Cholinesterase by the Numbers: AnUpdate on Washington State’sMonitoring Program

Allan Felsot

Washington State University

Department of Entomology

Food & Environmental Quality Lab

The Washington State Cholinesterase MedicalMonitoring Program started Feb. 1, 2004 & willcontinue through 2005

n Cholinesterase as a readily accessiblebiomarker of exposure

n How did we get “stuck” with theprogram

n How does the program work

n Results from the 2004 SAC Report

n Update on the 2005 program

Nervous SystemBasics

n Information carried by electricalsignals down nerves (axons)

n The information is transmittedfrom nerve to nerve and fromnerve to muscle

n At end of nerves, and wherenerves meet muscle, there arelittle gaps called synapses

Nucleus

Cell Body

Axon

DendriteSynapse

NerveSignal

Direction

Synapse

Synaptic Transmission

Neurotransmitters Carry the NerveSignal Across the Synapse

n The electrical nerve signal ischanged to a chemical signal tocross the gapn The nerve signal stimulates

the release of a chemical (a“neurotransmitter”) calledacetylcholine that is releasedinto the gap

n The chemical binds toreceptors on the adjacentnerve or muscles membranes& stimulates the electricalfiring of the adjacent nerve orstimulates muscle contraction

H3C C O CH2 CH2N CH3

CH3

CH3O Acetylcholine

Modulation of Nerve Signals

n Too much acetylcholinecauses nerves & muscles tofire uncontrollably

n The amount of acetylcholinein the synapse is regulatedby enzymatic breakdownn The enzyme is called

acetylcholinesterasen It is in the membrane of

the adjacent nerve ormuscle

Acetycholine Receptor

Acetylcholinesterase

Acetylcholine

Nerve/Muscle Membrane

Most insecticides adverselyaffect nervous system

3-D Structure ofAcetylcholinesterase

NeurotransmitterBinding Site

Organophosphates& Carbamatesblock binding site

n Only two classes of insecticides cause toxicityby binding to acetylcholinesterase andinhibiting its activityn Organophosphates & Carbamates

If Acetylcholinesterase is Partof the Nervous System WhyTest Blood?

n The acetylcholinesterase enzyme(AChE) is also present in the red bloodcells, circulating in the blood

Thus, overexposure to OP &carbamate insecticides

Can be determined by monitoring theactivity of AChE in the red blood cells

An alternative measure of OPexposure

n In addition to AChE in the red blood cells, bloodplasma (or sera) contains an enzyme calledbutyryl cholinesterase (ChE)

n Although a different enzyme with an unknownfunction, ChE is also inhibited by OP andcarbamate insecticidesn However, inhibition of ChE is only a measure of

exposure, not poisoning

Two for the price of one

n Thus, blood samplescan be separated intored blood cells andplasma (no cells)

n Activity of AChE andChE is then determinedin each fraction toestimate overexposureto OP & carbamateinsecticides

How Much Change in AChELevels Is Too Much?

n No exact depression (inhibition) level can bepredicted to cause poisoning

n However, studies suggest that individualsapproaching 40% or more depression of RBCAChE face a greater probability of havingsymptoms of poisoning

n So, a threshold of 30% inhibition is chosen inmonitoring programs to PREVENT furtherexposure from tipping you over the limitn For plasma ChE tests, the threshold is 40%

because it is less predictive of poisoning

Symptoms of PoisoningFollowing Overexposure

n Dizzinessn Blurred visionn Stomach achen Diarrhean Droolingn Unusual sweatingn Tightness in the chestn Muscle twitchingn Pinpoint pupilsn Difficulty breathing

The L&I Cholinesterase MonitoringRule--A Quick Historyn L&I was successfully sued by a two workers for

failing to implement a rule that would be protective ofthem in the “work place”n They requested L&I to implement a ChE

monitoring rule in 1986 and again in 1991n California was doing it, why shouldn’t WA?n L&I issued rulemaking recommending ChE

monitoring but not requiring it;n Workers sued

n Workers lost first suit, because court agreed withL&I in ‘93 that technology couldn’t be provenfeasible (reliable) enough to implement in WA

