48
37
38
10
8
10
Not datected
Not detected
202
565
Hot measured
1994
439
224
136
65
Not measured
51
72
45
59
9
7
7
100
88
80
.X*
ng DEDTPK
Peak height of DEHOTP/ml of urine
-------
K>
TABLE 22. EXCRETION OF METABOLITES AND ChE ACTIVITY IN A WORKER EXPOSED TO PHOSALONE
FOR TWEE DAYS
Hours After Exposure
Amount of Metabolite" ChE Activity
' : (K of preexposure value)
Expc>i!;<'»
Expo£
-------
The observed kinetic patterns show an obvious similarity In the rate and
duration of excretion for. alI three phosalone metabolites, the amount of
residues excreted In urine Increases gradually and the highest value Is reached
4 - 5 h after exposure Is ended. Subsequently, the amount decreases abruptly,
but at the start of the following working day It Is still measurable, and it
shows a systematic Increase when exposure Is prolonged for several following
days.
It Is also obvious (see Table 23) that metabolite quantities vary during
TABLE 23. METABOLITES IN URINE OF A WORKER EXPOSED TO PHOSALONE FOR THREE DAYS
Days After
Exposure
Exposure
1
2
3
4
5
DEP
13
60
82
27
4
Amount of metabol tte
. DETPK
19
128
80
13
Not detected
DEDTPK
16
188
118
24
Not detected
*Peak height of DENP. DEMTP or DENOTP
Peak height of DEMDTP/ng DEDTPK
mi of urine
the period of excretion.. The total amount of all metabolites excreted during
one day Is certainly a better Indicator of exposure than metabolite concentra-
tion In a single urine fraction. However, the difficulties involved In
collecting a total volume urine .sample make such sampling inconvenient in a
routine monitoring of exposure among workers engaged In the production and
application of OP pesticides. Instead of the total excretion of metabolites,
the concentrations of DEDTPK In urine sample taken the same period of time
after exposure can be used as a satisfactory Index of exposure.
Blood and plasma ChE activities were only slightly reduced during
exposure; 10 to 20J5 depression can^not be regarded as a significant indication
of exposure.
Although the rate of pesticide degradation varies from Individual to In-
dividual, and is probably different for different compounds, similar kinetic
patterns can be expected for other OP pesticides. It follows that in a
pesticide monitoring program designed for control of occupational exposure,
routine analysis of urine samples taken 4 - 5 h after exposure offers the most
reliable Information about pesticide absorption. However, such a sampling
procedure Is not convenient for routine control, and analyses of urines taken
before and after work have to be performed Instead. A systematic Increase in
the concentrations of pesticide residues in the morning urine demands that more
efficient measures of protection be taken or that workers be transfered to
other jobs.
:-.-. . 25
-------
Human Blood ChE Activities and Oulnalphos Pesticide Residues In Urineof
OccupafionaMy Exposed Persons ;;-r: ':". ' ! '' ~" '
The absorption of qulnalphos In a group of'workers from a pesticide
formulating plant was studied by determining the metabolic degradation product
(DEP and DETPK) of this OP compound In their urine samples. OEDTPK was also
detected In most of the samples because 1-2 days before starting to work with
qulnalphos the same workers were making phosaione.
The level of excreted aIkyI.phosphates In the urine was correlated with
the level of ChE activity In the blood of these Individuals. Urine samples
were collected at the beginning and at the end of the working hours of one day
at Intervals of 30 days during three months - May, June and July - and
Immediately after one month's vacation In September. The blood samples were
collected before the beginning of the work In the same working day as urine was
taken.
For the determination of DEP, OETPK and DEDTPK the urine samples were
treated according to Procedure Ml. The GC analysis of DEP methylated
derivative was performed on column Ml, and GC analysis of DETPK and DEDTPK
methylated derivatives on column IV.
Simultaneously with the eleven urine samples of occupationally exposed
workers, eleven urine samples of non-exposed persons were analyzed. No a Iky I
phosphates were detected In the samples from non-exposed workers.
The results of the three month measurements of the eleven workers exposed
to qulnalphos are listed In the Appendix Table A-2.. Note that workers B.D.
and U.B. did not have ChE activities measured.
Tables A-2 shows that the amounts of a Iky I phosphates In exposed workers
vary widely.
On the first working day after one month vacation small amounts of
residues were detected at the end of the work only In the urine samples of
workers K.F. (67 ng DETPK/ml) and K.M. (158 ng DEP/ml, 123 ng DETPK/ml and 37
ng DEDTPK/mI). The values of ChE activity of all workers were between 80-100£
with the exception of the worker K.M. whose whole blood and plasma ChE
activities were 28$ and 65!? of base values. The highest concentration of
residues (In K.M.'s urine sample collected In July) was followed by greatest
decrease of ChE activity.
Since the workers alternate every 3 to 5 days between work with OP
pesticides and work with some less toxic carbamates, and activity of blood
ChE In the exposed persons Is affected by both classes of compounds, a
comparison between the amounts of DEP, DETPK and DEDTPK and the whole blood and
plasma ChE activities can hardly be made. Moreover', the OP compounds and
carbamates are often formulated at the same time In the same room. Therefore,
In this case, ChE activity can^hot be used as a parameter Indicating the
Intensity of exposure to qutnalphos or OP pesticides only; because It Is caused
by absorption of ChE Inhibitors In general. On the other hand, tne auantiry o-f
26
-------
excreted residues specifically Indicates the absorption of the parent OP
pesticide, and Is a sensitive and selective method for early detection of harm-
ful exposure to these compounds.
SUMMARY
A GC determination of traces of alkali metal 0,0-dlethyl
phosphorodlthtoates Is described. The salts were converted Into a volatile
derivative by alkylatlon with dlazomethane. The product was Identified by GC-
MS. A linear relationship of peak height to amount of salt was obtained In
the range 0.04 - 1 ng with aqueous samples, and a calibration curve was
constructed for urine samples. The detection limit was 40 pg of salt. The
procedure was successfully used for monitoring phosalone absorption by occupa-
tional Iy exposed persons.
The absorption of malathlon and phosalone was followed In occupationaliy
exposed workers by determination of residues excreted In the urine. Because of
the high concentrations found In the morning urine samples,' the rates of
excretion of phosalone metabo11tes In the urine of a volunteer experimentally
exposed to phosalone during one, and then again during three subsequent,
working days were Investigated. The urinary excretion of phosalone metabolites
was most Intense 4 to 5 h after exposure. At the beginning of the next day',
the metabolites were still measurable In the urine. Blood and plasma ChE
activities were only slightly reduced during exposure. The analyses of 24 h
urine samples, or of urine samples taken 4 to 5 h after exposure , are not
convenient for the routine monitoring of occupational Iy exposed persons because
of sampling difficulties. Instead, analyses of samples taken Immediately
before and.after work hours have to be performed. A systematic Increase In the
concentrations of pesticide residues In -the mornfhg urine Indicates
the need for more efficient protection measures.
A study comparing blood ChE Inhibition to OP pesticides residues In urine
of occupational Iy exposed persons was carried out. The quantity of an excreted
residue Is a reliable Indication of the absorption of a given OP compound.
Blood ChE Inhibition, however, reflects the effect of any absorbed compound
which Inhibits these enzymes.
27
-------
SECTION 4.
RESIDUES OF CHLORINATED HYDROCARBONS IN HUMAN MILK AND BLOOD
The use of chlorinated hydrocarbons has recently been restricted In
Yugoslavia. In 1975 we Initiated a survey to establish the degree to which
these pesticides have been absorbed by the general population and by
occupationally exposed workers. The survey, which Includes
hexachlorocyclohexane and DDT, should provide a basis for a later study to
follow-up the effects of the recent restrictions. More recently, we also
started a survey. In collaboration with WHO/UNEP, of DDT found In human milk,
and In mothers' and cord blood serum.
We have previously published the results of our work In three papers
(Krauthaeker 1980a, b, Reiner 1977a) and two communications (Krauthacker 1977,
1978). For details of the experimental procedure, and for references to the
literature, the reader Is referred to these pub I leafIons.
MATERIALS AND METHODS
Blood samples from the general population and from occupational Iy exposed
workers were collected In four different parts of Yugoslavia. Human milk and
samples of mothers1 and cord blood were collected In one maternity hospital.
The pesticide residues were determined by GC. All compounds were
Identified by their retention times as compared with known standards.
RESULTS AND DISCUSSION
Residues In BIcod Serum of the General Population and In Occupationally Exposed
Workers
Levels of chlorinated hydrocarbon pesticide residues In serum samples of
the general population of four different parts of Yugoslavia, and In two qroups
of occupationally exposed workers were determined by GC (see Appendix, Table
A-3). Serum samples, 262 from the general population and 78 from exposed
workers, were analyzed for p,p1-ODT, p^l-ODEjp^-ODD, LIndane, and o-HCH. All
samples contained over I jig/I p,p1-ODE. The concentrations of residues In the
general pouplatlon fall within the range reported for other countries. Few
samples from the general pouplatlon contained LIndane or o-HCH; the means for
LIndane were between 3.1 ug/l and 3.8 ug/l and for a-WCH were between 1.3 ug/l
and 2.6 ug/l. However, the means for the exposed workers were much higher, 9.2
ng/l for one group and 38 ug/l for the other.
