United StatM Environmental Protection Agency Health Effects Research Laboratory Research Triangle Park. North Carolina 27711 EPA/600/1-90/001 Fbo'ruary 1990 Research and Development Indoor Air - Health LJ1 Neurotoxic Effects of Controlled Exposure to a Complex Mixture of Volatile Organic Compounds 38101 EJBD k ARCHIVE EPA . 600- > 1- 90- s, 001 ------- DISCLAIMER: This manuscript has been reviewed by the Health Effects Laboratory, U.S. Environmental Protection Agency, and approved for publication as an EPA document. Approval does not signify that the contents necessarily reflect the views and policies of the Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. ------- FINAL REPORT (Deliverable 2520) NEUROTOXIC EFFECTS OF CONTROLLED EXPOSURE TO A COMPLEX MIXTURE OF VOLATILE ORGANIC COMPOUNDS Principal Investigator: David A. Otto 2 Co-Investigators: Lars Molhave 3 H.Kenneth Hudnell George Goldstein John O'Neil1 4 Statistician: Dennis House Technical Support: Gregory Rose Jon Berntsen Wayne Counts Shirley Fowler1 Scott Meade Human Studies Division, Health Effects Research Laboratory, U.S. Environmental Protection Agency, RTF, NC 2 Institute of Environmental and Occupational Medicine, University of Aarhus, Aarhus, Denmark Neurotoxicology Division, Health Effects Research Laboratory, U.S. Environmental Protection Agency, RTF, NC Research and Regulatory Support Division, Health Effects Research Laboratory, U.S. Environmental Protection Agency, RTF, NC NSI Environmental Services, Inc., Research Triangle Park, NC 27711 CE Environmental, Inc., Chapel Hill, NC 27514 Sources of Support: EPA Contract 68-02-4179 (Combustion Engineering) EPA Contract 68-02-3800 (Subject Recruitment) Repository Material u H „ us EPA /„ ,, Headquarters and Chemical Libraries Pern lanent Collection EPA west a* Room 3340 Mailcode 3404T 1301 Constitution Ave NW Washington DC 20004 202-566-0556 ------- PREFACE In October 1986 Congress passed the Superfund Amendments and Reauthorization Act (SARA, PL 99-499) that Includes Title IV - The Radon Gas and Indoor Air Quality Research Act. The Act directs that EPA undertake a comprehensive Indoor air research program. Research program requirements under Superfund Title IV are specific. They Include Identifying, characterizing, and monitoring (measuring) the sources and levels of Indoor air pollution; developing Instruments for Indoor air quality data collection; and studying high-risk building types. The statute also requires research directed at Identifying effects of Indoor air pollution on human health. In the area of mitigation and control the following are required: development of measures to prevent or abate Indoor air pollution; demonstration of methods to reduce or eliminate Indoor air pollution; development of methods to assess the potential for contamination of new construction from soil gas, and examination of design measures for preventing Indoor air pollution. EPA's indoor air research program is designed to be responsive In every way to the legislation. In responding to the requirements of Title IV of the Superfund Amendments, EPA-ORD has organized the Indoor Air Research Program around the following categories of research: A) Sources of Indoor Air Pollution; B) Building Diagnosis and Measurement Methods; C) Health Effects; D) Exposure and Risk (Health Impact) Assessment; and E) Building Systems and Indoor Air Quality Control Options. EPA is directed to undertake this comprehensive research and development effort not only through in-house work but also in coordination with other Federal agencies, state and local governments, and private sector organizations having an interest in indoor air pollution. The ultimate goal of SARA Title IV is the dissemination of information to the public. This activity includes the publication of scientific and technical reports in the areas described above. To support these research activities and other interests as well, EPA publishes its results in the INDOOR AIR report series. This series consists of five subject categories: Sources, Measurement, Health, Assessment, and Control. Each report is printed In a limited quantity. Copies may be ordered while supplies last from: U.S. Environmental