ChE Monitoring--Brief Historyn L&I convened a technical advisory group (TAG) in ‘95

that also recommended monitoring but not feasible torequire it at the time

n Second request for ChE monitoring rule denied byL&I in ‘97

n Workers sue again; case becomes class-actionn New suit eventually heard by the WA Supreme Courtn The “Supremes” considered advances in the

technology as reported by the TAG, and said L&Iacted “capriciously” and therefore violated the WAIndustrial Safety & Hygiene Act (WISHA) in notimplementing a ChE rule

The Supremes Ruling

n “..we conclude that the pesticide handlersmet their burden of showing that theDepartment’s 1997 denial of the pesticidehandler’s request for rulemaking wasunreasonable.”

n “We therefore order the Department to initiaterulemaking on a mandatory cholinesterasemonitoring program for agricultural pesticidehandlers”

The L&I Rule for CholinesteraseMonitoring (Effective February 2004)

n Maintain accurate records of employees handling OPand CB insecticides

n Implement a medical monitoring programn Identify a physician who will provide ChE testing and

make recommendationsn Make ChE testing availablen Respond to depressed ChEn Provide medical removal protection benefitsn Maintain recordsn Provide trainingn Implement plan

Who is covered by the rule?

n All employers that have an employeethat handles organophosphates and N-methyl-carbamate insecticides, whichhave the signal words “DANGER” or“WARNING” on the label.

A pesticide “handler” is definedas anyone that:

n Mixes/loads/applies pesticidesn Disposes of pesticides or their contaminated

containersn Handles open contaminated pesticide

containersn Maintains contaminated application

equipmentn Acts as a flagger

Covered Insecticides(examples)

n Guthion

n Lorsban

n Imidan

n Diazinon

n Cygon

n Sevin

n Carzol

n Lannate

n Temik

Organophosphates Carbamates

Making ChE Testing Available:Overviewn Required for an employee handling OP & CB

insecticides for 50 h or more hours (1st year of rule) inany 30 day consecutive period

n Dropped to 30 or more hours of use in2nd year (i.e., during 2005)

n Annually monitor plasma ChE & RBC AChE at least30 days prior to handling covered insecticides(“baseline” test)

n Periodic blood monitoring within 3 days of meeting thehours worked exposure threshold or at least every 30days while exposure is expected to exceed thethreshold

n Worker can voluntarily opt out of testing

Rule Requirements

n Identify a medical provider (your choice) forcholinesterase testing

n Identify handlers that are likely to meet orexceed the exposure thresholdn 30 hours in any 30 consecutive day period

n Train employees on the health hazards, thepurpose & requirements of the ChEmonitoring

n Have qualifying handlers go to your medicalprovider for an exam & determination if theywant to participate in the program

Rule Requirements

n Employees not wanting to participate mustsign a declination form, documenting thatthey do not want to participate

n For those that want to & qualify to participate,a baseline blood test must be taken—eachyear

n Baseline tests should be done at least 30days prior to exposure

Rule Requirements

n Periodic (follow-up) blood tests must be doneeither:

n Within 3 days of meeting the hours of workexposure threshold

Or

n At least every 30 days from the day of the firstapplication of covered pesticides

n ChE results from follow-up blood tests arecompared to baseline blood tests to determinelevel of enzyme depression

Rule Requirements

n Keep track of each pesticide handler’shours working with the coveredinsecticides

Exemptionsn Employees that handle “only” N-methyl-

carbamate pesticides are exempt from themedical monitoring requirement, however theemployer must still track their hours

n The time spent using closed systems formixing and loading (water soluble packets)are used to determine when baseline testingshould be done but do not count toward theneed for a follow-up blood test