28
-------
Residues In Human Milk, and In Mothers' and Cord Blood Serum
All samples were collected In a continental town of Croatia from 1977 to
1979.
Two extraction methods for milk were used and compared. Method A was
recommended by the Environmental Protection Agency, USA (Thompson 1974) and
method B was described by Curley and Klmbrough (1969).
Method AOne ml milk was extracted three times with 2.5 ml acetonltrlle. To
the combined acetonltrlle extracts 25 ml 2# Na2S04 was added, and the
mixture was extracted three times with 5 ml hexane portions. Hexane
extracts were purified on a florlsll column (1 cm diameter) containing 1.6
g florlsll topped with 1.6 g N32S04 anhydrous. The column was prewashed
with 20 ml hexane. The organochlorlne compounds retained on the column
were then eluted with hexane until the collected eluates had a total
volume of 25 ml. The eluates were evaporated to dryness In a stream of
nitrogen, the compounds redlssolved In 1.0 ml hexane and analysed by GC.
Method B^-One half ml milk was partitioned with 2 ml methanol and 3 ml \%
I<2C03. The mixture was extracted three times with 5 ml hexane portions.
Hexane extracts were evaporated to dryness In a stream of nitrogen, the
compounds redlssolved In 1.0 ml hexane, and determined by GC.
Concentrations of p,p'-ODE, p-p1-000, and p,pi-DDT were determined In 34
samples of human milk obtained 3-5 days after delivery (Table 24) and In 37
samples obtained from 1.5 to 55 week after delivery (Appendix, Table A-4). AM
samples contained p,pi-ODE, but only a few samples contained p,pi-ODD and
p,p'-ODT. The mean concentration of p,pl-ODE was 31 ug/l In the samples
analyzed In the beginning of lactation, and 53 ug/l (By method A) for the
samples taken over a 55 week period . The concentration ranges for the first
group were 9 -97 ug/l and for the second 9.4-167 ug/l, and the difference In
the mean values is not significant.
The two methods of extraction of milk samples compare fairly well for p,pl
-ODE (Appendix Table A-4). Chromatograms of the samples extracted with method
B have fewer signals due to impurities than samples extracted by method A,
although the latter Includes purification on florisil columns.
The content of DDT derivatives in human milk In Yugoslavia falls within
, the middle of the range reported for non-European and other European countries
(WHO 1979). Comparison of our results with those of three other laboratories
In Yugoslavia may be found In the Appendix Table A-5.
For the extraction of residues from human serum the method of Dale et a I.,
(1970) was used. To 1.0 ml serum, 1 ml formic acid was added. The mixture was
extracted four times with 3 ml hexane portions by shaking on a Vortex mixer for
1 mln, and cantrifuglng for 5 min to ensure separation of phases. The combined
hexane extracts were washed with 1 ml 5% I<2C03 and then purified on a florisil
column (1 cm diameter) containing 1 g florisil topped with 1 g
29
-------
TABLE 24. CONCENTRATIONS OF CHLORINATED HYDROCARBONS
Compound
P.P'-ODE
p.p'-ODD
p, pi -DDT
SERUM
Mothers (Nt = 35) Non-Preqnant (N = 24) Cord (N = 35)
N* Cone* (iig/l) n Cone (ug/l)
18 20 >
35 (8.9 - 46) 24 (3.7 - 75)
2 2.9 3 8.3
(2.8 - 3.2) (6.7 - 12)
7 5.5 5 14
(3.8 - 8.5) (4.7 - 27)
n ' cone (M/|) n
6.8
35 (3.6 - 17) 34
Not 5
Detected
2 4.8 5
(4.0 - 5.8)
MILK*
N = 34
Cone (wg/1)
31
(9.0 - 97)
9.7
(7.0 - 11)
11
(4.4 - 20)
£
Samples of human milk taken 3-5 days past parturn, extracted by method A.
td = number of samples analyzed.
*m = number of samples containing the particular compound,
^Concentrations are expressed as geometric means,
-------
anhydrous*. The column was prewashed with 20 ml hexane. The organochlorlne
compounds retained on the column were then eluted with hexane until the
collected eluates had a total volumn of 25 ml. The eluates were evaporated to
dryness In a stream-of nitrogen, the compounds redlssolved In 1.0 ml hexane and
analyzed by GC.
SUMMARY
The concentrations of DOT and hexachlorocyclohexane residues In blood in
the general population of Yugoslavia fall within the range reported for other
countries. Workers engaged In the production, formulation and packing of
pesticides have a higher Incidence, and higher levels, of residues than the
general population.
Serum samples from non-pregnant'women have the same DDT content as serum
samples of mothers' at delivery.
Cord blood serum contains lower coneeo.tratIons of p,pi-ODE than the
mothers1 serum; the mean ratio for the Individual mother/child pair was 2.7.
Concentrations in mothers' milk were 2.1 times higher than In the mothers'
serum.
31
-------
SECTION 5
ASSESSMENT OF BIOCHEMICAL AND CLINICAL EFFECTS OF PESTICIDES IN HUMANS
Since the use of pesticides Is unavoidable, It Is necessary to protect
workers exposed to them from their adverse effects. The aim of this work was
to relate clinical symptoms In workers to the degree of exposure to a wide
variety of pesticides. We also measured. Vitamin A levels arid concentrations of
DDT In some workers and compared the results to the levels found In persons not
exposed to pesticides.
Some of the results of our studies have been published In two papers
(Wllhelm and Bradamante 1980, Wllhelm et a I.. 1979) and two communications
(Wllhelm 1979, Wllhelm et al. 1978).
ChE ACTIVITY
To evaluate the absorption of pesticides and prevent overexposure to antI-
cholInestrase pesticides, measurements of blood ChE activities of exposed '
workers were taken regularly.
During the period from 1970 to 1979 a. total of 567 Industrial workers who
worked at some time In any of the three different production lines (dust or
wettable powder, emulsion, and household sprays) In one plant were examined.
Only 170. of them were continuously employed In the production of pesticides for
a number of years (2-14). The other were seasonal workers employed only during
periods of Intense production.
The workers were exposed to different types of pesticides: OP compounds
(phosalone, dlmethoate, dlchlorvos, monocrotophos, naled, bromophos), carbamate
Insecticides (carbaryl, dloxacarb, carbofuran), some herbicides, fungicides and
other compounds. Plasma and erythroycte ChE activities were determined by a
spectrophotometrlc method (El I man et al. 1961). The measurements of blood ChE
activities were performed In the laboratory, while the sampling (by finger
prick) was done either In the laboratory or In the factory where the samples
were kept cool until transferred to the laboratory. The results of measure-
ments of ChE activity In the blood and their relation to symptoms and slans of
poisoning are given In Table 25. The observed complaints or symptoms were
weakness, fatigue,, headache, nausea, sweating and vomiting.
The production of OP insecticides Increased during the ten-year period of
observation. It was expected that with the increased exposure, the number of
blood samples with diminished enzyme activities would also Increase. However,
as Table 25 shows, the greatest number of blood samples with 'enzyme activity
32
-------
TABLE 25. RLOOD ChE ACTIVITY AND FVIDENrF OF POISONING
Year
Number of Individuals (Without Complaints/With Complaints)
1970
1971
1972
1973
1974.
1975
1976
1977
1978
1979
0-24$
Inhibition
46/1
59/1
99/0
47/4
56/0
83/1
89/3
167/3
155/3
183/4
25-49*
Inhibition
10/6
18/14
6/0
18/1
37/14
71/9
26/4
7/1
9/1
34/6
50-745? '
Inhibition
11/13
5/6
0/1
1/1
9/2
6/8
2/2
0/5
1/12
6/16
75-100*
Inhibition Total
1/5
0/3
0/0
0/0
0/1
1/1
0/0
0/3 ,
0/6
0/6
68/25
82/24
105/1
67/6
102/17
161/19
117/9
174/12
165/22
223/32
*
^
Comolatnts
87
23
.9
8
14
11
7
6
12
13
less than 505? of normal were recorded. In the first two years', as were the
greatest proportion of accompanying symptoms or signs of poisoning.
In the first two years, the workers were exposed to and handled mainly
dimethoate and chlorfenvlnphos, insecticides which are extremely toxic to
mammals. The technological procedures and working condition provided
Inadequate protection, and, In addition, there was a great deal of overtime
work during the peak of the season. After the cases of poisoning were .
analyzed,, working conditions were considerably. Improved- As a result, although
the extent of work remained the same, there were no cases of occupational
poisoning in 1972, except for one accidental intoxication at work. In 1973 the
situation did not change. Only two exposed workers had blood cholInesterase
activities below 505?, that is below the limit for safe occupational exposure.
In the next years there was an Increase In the production of insectlvtdes.
The number of seasonal workers with no previous experience with insecticides
also increased. From the results presented In Table 25 the number of .reduced
enzyme activities again seems .to have become higher. If, however, the
accompanying symptoms observed in some workers are expressed in percent .they
varied between 7 to 14?.
