Neurotoxicology and Teratology, Vol. 18, No. 4. pp. 449-453,1996 Copyright b 1996 Elsevier Science Inc.

Printed in the USA. All rights reserved 0X92-0362/96 $15.00 + .Wl

ELSEVIER PII SOS92-0362(96)00028-l

Organophosphates: The Relationship Between Chronic and Acute Exposure Effects

RICHARD STEPHENS,* ANNE SPURGEON? AND HELEN BERRY*

“Health and Safety Laboratory, Broad Lane, Sheffield, S3 7HQ, UK llnstitute of Occupational Health, University of Birmingham, Edgbaston, Birmingham Bl.5 2TT, UK

STEPHENS, R., A. SPURGEON AND H. BERRY. Organophosphares: The relationship between chronic and acute expo- sure effects. NEUROTOXICOL TERATOL 18(4) 449453, 1996.-The relationship between chronic (nonreversing) neuro- psychological effects and acute exposure effects was investigated in 77 organophosphate-exposed male sheep-dippers. Acute exposure effects were assessed prospectively using a purpose-constructed symptoms questionnaire administered pre-, and 24 h postexposure. Urine was analysed for dialkylphosphate levels to confirm recent exposure. Chronic effects were assessed in a cross-sectional neuropsychological study in the absence of recent exposure using computerised neuropsychological tests, the General Health Questionnaire, and the Subjective Memory Questionnaire. Simple correlation and multiple linear regres- sion analyses (adjusting for confounders) were used to assess relationships between the change in total symptom reporting from baseline to 24 h after exposure and chronic effect outcomes. There was no evidence of any association between reported symptom levels and chronic neuropsychological effects. This suggests that chronic effects of OP exposure appear to occur in- dependently of symptoms that might immediately follow acute OP exposure. This has implications for exposure control: indi- viduals may experience chronic effects without the benefit of earlier warning signs of toxic effects during acute exposures.

Organophosphate Chronic effect Acute effect Neuropsychological tests Dialkylphosphate Sheep-dip

ORGANOPHOSPHATES (OPs), although widely used as agricultural pesticides, may cause both acute and chronic ad- verse health effects in humans following exposure. Acute ef- fects of OP exposure relate to the inhibition of cholinesterase during neurotransmission, leading to a build-up of acetylcho- line (4). More recently, chronic (i.e., apparently nonreversing) neuropsychological effects of OP exposure have been re- ported, both as sequelae of acute OP poisoning (8-10) and following periods of chronic low-level exposure (3,6,11). The toxicological mechanisms involved in the development of chronic effects of low-level OP exposure are presently unknown. This study investigated whether any relationship exists between the occurrence of acute and chronic OP exposure effects.

POPULATION

The study group consisted of 77 male UK sheep farmers with long-term exposure to OPs as a result of sheep-dipping. At the time of the study, licensed OP sheep-dips in the UK contained either diazinon, a mixture of diazinon and chlorfen- vinphos, or propetamphos as their active OP ingredients. Farmers were recruited by selecting every 10th name on regis-

tration lists obtained from the Wool Marketing Board. lnitial contact was by letter, which produced a low response (33%). A further sample was recruited by telephone with the same selection procedure. This produced a response rate of 69%. Demographic and exposure variables of the samples showed no significant differences, except for age (telephone sample 5 years younger, mean age 45 years). The 77 farmers included in these analyses are a subgroup of the main study group (which consisted of 146 sheep farmers) for which acute effect data was available; acute data were not collected from all partici- pants.

Quarry workers were considered to be a suitable control group (11). Workers in quarries situated in the same geo- graphical areas were approached and the response rate over- all for the controls was 35%. Participants were male, aged 16 65 years, native speakers of either English or Welsh, and if farmers, had more than 50 sheep. Potential participants re- porting any nervous system disease, a prior head injury result- ing in loss of consciousness for more than 1 h, a current or prior alcohol problem, recent exposure to other neurotoxic agents, or if controls, current or previous regular exposure to pesticides, were excluded.