Record-keeping Requirements

n Track & Document the hours of exposure forall employees

n Retain pesticide-handling records for sevenyears

n Post on Bulletin Board the name, address,and phone number of the medical provider

n Document the results of all required workplace evaluations

Record-keeping Requirements

n Report to L & I the name and hours ofexposure for each employee that has afollow-up blood test

n Keep signed declination forms &provide a copy to the employee

Follow-up Blood Test:Benchmarks for Required Actions

n If RBC AChE or Plasma ChE drops to 80% ofbaseline (i.e., a 20% inhibition), then L&Iinitiates a voluntary investigation of workplacepractices

n Red blood cell AChE depression of 30% orplasma ChE depression of 40% (relative tobaseline levels) requires work removal untilenzyme levels are within 20% of baselinen L&I requires a mandatory workplace

evaluation

Work Removal

n Must not handle covered pesticides until ChEenzyme levels are within 20% of the baselinevaluen Plasma ChE levels may return to normal within

hours to several days.n RBC AChE levels return at about 1% per day

n Employee can be reassigned other work but at samepay rate, same benefits and seniorityn If no work available, the employee must be paid

until the medical provider says the individual isable to return to work

Record-keeping Requirements

n Document names of anyone medicallyremoved under the program plus:n Date of work removal

n Date returned to work

n Maintain medical providerrecommendations for each employee

Scientific Advisory Committeen Issued final first year (crop yr. 2004) report during

March 2005n The first of two reports (second within a year after

2005 crop season)n Reviewed lab procedures (compliance with SOPs,

QC performance, problems)n Reviewed working of system (timeliness of testing

and reporting)n Reviewed handler test results and significancen Reviewed variability and issues of false

positives/negativesn Answered concerns/questions from Stakeholder’s

Advisory Committee; made recommendations

First Year Report (2004 Crop Year)

n 370 employers had at least oneemployee submit a baseline blood test

n 2758 blood samples were received & testedfor baseline cholinesterase levels (includes 52non-worker ‘controls’)

n 666 blood samples were received & tested forat least one follow-up test after the prescribednumber of hours worked (includes 52 non-worker ‘controls’)n = “periodic” tests

Non-Worker Quality Control RBC & Serum ChE Samples

Plot of paired replicate blood samples; pairedsamples collected at two different times

-20%

+20%

RBC 1 vs RBC 2

Serum 1 vs Serum 2

2 3 4 5 6

2

3

4

5

6

Serum ChE Levels: Periodic Tests Relative to Baseline Tests

Baseline Serum ChE Level

Periodic ChE Level

Population Mean Change: -7.96%

8

8

10

12

14

16

18

10 12 14 16 18Baseline RBC ChE Level

Periodic RBC ChE Level

RBC ChE Levels: Periodic Tests Relative to Baseline Tests

Population Mean Change: 2.14%

n ~21% of follow-up blood samples indicated atleast 20% depression (which only indicatesexposure)n Represents 130 workers

n 26 workers (~4%) had cholinesterasedepression requiring work removal(compared to ~612 workers with follow-ups)n Four of these were rescindedn 78% of depressions >20% due to plasma ChEn Over 60% of removals triggered by depressions of

plasma ChE

First Year Report (2004 Crop Year)

Number of Pesticide Handlers Qualifyingfor Alert or Workplace Removal

3.619.6% of Total PesticideHandlers in Program

22120Total **

16101Serum ChE

1029RBC ChE

Number Alertedfor WorkRemoval

Number >20%Depression from

BaselineTest

** 9 Handlers had both RBC & Serum ChE Depressions >20%; 5 of these handlers had RBC > 30% depressed

False Positive & False Negative Statistical Analyses

Given a 10% coefficient of variation for ChE test results and an action thresholdof 20% depression, what is the expected proportion of the population of workerswho may be concluded as false positives (depression above threshold whenthere is none) and as false negatives (no depression when there is some).