Vitamin A Level
There are two references in the Literature which deal with the effect of
pesticides on the level of vitamin A in humans. Ember and co-workers (1972)
report that the amount of vitamin A is lower In the blood of workers exposed to
OP compounds, and Kell and co-workers (1972) claim that the amount of vitamin A-
is higher in persons exposed to chlorinated hydrocarbons. Although the
difference between the control and exposed groups reported by these authors Is
small, we though It worthwhile to check the data, considering the importance of
the role of vitamin A in the human body.
33
-------
U4
TA8LE 26. V,,N A W SERUM OF WORKERS EXPOSEO TO PEST.C.OES
Description of
the group
Workers exposed to
different pesticides
except cnlor.hydr.
Workers exposed to
different pesticides
Including OP and
chlor. hydr.
Control
Non-exposed workers
Control
Non-exposed workers
Duration of
exposure
(Mean Range)
2 years
(1 - 6)
14 years
(4 - 23)
14 years
(4 - 23)
Date of
blood
sampling
May
1976
May
1976
June
1977
. L
April
1977
April
1977
June
1977
Vitamin A, IU/«I
Mean t S.O. (n)
(Range)
1.54 (17)
i 0.32
(0.75 - 2.0)
1.40 148)
* 0.48
(0.25 - 2.25)
1.78 (55)
t 0.50
(1.0 - 2.6)
1.79 (98)
t 0.52
(1.0- 3.4 )
1.76 (33)
t 0.41
(1.0-2.25)
1.41 (33)
* 0.31
(1.6-2.0)
Mean Age
(Range)
25 yrs
(20 - 29)
44 yrs
(24 - 63)
38 yrs
(1956)
38 yrs
37 : yrs
(20 - 60)
37 'vrs.'-*
* * i * ** .. .'
(20 - 60);
-'. ' ' '. ;_-.
-------
In the course of 1976, the level of vitamin A was determined In .the serwrn
of 65 workers exposed to pesticides; In a number of these workers the vitamin A
level was again determined In 1977. In the control group, the vitamin A
content was also determined twice, In April and In June 1977. The control
group was selected from the same factory, but among workers who had no contact
with pesticides. All the examines were of the same social status and belonged
to the suburban population.
Vitamin A was determined In the serum by the spectrophotometric method
with antimonium trichloride (Can- and Price 1926, Klmble 1939). Ry this method
its total amount Is determined regardless of whether vitamin A Is an ester or
an alcohol. In some samples vitamin A was measured immediately after blood
sampling (after 3 to 4 hours), while the remaining samples were frozen and
stored at -18°C until analysis (a month at the most). From the samples in
which vitamin A was determined Immediately after sampling, a part of the serum
was separated and frozen. When the analysis was repeated (within a month after
freezing) there was no difference In the results. During each analysis a
calibration curve was made on the same day with retlnll acetate as standard
solution (U.S.P. - standard; INC-RochvI I le,J*J.D. USA).
The results of measurements of vitamin A are given In Table 26. They show
no effect of pesticides on the level of vitamin A." All values of the conrrol
and the exposed groups are within the same range; the standard levels found In
the literature (see Appendix Table A-6) are within this range too.
Besides vitamin A, the total DDT In the: serum and ChE activities In blood
and plasma were also measured In exposed workers In.order to verify that
pesticides were absorbed. All workers were exposed to pesticides for a long
time, but exposure was not continuous and each of them was without exposure for
a certain period of time during the year. ChE activity was therefore monitored
before the beginning of work with antlcholInesterase pesticides and In the
course of the work. In Table 27 ChE activities are expressed as percentage
values measured before contact with pesticides or after a longer break In the
work with pesticides (1-2 months). At the time of sampling ChE activities were
not reduced more than 2Q% In any of the groups. The mean activities were
within the standard deviation of preexposure values. However, several times In
the course of work with pesticides the same workers showed considerably reduced
ChE activities, proving that they absorbed a certain amount of the
compounds they were In contact with. The total DDT content In the exposed
groups Is given In Table 27. Workers not exposed to chlorinated hydrocarbons
(A in Table 27) have a lower level of DDT than those exposed (R). The
amount of total DDT in those not exposed to chlorinated hydrocarbons Is
comparable to the amounts of DOT found In the general population (Krauthacker
et al. 1980b).
-------
TABLE 27. ChE ACTIVITY AND DOT IN SERUM OF WORKERS EXPOSED TO PESTICIDES
A*
Mean ± SO (n)
Rt
May 1976
June 1977
Mean ± SO (n)
Total DDT
(ppb)
37 ± 17 (9)
108 ± 78 (41)
ChE activity (*)
- blood
" plasma
92 ± 12 (17)
92 ± 17 (17)
88 ± 12 (48)
91 ± 12 (48)
84 ± 23 (48)
84 ± 24 (48)
workers exposed to pesticides but not to chlorinated hydrocarbons
'''workers exposed to chlorinated hydrocarbons
SUMMARY
The surveillance of workers exposed during the formulation of anticho-
Mnesterase insecticides has shown that the use of prescribed protective
devices at work and the observation of hygienic and sanitary regulations
provides a satisfactory degree of protection and significantly reduces the
absorption. of antlchol Inesterase Insecticides.
Our study has confirmed that weekly measurement of ChE activity during the
period of Intense production Is a practicable method of determining the degree
of absorption. With this method, one can learn In time whether there are
workers at risk, and prevent the danger of further absorption^
Without exception, workers showing choltnerglc symptoms had their blood
ChEs Inhibited at least 50?. However, complaints from workers were
not always related to ChE Inhibition.
Absorption of pesticides does not seem to have an effect on the serum
level of vitamin A.
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SECTION 6
EFFECT OF PESTICIDES ON THE EYE AND ON VISION
The work described In this section Is a continuation of the study of the
alleged effect of pesticides on the eye and on vision which was described In
our previous EPA report (Reiner 1977b). The literature concerning this topic
has recently been extensively reviewed (Plestlna and Ptukovlc-Plestina 1976,
1978).
SUBJECTS AND METHODS
Fifty-seven workers who never suffered from an eye disease were selected
for this Investigation from those on whom the effects of anticholInesterase
pesticides on blood ChE were monitored (see Section 5 for details of
their exposure). Group A (47) engaged In the production of pesticides and
group B (10) engaged In their application. Age, sex and years of exposure of
the 57 are presented in Table 28.
TABLE 28. EXPOSURE TO PESTICIDES FOR TWO GROUPS TESTED FOR EYE DEFECTS
Aqe (years) Exposure (years)
Group N Description of work X ± S.D. Range X ± S.D. Range
A
Men 25
Women
B
Men TO
Production and formula- 37.2*10.9 19-57
tion of pesticides,
most ly anticholin-
esterases
35.6±12.0 21-55
Application of pesticl- 34.8±10.2 21-52
des in agriculture and
for public health pur-
poses
1t.3±6.7' 1-24
11.4+6.6 1-23
6.4±6.8 2-20
Only permanent workers, preferably with lonq exposure to pesticides, were
selected. Among them, 18 were engaged In work with pesticides between one and
' '' '": :' 3?., .-.-': .
-------
fl-ve years, the remaining 39 for over five years. Only 11 workers were older
than 45. . ....
The pphthalmologlcal examinations were performed at the same University
Hospital, by the same team of ophthalmologists, and with the same equipment as
In previous years, so that the diagnostic criteria were Identical throughout
the study.
Besides a detailed history of past and present Illnesses, possibly
connected with the workers' eyes and sight, each worker underwent detailed
opthalmologfcal examination which Included tonometry, ophthalmoscopy,
slit-lamp blomlcroscopy, keratometry, and visual acuity tests. In most of the
workers, peripheral vision was measured on a Goldmann perimeter; In some
workers, dark adaptation ability was assessed on a Goldmann-Weekers
adaptometer. Whenever an abnormality was found, the worker was examined
further as a patient, and all the tests were used to clarify the diagnosis and
the etiology of the disease. These patients were then treated with the standard
therapeutic procedures.
The details of all ophthaImological examinations were described In the
previous report (Reiner 1977b).
RESULTS AND DISCUSSION
Out of the 47 workers from group A, 23 complained of some eye troubles
( lacrlmatlon, 13; photophobia, 8; Itching and burning of the eyes', 3; other
complaints, 2). Among the 10 workers from group B, only three complained of
eye problems (photophobia or lacrlmatlon).
The most frequent abnormal Ity of the frontal eye segment was dilated
and/or tortous eplscleral blood vessels. The other eye problems found as a
result of external eye examination are summarized In Table 29.
TABLE 29. EXTERNAL EYE EXAMINATION IN WORKERS EXPOSED TO PESTICIDES
Group Dilated eplscleral Conlunctlval Abnormal Lens
blood vessels Injection pupils opacities
A 23 3 3.3
(N=47)
B
(N=10)
38
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Keratometrlc measurements revealed no great abnormalities In most of the
workers. The results of these measurements are presented In Table 30. In four
TABLE 30. KERATOMETR1C MEASUREMENTS OF WORKERS EXPOSED TO PESTICIDES
Number of eves with difference In meridians of
Group
A
Right eye
Left eye
B
Right eye
Left eye
Mean±S.D. <0.5 0.6-1.0 1.1-1.5 1.6-2.0 >2.0
1.0110.96
27 38 18 47
1.07i1.09
1.08±0.57
5 ' 3 5 3 2
1.02±0.60
Expressed as a difference between Horizontal and vertical meridian in diopters.
workers from group A,, and In one worker from group B, a pronounced astigmatism
(I.e. the difference between the two vertically placed meridians of over 2
diopters) was found.