Request for reprints should be addressed to Richard Stephens, Health and Safety Laboratory, Broad Lane, Sheffield. 53 7HQ, UK. Tel: 0114 289 2811; Fax: 0114 289 2.500; E-mail: rich@ewps-hsl.demon.co.uk

449

450 STEPHENS. SPURGEON AND BERRY

METHOD

Acute effects were assessed prospectively, with measures taken before (baseline) and 24 h after exposure. A purpose- constructed symptoms questionnaire was employed. Validity was ensured by compiling OP-related symptom items from a literature review, producing an index of reported acute, de- layed, and chronic effects of OP exposure (12). This index was compiled into a 73-item questionnaire. The time frame of symptom occurrence was set to “within the last 24 hours.” Re- sponses were “not at all,” “some of the time,” “most of the time.” and “all of the time” for each item, and these were re- spectively scored 0, 1, 2, and 3. The outcome was the change in the total reporting of symptoms from baseline to 24 h after dipping, the potential range of which was ? 219.

of mental ill-health, and the Subjective Memory Question- naire (2). Summary outcomes of the tests and questionnaires used in the current study are shown in Table 2.

The data relating to acute effects were collected before and after each participant’s first dip of 1993. The data relating to chronic effects were collected during the first half of lY94. Data were collected at the workplace, generally in the farm- house. Each acute data collection session lasted for approxi- mately 20 min; the chronic effect data collection sessions lasted up to 2 h.

Urine samples were taken the morning after dipping. The extent of recent exposure was evaluated by measuring the lev- els of the metabolites diethylphosphate (DEP) and diethylthi- ophosphate (DETP) in these samples. This technique allows the assessment of any recent exposures to diazinon and chlor- fenvinphos. although not exposures to propetamphos (7). Therefore, only a subgroup of dippers using these detectable OPs (n = 43) could be included when assessing recent exposure.

Analyses

Chronic effects were assessed in a cross-sectional neuro- psychological study, at least 2 months after any exposure, us- ing computerised neuropsychological tests and questionnaires (11). Neuropsychological tests were taken from the Neurobe- havioral Evaluation System (NES) (1) and the Automated Cognitive Testing system (ACT) (13,14), and were selected to assess performance over a range of cognitive processes re- portedly affected by OP exposure. Also included were the General Health Questionnaire (GHQ) (5) to assess symptoms

Comparisons were first made between this study’s sub- group (with acute and chronic data, 17 = 77). and the remain- der of the main study group (n = 69) across demographic, life- style, and exposure variables, using appropriate parametric and nonparametric tests. Hence, a r-test was used for compar- isons across age, a Mann-Whitney U-test was used for com- parisons across educational level, lifetime alcohol consump- tion, self-reported number of years of OP exposure, annual dipping frequency. and average dipping session duration, and a chi-square test was used across first language, computer fa- miliarity, time of day of testing, and smoking status. This was to ensure that the subgroup from the current study is repre- sentative of the main study group, and hence of the popula- tion from which the main study group was sampled. Further analyses were undertaken to investigate the association be- tween the change in total reported symptoms following acute

TABLE 1

CHARACTERISTICS OF SHEEP FARMERS IN THE CURRENT STlJDY (}I = 77). COMPARED WITH THE REST OF THE MAIN STlJDY GROIJP 01 =6’))

Characteristics C‘urrcnt Study Rest OI Main

Subgroup Group I’

Mean age (SD) in years

Mean educational level (SD)

Lifetime alcohol consumption

(10’units) (SD)

Smoking

Never

Previous Current

Computer familiarity None Some A lot

Time of day of tcstinp

Morning

Afternoon Evening

First language (% English)

History of self-reported OP exposure

(years) (SD)

Annual dipping frequency (SD) Average dipping session duration

(days) (SD)

47.1 (9.6)

3.9 (2.5)

13.3 (14.7)

42

I7

18

57

20

17 31

29

75%

Ii. I (7.6) 14.x (6.2)

2.X (1.9) 2.8 (2.4)

2.x (2.2)

463 (9.8) 0.679

4.3 (2.4) 0.324

11.4 (13.3) 0.729

3’1

17

13

41

2X

17 35

IS 79%

2.1 (2.0)