% Change in ChE from Baseline Relativeto Hours Worked

-7.40 (657)1.73 (664)All Handlers

-2.86 (15)4.81 (15)100+

-8.69 (148)3.08 (151)51 - 100

-10.63 (104)2.02 (104)31 - 50

-8.63 (49)1.65 (50)1 - 30

-5.90 ( 341)0.93 (344)None

reported

Serum ChE %Change from

Baseline (n workers)

RBC ChE % Changefrom Baseline

(n workers)

Hours ofWork

Interesting Associations (2004)

n Almost all workers showing depressions atany level were working in the pome fruitindustry

n Cholinesterase depressions seen in someworkers who had only been usingcarbamates (Sevin thinner)

n Chlorpyrifos (Lorsban) was the only OP withsignificant association with depressions

n By the end of May, very few workers hadblood tests showing threshold depressions

Number of Pesticide Handlers with RBC or SerumChE Levels Requiring Alert and Associated

Pesticide Use (n = 51 interviews)

2.010Diazinon

2.001Guthion

13.761Lorsban + Other

OP/Carzol

23.584Sevin + OP

27.5104Lorsban Alone

43.1175Sevin/Sevin+Carzol

% ofInterviews

Serum ChEAssociated

RBC ChEAssociated

Pesticide

Number of Handlers

2.001Guthion

2.010Lorsban + Carzol

19.646Sevin + OP

7.813Lorsban Alone

9.832Sevin/Sevin+Carzol

% ofInterviews

Serum ChEAssociated

RBC ChEAssociated

Pesticide

Number of Pesticide Handlers with RBC or SerumChE Levels Requiring Work Removal and

Associated Pesticide Use(n = 51 interviews)

Number of Handlers

0 10 50 10020 30 40 60 70 80 90

Days Until Testing

Num

ber

of S

ampl

es

100

200

300

400

500

600Frequency Distribution of Time from LabReceipt of Blood Sample Until Testing

Serum ChE Data

Average Delay: 27.7 days

Time Periods for Selected Steps inCholinesterase Monitoring System

34.5 / 3514 / 7WISHA Consultation

7.8 / 7.28 / 8L&I Informs WISHA Consultants

3.6 / 3.93 / 3L&I Informs Medical Provider

Time Period for Periodic Tests Requiring WorkplaceEvaluation/Removal

Time Period for Baseline Testing

3.62Lab Test to Mailing Result to Provider

1.11Time to Testing

1.11 - 1.5Blood Draw & Receipt by Lab

Time Period for Periodic Tests

24.61Time to Testing

1.11 - 1.5Blood Draw & Receipt by Lab

ActualTime (d)

ExpectedTime (d)

Activity Time Period

Labor & Industries Work PlaceEvaluation Findings

n Most employers were in compliance with theWorker Protection Standards

n Some PPE, sanitation, and posting problemswere noted

n In general, however, work place observationsrevealed no clear links to observed cases ofcholinesterase depressions requiring an L&Iconsultation or an employee work removal

Problems with PPE(Personal Protective Equipment)

n No respirator fit testn Failure to properly change respirator filters.n Failure to clean respirator &/or other PPE

after use.n Improper storage of respiratorn Applicator removing respirator, at some time

during application process.n Bulky gloves removed to unplug spray

nozzles & or repair sprayer, etc.

Decontamination Problems

n Baseball caps &/or bandana’s worn duringapplication, then worn home

n No eyewash or emergency shower facilitiesat mixing & loading station

n Failure to wash hands, face and neck afterremoving part or all of PPE (smoke &bathroom breaks, lunch, & end of application,etc.)

n One employee wore spray suit to lunch

2005 Preliminary Data (as ofApril 17, 2005)

n 2315 baseline blood samples received andanalyzed

n 341 periodic (follow-up) blood samplesreceived and analyzed

n 115 samples rejected (did not meet SOPguidelines)

n 12 workers >20% depression (alerted;workplace evaluation)

n 2 workers on work removaln This time last year, 42 worker alerts

For More Information

n http://feql.wsu.edun Food & Environmental Quality Lab

n http://wsprs.wsu.edu/n WA State Pest Management Resource Ctr.

n http://aenews.wsu.edun Agrichemical & Environmental News

n afelsot@tricity.wsu.edu