In all but one worker from group. B, visual acuity, after correction with
lenses, was 1.0 or over. Accomodatlon ability and focussing power, tested with
Jager's reading charts, were found normal or appropriate to the age of the
worker.
Intraocular pressure was found to be considerably above physiological
standards In six workers. However, after thorough examination (repetitive
applanatlon tonometry, gonloscopy, water load tests) an open angle glaucoma was
diagnosed In one of the workers. The cause of the increased Intraocular
pressure In other five workers remained unexplained. The results of
Intraocular pressure measured by the Schiotz's tonometry or applanation
tonometry, are presented In Table 31.
Eyeground abnormalities were found In the patient with glaucoma, as
expected, and in several other workers. These abnormalities were mostly mild,
degenerative changes of the retina or slight abnormalities of retinal blood
vessels, which are as prevalent In the general population as among these
workers.
Peripheral visual fields were determined In most of the workers from group
A and In four subjects from group B. Peripheral visual fields were found
normal In 28 workers from group A and In two workers from group B.
Slightly constricted visual fields, mostly for the first and the second
Isopters, were found In 11 subjects from group A and In two from group B. In
five workers from group A, visual-fields-were considerably constricted in all
the four Isopters.
",*.. - ..-. 39"M;.': "-' - ' '-..
-------
TABLE 31. INTRAOCULAR PRESSURE* IN WORKERS EPXOSED TO PESTICIDES
Right eye Left eye
Group N Mean S.D. Mean S.D.
A 44 17.0 2.89 16.4 2.83
B 10 13.9 2.47 13.4 2.95
Measured by Schlotz's tonometry and/or applanatlon tonometry (In mm Hg).
No feasible explanation for constricted visual fields was found (except
for the patient suffering from glaucoma). In the patient with glaucoma, the
shape of the constriction of the visual fields was typical for glaucoma. In
the others, the visual fields were constricted concentrically for all the four
Isopters, but mostly for the first and the second.
Dark adaptation ability was measured fn 16 subjects from group A. In two
workers, adaptation ability was remarkably slower. In the others, no
significant differences from standard values was noticed.
SUMMARY AND CONCLUSIONS
Since no group of unexposed workers served as a control In this study, the
results of these examinations must be compared with those obtained on control
subjects In the previous study. In addition, the evaluation of the results and
comparison of the data obtained should be based on the normal physiological
values for our population as Judged by the experienced optha Imolegists.
Considering all the results, from the previous and the present Investiga-
tions, we did not find that exposure to pesticides had the effect on refraction
reported by many Japanese workers In the past. Recently, however there have
been several reports from Japan denying any connection between the high
Incidence of myopia and exposure to pesticides (Sato 1977).
The very high prevalence of dilated and/or torfous eplscleral blood
vessels Is very Intriguing since It Is considerably higher than normally found
In general populations of the same age or among workers not exposed to
pesticides (PIukovio-PlestIna, unpublished results).
The most consistent abnormality found among the workers exposed to
pesticides was constriction of the visual fields of unknown etiology. Any
assumption regarding Its pathophyslologlcal basis would be speculative.
40
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REFERENCES
Aldrldge, W.N., and E. Reiner. 1972. Enzyme Inhibitors as substrates.
In: Frontiers of Biology, A. Neuberger and E.L. Tatum, eds.,. Vol. 26;
North Holland, Amsterdam, pp.
Blair, D. and H.R. Roderick. 1976. An Improved method for the determination
of urinary dimethyl phosphate. J. Agrlc. Food Chem. 24:1221-1222.
Bradway, O.E. and T.M. ShafIk. 1977. . Malathlon exposure studies. Determina-
tion of mono- and dlcarboxyllc acids and alkyl phosphates in urine.
J. Agric.-Food Chem. 25:1342-1344.
Carr, F. H. and E.A. Price. 1926., Colour-reactions attributed to vitamin A.
Btochem. J. 20:490.
Curley, A. and R. Kimbrough. 1969. Chlorinated hydrocarbon Insecticides In
plasma and milk of pregnant and lactating women. Arch. Environ. Health
18:156-164.
Dale, W.F., J.W. Miles and T.B. Gaines. 1970. Quantitative method for
determination of DOT and DDT metabolites In blood serum. J. Assoc. Off.
Anal. Chem. 53:1287-1292.
Oaughton, C.G., D.G. Crosby,. R.L. Garnas, and D.P.H. Hsieh. 1976. Analysis
of phosphorus-containing hydrolytjc products of organophosphorus
Insecticides in water. J. Agric. Food Chem. 24:236-241.
Drevenkar, V., K. Fink, V. Kovac, R. Radic and Z. Vast lie. 1976a. The fate
of pesticides In aquatic environment. III. Thin layer chromatograph.Ic
method for the determination of abate. Arh. hlg. rada 28:163-169.
Drevenkar, V.,-K. Fink, V. Kovac, B. Radic and Z. Vasfllc. 1977.. Thin-layer
chromatographtc method for the determination of abate. Abstracts No. 22,
Scientific Session on Environmental Analysis, Szombathely, Hungary.
Drevenkar, V., K. Fink, M. Stipcevlc, and B. Tkalcevlc. 1976b. Some factors
affecting the hydrolysis rate of d.ichlorvos In river waters. Abstracts
No. 17, Scientific Session on Environmental Analysis, Szombathely, Hungary.
Drevenkar, V., K. Fink, M. Stipcevlc and B. Tkalcevic. T976c. The fate of
pesticides in aquatic environment. II. Hydrolysis of dlchlorvos In a
model .-system and In river water. Arh. hig. rada 27:297-305.
41
-------
Drevenkar, V.. Z. Frobe, J. Meczner and R. Tkarlcevlc. 1979a. netermlna-
tlon of The extent of exposure to various organophosphorus pesticides,
Abstracts, 6th Meettng of Chemists of Croatia, Zagreb, Yugoslavia,
p. 209. ' :
Drevenkar, V. Z. Frobe, Z. Vaslllc, B. Tkalcevlc and Z. Stefanac. T979b.
The rate of urinary excretion of phosalone residues In occupationally
exposed persons. Scl. Total Environ. 13:235-243.
Drevenkar, V., M. Stlpcevlc, B. Stengl and Z. Stefanac. 1979c. Gas
chromatographlc determination of alkali metal 0-0-dtethyl phosphoro-
dlthloate present In trace amounts. Mlkrochlm. Acta (VIENNA) I
385-394.
Drevenkar, V., M. Stlpcevlc, B. Stengl, Z. Vaslllc and Z. Stefanac. 1978.
Determination of dlthlophosphorlc acid ester residues In urine samples.
Abstract No. VI-811, 4th International Congress of Pesticide Chemistry,
Zurich, Switzerland.
Drujan, B. D., R. CastI Ion and E. Guerrero. 1968. Application
of ftuorometry In the determination of vitamin A. Anal. Blochem.
23:44-52.
Ellman, G. L., K. D. Courtney, V. Andres-and R. M. Featherstone. 1961. A
new rapid colortmetrlc determination of acetylcholInesterase activity.
Blochem. Pharmacol. 7:88-95.
Ember, M., L. Mlndszenty,. B. Rengel., L. Csaszar and M. Czlna. 1972. Secon-
dary vitamin A deficiency In organophosphate formulators and spray
workers. Res. Commun. Chem. Pathol. Pharmacol. 3:145-154.
Frobe, Z. 1977. Preparation of sllyl derivatives for the gas chromatographlc
analysis of organophosphorus pesticide residues. B. Sc. Thesis,
University of Zagreb, Yugoslavia.
Frobe, Z. and Stlpcevlc, M. 1977. Preparation of sllyl derivatives for the
gas chromatographlc analysis of organophosphorus pesticide residues.
Abstracts of papers, VI Yugoslav Meeting of Students of Pure and Applied
Chemistry with International Participation, Novl Sad, Yugoslavia.
Holmstedt, B., I. Nordgren, M, Sandoz and A. Sundwall.. 1978. Metrifonate,
Arch. Toxlcol. 41:3-29.
Kell, J. and H. S. Sandlfer. 1972. Serum vitamin A elevation In DDT exposed
volunteers. Bull. Environ. Contam. Toxlcol. 3:317-320.
Kimble, M. S. 1939. The photocolorlmeter determination of vitamin A and
Carotene In human plasma. J. Lab. Clln. 24:1055-1058.
Kirk, E. and M. Chleffl. 1948. Vitamin studies In middle-aged and old
Individuals. J. Nutr. 36:315-322.
42
-------
Krauthacker, B. 1977. Determination of chlorinated hydrocarbons In human
serum. Book of Abstracts, Meeting of Chemists of Croatia, Zagreb,
Krauthacker, 8., T. AlebJc-Kolbah, A. Buntlc, R. Tkalcevlc and E. Reiner.