0.7X6

0.06 I

0. I34

MSR

0.975

0.079

0.006

CHRONIC AND ACUTE OP EXPOSURE EFFECTS 451

TABLE 2

CHRONIC EFFECT OUTCOME MEASURES AND MEAN SCORES FOR FARMERS AND CONTROLS, AND PEARSON CORRELATION COEFFICIENTS BETWEEN ACUTE SYMPTOMS (THE CHANGE IN TOTAL SYMPTOM REPORTING FROM

BASELINE TO 24 h AFTER DIPPING) AND CHRONIC EFFECT OUTCOMES WITHIN THE FARMERS

Chronic Effect Measures TEAI Summary Outcome

Farmers‘ Mean, n = 71 (Controls‘ mean, n = 130)

Farmers‘ SD (Controls’ SD)

Correlation Between Chronic and Acute

Outcomes for Farmers (n)

Tests from the NES

Simple Reaction Time

Mean RT, 80 trials (ms)

Symbol-Digit Substitution

Mean trial RT (5 trials of 9 items) (s)

Digit Span

Max no. items, forwards

Tests ,from the A CT

Syntnctic Rerrsoning

Mean correct RT per item (64 items) (s)

Grtc~ory Search Classification

Mean correct RT per item (20 items) (ms)

Category Search Recognition

Mean correct RT per item (40 items) (ms)

Spatial Recognition

Mean correct RT per item (60 items) (ms)

Seriul World Learning

Mean No. items recalled over three

identical trials of 15 items

Other measures

Genrral Health Questionnaire

“Normal” GHQ scoring method

Subjective Memory Questionnaire

376 (353)

24.22 (21.01)

5.72 (5.79)

6.79 (4.73)

1.55 (1.55)

1.30 (1.25)

1.45 (1.31)

X.59 (X.63)

3.52 (2.27)

136.74 (142.08)

56 (37)

6.1 (4.6)

1.6 (1.3)

2.9 (1.7)

0.6 (0.6)

0.4 (0.4)

0.4 (0.2)

2.6 (2.0)

4.x (4.1)

15.7 (15.6)

0.09 (72)

-0.06 (72)

0.03 (72)

-0.12 (5X)

-0.10 (70)

0.01 (66)

0.03 (65)

0.03 (6X)

0.07 (73)

-0.04 (77)

OP exposure through sheep-dipping, and neuropsychological

test performance at the time of assessment for chronic effects. To do this, Pearson correlation coefficients were calculated, and a series of multiple linear regression analyses, including potential confounding covariates (age, educational level, first language, smoking status, and time of day of neuropsycholog- ical testing), were performed. Covariates were entered, step- wise, if statistical significance was 0.05 or greater, and re- moved if statistical significance dropped to 0.10 or less at

subsequent steps. All analyses were performed using the SPSS for Windows statistics package.

RESULTS

Recruitment of the group of 146 farmers who participated in the study of chronic neuropsychological effects has been described elsewhere (11). Acute symptom data were available for a subgroup of 77 of the main study group of 146 farmers.

TABLE 3

REGRESSION COEFFICIENTS OF COVARIATES APPEARING IN THE REGRESSION EQUATION AT THE FINAL STEP OF STEPWISE MULTIPLE LINEAR REGRESSION ANALYSES 0, IN O.OS:(, OUT 0.1)

Chronic Effect Measure Summary Outcome

Acute Effects

Educational First Smoking Time of

Age Level Language status Day

Simple Reaction Time (NES)

Symbol-Digit Substitution (NES)

Digit Span (NES)

Syntactic Reasoning (A CT)

Category Search Classification (ACT)

Category Search Recognition (ACT)

Spntial Recognition (A CT)

Serial Word Learning (A CT)

Gene& Health Questionnaire

Subjective Memory Questionnaire

-

34.4

0.22 -0.73 2.97

0.15 -

- 1.3

- 0.01 0.3

0.3 -

-

452 STEPHENS, SPURGEON AND BERRY

There were no statistically significant differences between this subgroup and the remainder of the main study group across demographic, lifestyle, and exposure variables (illustrated in Table 1). Of this study subgroup, 43 dippers used a dip con- taining an OP compound, which was detectable in the urinaly- sis. Mean combined DEP/DETP for this group the morning following exposure was 44.9 nmol/mmol creatinine (95% CI for the mean 26.5 to 63.3). This compares with control levels of 4.7 nmol/mmol creatinine (95% CI for the mean 1.0 to 8.3; n = 22) and confirms recent exposure.