1980a. DDT residues In samples of human milk, and In mothers' and cord
blood serum. In a continental town In Croatia, Yugoslavia. Int. Arch.
Occup. Environ. Hlth. (In press).
Krauthacker, 8., T. Aleblc-Kolbah, M. Kralj, R. Tkalcevlc and E. Reiner.
1980b. Organochlortne pesticides In blood serum of the general Yugoslav
population and In occupationally exposed workers. Int. Arch. Occup.
Environ. Hlth. 45:217-220.
Krauthacker, B., A. Buntlc, M. Kralj, R. Tkalcevtc and E. Reiner. 1978.
Monitoring of pp -DDT, pp -ODE and pp -ODD In human milk,, and In maternal
and placenta! blood, Abstract No. Vl-804. 4th International Congress
of Pesticides Chemistry, Zurich, Switzerland.
Krupka, R. M. 1966. Hydrolysis of neutral substrates by acetylcholInester-
ase. Biochemistry 5:1983-1988.
Lores, E. M. and D. E. Bradway. 1977. Extraction and recovery of
organophosphorus metabolites from urine using an anlon exchange resin.
J. Agrlc. Food Chem. 25:75-79..
Mandel, H. G. 1971. In: The Pharmacologjcal Basis of Therapeutics,
L. S. Goodman and A. Gllman, eds. MacmlI Ian, Co., London pp. 1672-1679.
Meezner, J. 1978. Simultaneous gas chromatographlc determination of alkali
metal salts of O-O-dlethyl-, 0,0-dtmethyldithlo-, and 0,0-dfethyldIthlo-
phosphorlc acid. B.Sc. Thesis, University of Zagreb, Yugoslavia.
Neeld, B. J. and.W. N. Pearson. 1963. Macro^ and mlcromethods for. the
determination of vitamin A using trIfIuoroacetIc acid. J. Nutr.
79:454-458.
Nordgren, I., M. Bergstrom, B. Holmstedt and M. Sandoz. 1978.
Transformation and Action of Metrifonate, Arch. Toxlcol. 41:31-41.
Plestina, R. 1976a. Toxlcological problems In war-time (In Croatian), In
"Zdravstvo u opcenarodnoj obranl", Zagreb,. Yugoslavia, pp. 219-227.
Plestina, R. 1976b. Toxic effects of metrlfonate in mammals. Veniam Legend!
Thesis, Medical Faculty, University of Zagreb, Yugoslavia,
Plesttna, R. 1978a. Toxlcity of pyrethrolds. Working paper No. PDS/EC
25/78.31. Meeting of the Expert Committee on the Safe Use of Pesticides,
Geneva, Switzerland.
Plestina, R. 1978b. Effects of OMS-43 on Spraying Operators..Our Ing Stage
VI AM Trial. Working paper No. PDS/Ec 25/78.20. Meeting of" the Expert
Committee on the Safe Use of Pesticides, Geneva, Switzerland.
43 ., - ' . '
-------
Plestlna, R. 1978c. Field Evaluation of the Spectrpphotometrlc Kit for
Measuring ChoiInesterase Activity. Working paper-No. PDS/EC 25/78.26
Meeting of the Expert Committee on the Safe Use of Pesticides,
Geneva, Switzerland.
Plestlna, R. 1978d. Temephos. Metrlfonate Combined Exposure. Working paper
No. POS/EC 25/78.32. Meeting of the Expert Committee on the Safe Use of
Pesticides, Geneva, Switzerland.
Plestlna, R. 1978e. Use of pyrethrolds In dlslnsectlon In the navy (In
Croatian). Book of Abstracts, 2nd Yugoslav Congress on Naval Medicine,
Hvar, Yugoslavia, p. 24.
Plestlna, R. 1978f. lexicological properties of pyrethrolds (In Croatian)
Abstracts, 6th Symposium on Disinfection and DIstnfestatlon of Insects
and Rats. Benja Luke, Yugoslavia, p. 11a.
Plestlna, R. I978g. Pesticides. .(In-Croatian), In: Occupational Health,
D. Stankovlc, ed., Yugoslav Association of Occupational Health and Pub-
lishing House, Zenlca, Sarajevo, Yugoslavia, pp. 319-331.
Plestlna, R. 1979a. Experimental model for the estimation of, tolerance of
some Insecticides at slow Intake In the organism (In Croatian). 7th
Congress on Disinfection, Dlslnfestatlon of Insects and Rats. Ohrld,
Yugoslavia. In: Makedonskl veterlnaren pregled. Skopje, Yugoslavia.
pp...206-213.. . .... ' ...-. . . ,. ....... . . ,:.. ,.. ,.. .. ... ,
Plestlna, R. 1979b. Modern views on toxicology problems relevant to the safe
use of pesticides (In Croatian) Slgurnost,, 21:45-50;
Plestlna, R. ed. 1980. Chemical Hazards. Proceedings of the XIX Interna-
tional Congress on Occupational Health, Institute for Medical Research
and Occupational Health, Zagreb, Yugoslavia.
Plestlna, R. and M. Plukovlc-Plestlna. 1976. Effect of pesticides on the eye
and on vision (In Croatian). Arh. hlg. rada tokslkol. 27:321-33.
Plestlna, R. and M. Plukovlc-Plestlna. 1978. Effect of antIchoIInesterase
pesticides on the eye and on vision, C.R.C. Crlt. Rev. Toxtcol. 6:1-23.
Radio, Z. 1979. Mechanism of Inhibition of acetylcholInestarase by some
oxlmes. B.Sc. Thesis, University of Zagreb, Yugoslavia.
Reiner, E. ed. 1975. ChoiInesterases and Chollnerglc Receptors.
Proceedings of the International Meeting Split, 1975. Croat. Chem. Acta
47:163-505.
Reiner, E. 1977a. Comparison between mammalian and parasitic choltnester- .
ases. Book of Abstracts, Yugoslav Symposium on Enzymes, Zagreb,
Yugoslavia, pp. 520-521.
44
-------
Reiner, E. ed. 1977b. Toxicology of antIcholInesterase pesticides.
EPA-600/ 1-77-031, U.S. Environmental Protection Agency, Washington, D.C.
Reiner, E. 1980. Kinetic properties of aryIesterases and chotInesterases In
parasitic helminths and mammals. In: Trends In Enzmology, Industrial
and Clinical Enzymology, Proceedings of the FERS Special Meeting on
Enzymes, Dubrovnlk 1979. LJ. Vltale and V. Simon, eds., Pergamon Press,
Oxford, Vol. 61pp. 325-330.
Reiner, E., 8. Krauthacker, M. Stlpcevlc and Z. Stefanac. 1977a.
Blood levels of chlorinated hydrocarbon residues In the population of a
continental town In Croatia (Yugoslavia). Pestle. Monft. J. 11:54-55.
Reiner, E., and R. Plestlna. 1979. Regeneration of cholInesterase
activities In humans and rats after Inhibition by
0,0-dImethyl-2,2-dichlorovtnyl CDDVP) phosphate. Toxlcol. Appl.
Pharmacol. 49:451-454. . -
Reiner, E., B. Pellcarlc, A. BuntIc and S. Krvavlca. 1979a. ChoiInesterases
In PARANPHISTOMUM MICROBOTHRIUM ASCARIS SUUM and NEOASCARIS VITULORUM;
Comp.. Biochem. Physio I'.,. 63C:73-74.
Reiner, E., 2. Radlc, A. Buntlc and V. Simeon. 19791. Mechanism of
Inhibition of acetyIcholInesterase by P2AM and toxogonln. Abstract No.
S7-80, FEBS Special Meeting on Enzymes, Dubrovnlk-Cavtat, Yugoslavia. ,
Reiner, E. and V. Simeon. 1977. Competition between substrates for
actyIcholInesterase and cholInesterase. Slochlm. Blophys. Acta 480:
137-142.
Reiner, E., V. Simon and M. SkrlnJarlc-Spoljar. 1980. Hydrolysis of
0,0-dImethyl-2,2-d1chlorovtnyl phosphate (DOVP) by esterases In parasitic
helminths, and Invertebrate plasma and erythrocytes. Comp. Biochem.
Physiol. (In press)
Reiner, E., M. Skrfnjarlc-Spoljar, M.. KralJ and S. Krvavlca. 1978. Kinetic
properties of the cholInesterase In MetastronqyI us aprl (Nematoda):
Substrate hydrolysis and reaction with organophsophorus compounds. Comp.
Biochem. Physiol. 60C:155-157.
Reiner, E., M. Skrlnjarlc-Spoljar and Dj. Mrvos. 1976. Decomposition of
0,0-dImethyl-2,2-dichtorovinyl phosphate (DDVP) by human and rabbit
plasma, and human erythrocytes.- Abstract No. 07-5-151, 10th Inter-
national Congress of Biochemistry, Hamburg, W. Germany.
Sato, C. 1977. Criticism on the recent theory of myopia. Part 5.
Principles of countermeasures against acquired myopia. Jpn. Rev. Clin.
Ophthalmol. 71:1028-1029. From: Pestle. Abstr. 10 (1977) no. 77-2326.
Sauberltch, E. H., P. R. Dowdy and J. H. Skala. 1973. Laboratory tests for
the assessment of nutritional status. CRC Crit. Rev. Clin, Lab. Scl.
pp. 215-226. -..-. .' ' V :' : ' ' : :''''"'
' ' ' 45 ' :
-------
f.
Shaflk, T., 0. E. Rradway, H. F. Enos and A. R. Yobs. 1973. Human'exposure
to organophosphorus pesticides. A modified procedure for the^gas-
llqutd chromatographIc analysts of alkyl phosphate metabolite^In urine.
J. Agrlc. Food Ghetn. 21:625-629.
Simeon, V. and A. Buntlc. 1978. Testing of some oxtmes In experimental
poisoning by highly toxic organophosphorus compounds. I. Reactivation
In vitro of dImethoxy-and ethoxymethylphosphorylated
acetylchollnesterase by 1,3-blspyrId In Iumd!methyl ether mono and
dloxtnes. Abstracts, 19th International Congress on Occupational Health,
Dubrovnlk, Yogoslavla. p. 194.
Simeon, V., Dj. Kobrehe! and E. Reiner. 1977. Effect of temperature on
Inhibition of acetylchollnesterase by dI-(2-chloroethyl)-3-chloro-4-
methyI-coumarIn-7-yI phosphate (Haloxon), Groat. Chem. Acfa-. 50:331-334
Simeon, V., M. SkrlnJarlc-Spoljar, A. Buntlc and E. Reiner. 1978. Hydrolysis
of DOVP (0,0~dlmethyl-2, 2-dlchlorovInyl phosphate) by human
erythrocytes. Abstract No. 0831, 12th Meeting of the Federation of
European Biochemical Societies, Dresden, E. Germany.
Simeon, V., K. Wl Ihelm,. A. Granov, S. Besarovlc-Lazarev, A. Bunttc, A.
Fajdetlc and Z. Blnenfeld. 1979.. 1,3 - BIspyrId In turn-dimethyl ether
mono-and dloxlmes: synthesis, reactivating potency and therapeutic
effect In experimental poisoning by organophosphorus compounds. Arch.
Toxlcol. 41:301^-306.
SIPRI. 1976. Medical Protection against Chemical Warfare Agents, Almqufst
and Wlksell International,. Stockholm^ Sweden.
SkrlnJarlc-Spoljar, M. and M. Kralj. 1979. Protection and reactivation
effect of some pyrldlnlum compounds In acetylchollnesterase Inhibition
by two organophosphates. Abstract No. S7-78, FEBS Special Meeting on
Enzymes, Dubrovnlk-Cavtat, Yogoslavla.
Skrlnjarto-Spoljar, M. and M. Kralj. 1980.. Reactivating and protective
effects of pyrId Inlum compounds In human erythrocyte acetylchollnesterase
Inhibition by organophosphates In vitro Arch. Toxlcol, 45:21-27.
Skrinjarlc-Spoljar, M., V. Simeon, M. Kralj and E. Reiner. 1978. Hydrolysis
of DOVP (0,0-dimethy!-2,2-dlchlorovlnyl phosphate) and paraoxon
(0,0-dIethyl 4-nttrophenyl phosphate) by vertebrate plasma and
erythrocytes.' Abstract No. 0830, 12th Meeting of the Federation of
European Biochemical Societies, Dresden, E. Germany.
Stefanac, Z., B. Stengl, and Z. Vast lie.. 1976. Quantitative determination
of organophosphorus pesticides by thin-layer densltometry. J. Chromatogr.
124:127-133.
Stlpcevic, M. 1978. Determination of organophosphorus pesticide residues by
gas chromatography, M.Sc. Thesis, University of Zagreb, Yugoslavia.
46
-------
Svetltclc, B. and K. Wllhelm. 1973. 'Methods of measuring exoosure to
anticholInesterase Insecticides. Arh. hlg. rada 24:357-365.
Thompson, J. F. ed. 1974. Manual of. analytical methods for the analysis
of pesticide residues In human and environmental samples. Research
Triangle Park, N.C. U.S. Environmental Protection Agency, Environ-
mental Toxicology Division 1974, Section 5, A, (2), (a) A (b).
Vasillc, Z. 1979. Organophosphorus pesticides In surface waters. M.Sc.
Thesis, University of Zagreb, Yugoslavia.
Vasillc, Z. V. Orevenkar, B. Stengl and B. Tkalcevlc. 1978. Determina-
tion of the total pollution of surface waters by esters of organopho-
sphoric acid and their residues. Abstracts No. 13, Scientific Session
on Environmental Analysis, Szombathely, Hungary.
Vasillc, Z., V. Drevenkar, B. Stengl and B. Tkal'cevlc. 1979. Water Pollu-
tion with regard to organophosphorus Pesticides. Abstracts No. 25,
Sixth Scientific Session on Environmental Analysis, Szombathely, Hungary.
VIdacek, S. and K. Mates Ic. 1978a. Psychometorics ,of workers exposed to
pesticides (In Croatian). In: Psychological aspects of work under
special conditions. V. Vlzek-Vldovlc. ed., Zagreb, Yugoslavia.
pp. 25-34
Vidacek, S., K. MatesIc and N. Marlon 1978b.. Psychomotorlcs of workers
chronically exposed to pest pesticides. Abstract 19th International
Congress on Occupational Health, Dubrovnlk, Yugoslavia.
Vltale, LJ. and V. Simeon, eds. 1979.. Industrial and Clinical Enzymology.
Proceedings of the FEBS Special Meeting on Enzymes, Dubrovnlk,
Yugoslavia. Trends In Enzymotogy, Vol. 61. XIII Pergamon Press, Oxford
1980. pp. 363. .
Yogel, A. I.- 1956. Practical Organic Chemistry. Longmans, Green and Co.,
London, New York, Toronto, pp. 971.
Vragovlo, V. 1976. Quantitative determination of some.larvlcides by thin
layer chromatography, B.Sc. Thesis, University of Zagreb, Yugoslavia.
WHO. 1979. DDT and Its derivatives Environmental Health Criteria, No. 9.
World Health Organization, Geeva, Switzerland. Table 15.
Wllhelm, K. 1979. The measurement of acetylene IInesterase activity in
haemolysed and nonhaemolysed human erythrocytes by the spectrophoto-
metric method. Abstract, SZ-79. FEBS Special.Meeting on Enzymes.
Dubrovnik-Cavtat, Yugoslavia.
Wllhelm, K. and V. Bradamante. 1980. Blood choIInesterase activity in
workers exposed to anticholInesterases: A ten-year follow-up. Arh. hlg.
rada tokslkol. 31 (In press)
47
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Wllhelm. K., A. Fajdettc, V. Oeljac and 7. fl1.nenfe.JdV 1979,. Protective,
effect of Dexetlmlde and HI-6 In poisoning with highly toxic organo-
phospHorus compounds. Arb. hlg^ rada tokslkol. 30:147rl5t.
Wllhelm, K., A. Granov, S. Besarovlc-Lazarev, Z. Blnenfeld and A. Fajdetlc.
1978. Jesting of some oxlmes In experimental poisoning by highly toxic
organophosphorus compounds: II Synthesis, acute toxic?ty and therapeutic
effect of 1,3-blspyrId In turn-dimethyl ether mono-end dloxtmes. Abstracts;
19th International Congress on Occupational Health, Dubrovnlk,
Yugoslav la» p. 200
Wright, A. S., D. H. Huston and M. F. Wooder. 1979. The chemical and
biochemical reactivity of dlchlorvos. Arch. Toxlcol. 42:1-»18.
48
-------
APPENDIX A
TABLE A-I. AMOUNTS OF DEDTPK IN URINE OF WORKERS EXPOSED TO OPs
Subject
I.P.
S.B. .
R.V.
S.D.
M.S.
S.V.
F.T..
J.S.
F.K.
I.D.
M.M.
S.S.
Amount found, ng/ml
1st sampling 2nd sampling
70 ' 254* .
80' ': 266* V \
153 268
178 272
156
157
268
210
113
148
.115
157 .
140 :..-
150
70
72
741 115*
906 . 147*
283
389
123 - . :;. --... ...--.. .. .
128
55 , .. , ... . . ; : .
46
Gas chromatographic analyses were performed on two columns of different
polarities (column la and II).
The urine samples of exposed persons were taken at the end of a working day
and independent analyses performed with two allquots.
49
-------
TABLE A-2. METABOLITES IN URINE OF WORKERS EXPOSED TO QUINALPHOS
DEP
nq of
metabolite/ml of urine
DETPK DEDTPK
WORKERS «
INITIALS A
May
A.M
B.O.
J.S.
K.J.
U.B.
Z.S.
J.A.
K.S.
K.F.
K.M.
P.S.
June
A.M..
B.O.
J.S.
K.J.
U.B.
Z.S.
J.A.
K.S.
K.F.
K «M«
P.S.
July
A.M.
B.O.
J.S.
K.J.
U.B.
Z.S.
J.A.
K.S.
K.F.
K.M.
P.S.