The mean level of acute effects (the change in levels of to- tal OP exposure symptom reporting from baseline to 24 h post- exposure) was 0.3 (95% CI -1.1 to 1.7) for the sheep farmers in this study, and -2.1 (95% CI -4.1 to -0.1) for controls (n = SO). Mean scores on chronic effect outcome measures are shown in Table 2. The Pearson correlation coefficients il- lustrating the relationships between the acute and chronic ef- fect outcomes are also presented in Table 2. There were no statistically significant correlations. Multiple linear regression models fitted for the same variable relationships as in each bi- variate correlation analysis, adjusting for the already stated potential confounding covariates, also showed no statistically significant associations between acute effects and chronic ef- fect measure summary outcomes. Regression coefficients for the covariates appearing in the regression equation at the fi- nal step of the multiple linear regression analyses, and there- fore achieving statistical significance (with stepwise addition and removal of covariates), are presented in Table 3.

DISCUSSION

There was no evidence of any association between acute effects (the change in levels of total OP exposure symptom re- porting from baseline to 24 h postexposure) and chronic ef- fects (neuropsychological test and questionnaire outcomes) of OP exposure in this group. This group had confirmed recent exposure (illustrated by the urinary dialkylphosphate levels), and was part of a larger group that was found to have statisti- cally significant decrements in two of the neuropsychological performance outcomes used in the study to assess chronic ef- fects: mean Symbol-Digit Substitution latency (farmers 23.58 s; controls 21.01 s), and mean Syntactic Reasoning latency (farmers 6.60 s; controls 4.73 s) (11).

To the best of our knowledge, no previous studies of

chronic effects of low-level OP exposure have examined the relationship between such effects and acute effects. One study of sequelae of acute intoxication, however, examined the rela- tionship between chronic effects and acute poisoning severity, assessed via “days taken off from work” as a result of the poi- soning episode, and found significant relationships for a num- ber of chronic effect outcomes (10). Our results neither con- firm nor contradict this finding, because our study was concerned with a different OP exposure profile, namely chronic low-level exposure.

This study group consisted of a representative group of sheep-dippers. Rigorous exclusion criteria were applied, to ensure that those with confounding health problems or lack of exposure were not included. The subgroup of 77 farmers whose data were included in the present study was not statisti- cally significantly different from the remaining participants from a larger group, which was found to be representative of sheep-dippers (11) across a number of demographic, lifestyle, and exposure variables. The exception to this was for the vari- able “average dipping session duration,” which was on aver- age approximately half a day longer in the current study group, indicating that the current study group might have slightly increased levels of OP exposure compared with sheep-dippers generally. Statistical control was made for a number of potentially confounding variables in the multiple linear regression analyses, including the effects of age, educa- tional level, smoking, time of day of chronic effect testing, and whether participants’ first language was English or Welsh. These analyses confirmed the conclusions of the bivariate cor- relations, that there was no association between acute and chronic effects as measured in this study.

In conclusion, the chronic effects found in this group, which might have slightly higher long-term OP exposure than sheep-dippers in general, appear to occur independently of symptoms that might immediately follow acute OP exposure. Thus, individuals with chronic effects of OP exposure do not appear to also necessarily experience acute effects. This has implications for exposure control, such that individuals expe- riencing chronic effects may do so without the benefit of earlier warning signs of toxic effects, manifest as acute symptoms at the time of exposure. This absence of immediate negative feed- back during exposure is likely to passively influence measures taken to reduce exposure, such that they are fewer than if feedback, in the form of symptoms of exposure. were present.

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