D =
NO =
NM =
1979
592
475
232
119
249
193
424
NO
228
680
731
1979
99
414
ND
NO
71
ND
708
ND
206
NM
NM
1979
1896
564
247
113
147
213
NM
357
477
1807
422
detected
not detected
not measured
B
308
275
86
ND
293
193
359
ND
134
319
245
96
214-
ND
ND
ND
67
704
ND
73
NM
NM
656
749
106
ND
325
ND
NM
168
238
1245
828
A
292
59
83
ND
94
82
276
44
NO
163
291
94
206
37
ND
74
45
261
36
81
NM
NM
349
208
83
68
120
100
NM
197
203
759
245
B
187
145 .
139
ND
153
112
266
57
65
181
152
73
58
ND
NO
ND
63
391
68
D
NM
NM
381
657
103
ND
352
128
NW
181
139
1752
616
A
285
69
NO
ND
54
83
137
NO
ED
201
- 94
49
82
NO
ND
ND
37
86
NO
42
NM
NM
61
NO
54
ND
NO
ND
NM
D
57
50
0
B
151
117
43
D
80
59
114
26
60
192
53
ND
D
ND
ND
ND
38
74
41
0
NM
NM
48
79
43
ND
ND
ND
NM
NO
D
78
86
ChE
activity (# of
preexposure value)
Blood
77
74
85
88
57
86
83
88
100
84-
73
68
93
80
46
34
NM
NM
79
80
68
86
NM
72
59
68
NM
Plasma
58
70
82
88
35
100
56.
77
82
58
70
NM
83
47
76
42
NM
NM
61
73
65
87
NM
76
48
45
NM
50
-------
TABLE A-3. SERUM CONTENT OF ORGANOCHLORINE COMPOUNDS IN YOGOSLAVIA
Gencrnl Population
Plocr
yoer
Continental north
1975
Conl (rental north
1976-1978
Continental central
1979
Continental south
1976
Island
1977
Nw
(«.n
147
(69,82)
11
(6,5)
41
(14,27)
19
119.-)
44
(29,19)
Mean age
Range
42
8-92
34
21-47
35
6-79
37
20-69
46
17-78
Concentration In uq/l (n)T
Mean
Range
Mean
Range
Mean
Range
Mean
Range
Mean
Range
o-HCH
1.8 (57)
0.1 13
n.d.
2.6 (4)
1.4-8.3
1.3 (8)
0.7-2.9
Llndan*
3.1 (23)
0.3-19
n.d.
3.8 (2)
2.1-6.7
n.d.
P.P'-OOE
30 (147)
8.4-1 18
27 (11)
3.1-70
6.9 (41)
1.2-31
29 (19)
9.2-118
18 (44)
4.8-83
p,p'-ODO
9.4 (7)
3.0-23
4.3 (9)
1.3-8.6
1.1 (34)
0.9-2.1
3.2 (10)
3.4-17
6.4 (20)
2.9-23
p.p't-GOT
19 (20)
2.2-81
4.0 (4)
0.4-10
2.2 (32)
1.1-5.3
6.1 (8)
3.7-17
7.2 (17)
3.1-39
Expo««-d Workers '
Factory ;
year '
Continental north
1976
Conllnontal south
1976
"N » number of sample*
tn » number of samples
*(«.(> male, female
50
(31.19)
28
(25.3)
analyzed
containing
41
20-62
33
21-32
Mean
Range
Mean
Rang*
9.2 (7)
3.6-29
38 (28)
9.2-137
' 10.4 (29)
3.6-30
26 (28)
4.3-98
33 (50)
7.1-213
23 (28)
4.1-93
It (39)
3.1-318
6.2 (B)
3.6-11
16 (32)
3.7-96
11 (7)
4.7-29
the particular compound
-------
TABLE A-4. CONCENTRATIONS OF P,P'-ODE IN HUMAN MILK AT LACTATION PERIODS
UP TO 55 WEEKS; '
Initials time After Delivery p.p'-DDE (uq/l)
M.B.
M.M.
M A»
P.Lj.
H.M.
S.M.
P.M.
8.V.
P.K
S.D.
S.Z.
B.Bo.
L.B.
N.K.
M.D.
C.V.
B.8I.
V.M.
M.O.
S.K.
H.LJ.
S.A.
P.S.
M «R»
J.D.
(weeks)
1.5
3
3
3
3
4
5
6
6
7
. 7 ' -
8
9
9
11
12
15
16
32
55 .; . -' .-. .
7
20
24
32
45
3
7
11
15
16
23
31
39
3
10
18
27
Extraction
A
86
9.4
ITS
102
58
84
51
75
69
34
135
19
56
109
120
87
67
53
89
73
80
51
63
20
9.8
167
74
44
77
12
43
45
49
56
39
16
37
method :
B
130
13
125
140
104
141
64
97
77
64
163
26
69
75
139
48
98
. 71
119
79
121
57
57
30
11
77
83
57
103
13
56
28
42
56
49
15
39
Mean age-of 25 mothers: 30 yrs (range 24-42). Mean concentrations (expressed
as geometric means of 37 samples: method A 52.6 ug/1 (range 9.4-167), and
method B 60.6 pg/l (range 11-163).
52
-------
TABLE A-5. CONCENTRATIONS OF P.P-DDE AND P,P«-DDT IN HUMAN MILK IN YUGOSLAVIA.
Ul
01
Republ Ic
$ 1 oven 1 a
Serbia
Croatia
Year of Sampling
1973
1973
1968
1971
1971
1974
1976
1976
1977
1979/79
Number of tothers
14
14
Not Stated
3
Is 2*
Si 5*
27
27
34
25
P,P'-OOE
3000*
60t
102
411
126: 559*
192: 142* t
1537"
3lt
31
53
Concentration
Range
1700-6950*
34-l39t
Not Stated
104-720
104-720
Not Stated
28-6900*
0.6-U8t
9-97
9.4-167
(liq/l)
P.PMJOT
850*
I7t
94
96
48: 120*
48: 36*
256*
5.lt
II
10
Range
280-2760*
5-55t
Not Stated
40-216
40-216
Not Stated
0-590*
0-12+
4.4-20 This Report
2.1-22 This Report
Concentration expressed on fat basis.
tRecalculated from (a) based on 2* fat In milk.
*The first number refers to mothers of a single child, and the second to mothers of twins.
-------
TABLE A-6. VITAMIN A LEVELS IN HUMAN SERUM
Authors
Kirk
(1948)
Nee Id et al.
(1963)
Or u Jan
(et al. (1968)
Mandel
(1971)
Ember
(1971)
Kell et al.
(1972)
Vttamtn-A
lU/ml serum
0.5-0.8
1 .2
1.5
1.3
1.2
0.8
1.4
2.3
Number of
Individuals
202
10
50
'
. 23
23
21
21
Population
Non-exposed
Non-exposed
Non-exposed students
Non-exposed
Workers 40 days after
exposure
Exposed to OP compounds
Non-exposed workers
Exposed to chlorinated
SauberlIch
et al. (1973)
1..5-2.5
hyrocarbons
Non-exposed
54
-------
APPENDIX B
SUMMARIES OF THESES
Z. Probe; Preparation of Sllyl Derivatives for the Gas Chromatoqraphlc
Analysis of Organophosphorus Pesticide Residues, B.Sc. Thesis, University
(1977).
OP pesticides decompose easily, producing salts of corresponding
organophosphorlc acids. While the original compounds can be directly
determined by GC analysis, their residues, organophosphates, have to be first
transformed to some more volatile form. The possibility of quantitative
determination of OP pesticides residues by GC of sllyl derivatives was
investigated.
The derivation of 0,0*-dlmethyI phosphate (DMP> and 0,0-dlethylphosphate
(DEP) was carried out In acetonltrlle with hexaraethyldlsl lazane as sllylatlng
reagent. The product obtained was dissolved In carbon tetrachlorlde and n-
hexadecane was added as Internal standard for GC. The sllyl
derivatives were Identified with MS Interfaced directly with the GC.
Using the ratio of Internal standard to sllyl derivative peak height
as quantitative parameter, linear dependence of detector response on the
concentration change of both DMP and DEP were obtained for the tested range 1-6
mg/ml.
The sensitivity of detection with flame lontzatlon detector was 70 vg/ml.
The developed procedure Is not sensitive enough for most residue analysis
problems In biological samples. However, It Is suitable for the monitoring of
polluted or surface waters with an accumulation procedure from bigger sample
volumes Included In the sample treatment.
J. Meczner; Simultaneous Gas Chromatographlc Determination of Alkali Metal
Salts of 0,0-Qlethyl-, 0,0-Dlmethyldlthlo-, and 0,0-Oiethyldlthlo-phosphorIc
Acid, B.Sc. Thesis, University of Zagreb (1978).
Monitoring procedures of persons occupational Iy exposed to OP pesticides
have been improved by a simultaneous GC determination of alkali metal salts of
0,0-diethyl-, 0,0-dimethyldithio-, and 0,0-diethyldIthlo-phosphoric acid.
In the following concentration ranges of tne originally present salts, a
linear relationship has been found; 63-1895 ng/mg urine (b-Q.0158, a=0.76)
for 0,0-diethyl-phosphoric acid K-salt, 100-1555 ng/ml urine (b=0.053, a=4.61)
for 0,0-dimethyldithlo^phosph. ac. K-salt. 47-1405 ng/ml urine (b=0,0738,
55'. . . ...-- ... .,.-- "
-------
a= 3.25) for 0,0-dlethyldlthlo-phosph. ac. K-salt. The Inorganic phosphates
present In urine samples Interfere with .the determination of the salts of 0,0-
dImethyl-phosphoric acid, because the methylatlon products of the acids
obtained by acidification of the salts are gas chromatographlcally determined
species.
R. Plestlna; Toxic effects of metrlfonate In mammals, Venlom legandl Thesis,
University of Zagreb (1976).
Metrlfonate, an antlparasltlc drug, Is an OP compound known also as an
Insecticide under the name dtpterex. In this work, the author presented the
results of his Investigations In humans and In experimental animals. The
effects of metrlfonate was studied In children treated against' S. haematoblum
Infection and In adults who had the same parasite but also had various types of
hemogloblnopathles. Besides very pronounced Inhibition of blood ChE no
.untoward effects of metrlfonate were observed In the treated patients.
The experiments on rats revealed that after an Intravenous Injection of
metrlfonate, chollnerglc symptoms appeared with some delay. This suggest that
metrlfonate Itself Is devoid of direct chollnerglc action and that a conversion
of metrlfonate to a biologically active compound takes place In the animals
organism. This observation was supported by the results of experiments with
Intestinal sacs In which It was shown that the Inhibitory power of metrlfonate
Increases with an Increase of pH of the media. In rats treated with
metrlfonate, the pattern of plasma and brain ChE Inhibition was similar to that
In rats treated with sublethal doses of dlchlorvos. In rats fed on a diet
containing 500 ppm of abate, toxlclty of metrlfonate remained unchanged,
-Indicating that the potent I at Ion of toxlclty for the two OP compounds did not
exist.
Z. Radlc: Mechanism of Inhibition of AcetyIcho11nesterase by Some Oxtmes, R.
Sc. Thesis, Un Ivert sty of Zagreb (1979).
The activity of purified bovine erythrocyte AChE (EC 3.1.1.7.) was
measured with acetylthtdcholIne as substrate (0,10, 0,40, 1,0, 5,0 and 10,0
mM). The maximum activity was at 1.0 mM acetyIthiochoI Ine. The evaluated
Michael Is constant (Km) and substrate Inhibition constant (Kss) were 0.036 mM
and 11.13 mM respectively, and the maximum rate of substrate
hydrolysis (V) was 1.45 jimol mln-1 mg~1.
The inhibition of AChE by 2-PAM (0.10/0.25, 1*0, and 2.0 mM) and
toxogonin (1.0, 2.0, 4.0, 8.0, and 12.0 mM) was studied with acetyIthIo-
choline as substrate. The concentrations of 2-PAM were 0.10, 0.25, 1.6 and
2.0 mM, and of toxogonin 1.0, 2.0, 4.0, 8.0 and 12.0 mM; the substrate
concentrations were as stated above.. The Inhibition of AChE by both
Inhibitors was slightly competitive with respect to acetyIth5ocholine. The
Hunter and Downs plots for the degree of Inhibition as a function of substrate
concentration were slightly curved. The hypothesis of Aldrlge and Reiner was
applied to interpret this result. According to this hypothesis, the enzyme has
two binding sites, one Is the active site and the other an allosterlc site.
56
-------
Both sites are assumed to blnd"*both the. substrate and the Inhibitor. Rl-ndlng
of the Inhibitor to either site causes enzyme Inhibition. The Inhibitor
constants were evaluated In the following way. Extrapolation of the Hunter and
Downs plots to zero substrate concentration gave the dissociation constants for
th-3 i?nryme-inhibitor comol^x at the active site (K3) t and th«se wers 0.178 mM
for 2-PAM and 4.39 mM for toxogonin. The dissociation constants for the
enzyme-Inhibitor complex at the allosterlc site (Kj) were calculated from the
theoretical equation of Aldrldge and Reiner for eleven pairs of substrate and
Inhibitor concentrations. The mean value for 2-PAM was 0.37 mM and for
toxogenln 7.9 mM. The relative standard deviation of each constant was about
5%. The narrow range within which the Kj values varied, and Is considered
evidence that the theoretical equation describes well the Inhibition of AChE by
the two oxImes.
Two techniques were used to measure the enzyme activities, the
conventional technique and. the stopped flow technique. For the latter :
technique, the enzyme concentration during assay was TOO times higher than In
the conventional technique. The activities of the uninhibited enzyme
(expressed per unit weight) were the same Irrespective of the technique used.
The degree of enzyme Inhibition however, was smaller when measured by the
stopped flow technique. This.was attributed to non-specific binding of the
Inhibitors onto the enzyme preparation. The amount of absorbed Inhibitor was
calculated to be 37? for 2-PAM and 45?; for toxogonin.
2-PAM and toxogonin catalyze the nonr-enzymIc hydrolysis of
acetylthlocholIne. These reactions are bimolecular second-order reactions, and
the fol lowing rate constants (k) were evaluated: 14.1 mln-1 mol~'dm3 for 2-PAM
and 3.9 mln~' mol'^dnP for toxogonin.
The spontaneous hydrolysis of acetylthlochol Ine was measured over a wide
range of time Intervals. The reaction was first order and the corresponding
rate constant was evaluated as 5,7 x 10-5 mln~'.
All experiments were done In 0.1 M phosphate buffer pH 7.4 at 25°C.
M. Stlpcevfc; Determination of Organophosphorus Pesticide Residues by Gas
Chromatography, M.Sc. Thesis, University of Zagreb (1978).
The quantity of residues excreted with human urine Is a useful parameter
that Indicates the extent of exposure to the parent OP pesticides. The
residues are salts of dimethyl or dlettiyl phosphate, phosphorothfoate or
phosphorodlthtoate. For GC analysis they have to be converted Into volatile
derivatives. The treatment with diazoalkanes and conversion Into trIa Iky I
phosphate is widely applied for this purpose.
When dlazomethane or dtazoethane are used for alkylatlon the interference
of Inorganic phosphate present In urine samples might be a serious problem. It
can be eliminated by the alkylatlon with dlazopentane, which yields derivatives
more easily resolved by GC.
57
-------
We applied.the latter procedure first for the monitoring of persons*
occupational Iy exposed to phosalone by determining Its residue* 0,0-dlethyl
phosphorodlfhloate salt. In addition to the difficulty caused by
Inaccessibility of the reagent for dlazopentane preparation, the Impurities
present In Its ethereal extract could not be removed efficiently enough and
they spoiled the gas chromatograms. For this reason we chose the a IkyI atIon
with distilled ethereal diazomethane. The presence of Inorganic phosphate did
not represent a problem In our case. . - '
In all urine samples of occupational Iy exposed persons, 0;0-d!ethyl-S-
methyl phosphorodlthloate was found, and the highest values were accompanied by
the lowest values of AChE activity In blood. Analyses of 22 urine samples of
unexposed persons did not show the present of 0,0-dtethyl-S-methyl
phosphorodIthloate.
Z. VasII let Organophosphorus Pesticides In Surface Waters,, M.Sc. Thesis,
University of Zagreb (1979). . .
The degradation rate of OP pesticides has to be considered for proper
evaluation of the results obtained by analytical methods for the determination
of these compounds in surface waters.
The experiments performed with model systems of surface waters with
various levels of pollution from various sources, have shown that the degrada-
tion rate depends not only on the chemical factors ,but also on the
bacteriological profile of the water supply*
The determination of the total amount of OP pesticides. Is proposed as an
adequate parameter for measurement of polllutlon of surface waters. This
approach requires the determination of diverse compounds of this class taken
together and of their degradation products.. The analytical method proposeed
for pollution control Includes 1) hydrolysis of all OP pesticides and of their
phosphorus-containing degradation products to phosphoric acid; 2) alkylatlon
with diazomethane and 3) GC determination of trlmethyl phosphate. The most
critical step Is the accumulation of the species to be analyzed along with a
complete elimination of Inorganic phosphates prior to hydrolysis. The chromato-
graphy on an Amberllte XAD-4 column Is shown to be promising for this purpose.
V. Vraqovlc; Quantitative Determination of Some Larvlcldes by Thin Layer
Chromatography, B. Sc. Thesis, University of Zagreb (1976).
The spot detection based on the AChE Inhibition with Indophenyl acetate as
substrate has been successfully applied for the determination of malathion and
parathion on thin-layer chromatography. A linear relationship of the spot
area, measured densltometrically, to the applied quantity of the pesticide was
been obtained in the range 0.9 - 3.5 ng for parathion and 0.2 - 1.0 uq for
malathion. Enzyme Inhibition could very well be used, for qualitative
detection of abate on a fluorescent layer even If only 3 ng was present. For
the quantitative determination of abate extracted with chloroform from surface
water samples, the measurement of the quenching of the fluorescence of the
58
-------
spots on a-fluorescent layer was shown to be*-satisfactory. In this way
the linearity range for 0.9 to 5 pg of abate was obtained.
59
------- |