Protection Agency Center for Environmental Research Information 26 West Martin Luther King Drive Cincinnati, OH 45268 When EPA supplies are depleted, copies may be ordered from: National Technical Information Service U.S. Department of Commerce 5285 Port Royal Road Springfield, VA 22161 ------- TABLE OF CONTENTS A. EXECUTIVE SUMMARY J B. BACKGROUND AND RATIONALE * C. METHODS ; C.I Subject Selection and Characterizaton / C.2 Physical Facilities 8 C.3 VOC Generation and Monitoring 9 C.4 Equipment Readiness and Pilot Study 9 C.5 Experimental Design 1° C.6 Behavioral Battery 11 C.7 Subjective Reactions 11 C.8 Confirmatory and Exploratory Hypotheses 12 C.9 Data Analysis Plan 13 C.10 Risks and Safeguards 1* D RESULTS 15 D.I Comfort Meter (Potentiometer) 15 D.2 Symptom Questionnaire 15 D.3 Auditory Digit Span (ADS) 16 D.4 NES Main Test Results 17 D.4a Finger Tapping 17 D.4b Visual Digit Span (VDS) 17 D.4c Continuous Performance Test (CPT) 17 D.4d Symbol Digit Substitution (SDS) 18 D.4e Serial Digit Learning (SDL) 18 D.4f Pattern Memory (PM) 18 D.4g Switching Attention (SWATT) 18 D.4h Mood Scales 19 D.5 NES Secondary Test Results 19 D.5a Associate Learning/Recall 19 D.5b Pattern Comparison 19 D.5c Simple Reaction Time (SRT) 20 D.5d Grammatical Reasoning 20 D.5e Horizonal Arithmetic 20 D.6 Water Consumption 20 D.7 Learning/Practice Effects 20 E. DISCUSSION 22 E.I Confirmatory Hypotheses ^ E.2 Exploratory Hypotheses 22 E.2a Sensory Irritation 22 E.2b Controlled Climate Variables 23 E.2c Motor Performance 23 E.2d Mood Scale Alterations 24 E.2e Cognitive Performance 24 E.3 Effects of Practice and Learning 25 F. REFERENCES 28 ------- LIST OF TABLES 1. Symptoms of Sick Building Syndrome 30 2. Subject Selection Criteria 30 3. VOC Mixture 31 Experimental Design 31 5. Measurement Variables 32 6. Comfort Ratings (Potentiometer Readings) 34 7. Symptom Questionnaire 35 8. Symptom Response Changes Over Time 37 9. Yes-No Question Responses 38 10. Auditory Digit Span (without 12 subjects) 39 11. Auditory Digit Span (All subjects) 40 12. Finger Tapping 41 13. Visual Digit Span 41 14. Continuous Performance Task 42 15. Symbol Digit Substitution 42 16. Serial Digit Learning 43 17. Pattern Memory 43 18. Switching Attention Test 44 19. Mood Scale 45 20. Associative Learning 46 21. Pattern Comparison 46 22. Simple Reaction Time 47 23. Grammatical Reasoning 47 24. Horizonal Arithmetic 48 25. Serial Order Effects: Auditory Digit Span 49 26. Serial Order Effects: Visual Digit Span 50 27. Serial Order Effects: Pattern Memory 50 28. Summary of Serial Order Effects 51 LIST OF FIGURES 1. Schematic of Test Protocol 53 2. Comfort Rating Scale Results 54 3. Headache, Odor and Air Quality Responses 55 4. Odor, Eye and Throat Irritation 56 5. Control Question Responses 57 6. Yes-No Question Responses 58 7. Auditory Digit Span 59 8. Visual Digit Span 60 9. Continuous Performance Task 61 10. Switching Attention Test 62 11. Mood Scale Difference Scores 63 12. Serial Order Effects: Auditory Digit Span 64 13. Serial Order Effects: Visual Digit Span 65 14. Serial Order Effects: Confusion Scale 66 15. Serial Order Effects: Pattern Memory 67 ------- APPENDICES A. VOC Generation and Monitoring Methods .68 B. Instructions for Administering and Scoring the Auditory Digit Span Test 79 C. Description of Neurobehavioral Evaluation System (NES) Tests 84 D. Symptom Questionnaire 91 E. Linear Analysis Rating Scale (Potentiometer) 93 ------- A. EXECUTIVE SUMMARY Growing public awareness of the potential health hazards of chemicals found in the indoor environment has given rise to the definition of a new problem designated as sick (or tight) building syndrome (SBS). SBS is characterized by irritation of the eyes, nose and throat. Preliminary evidence from Danish studies (Molhave et al, 1986; Kjaergaard et al, 1989) also indicates effects on cognitive functions such as memory. Many of the chemicals found indoors that have been linked with SBS are volatile organic compounds (VOCs). Congress has mandated EPA to characterize the sources, real-world exposure levels and health risks of indoor pollutants. Little information is currently available on the health effects of exposure to VOC mixtures found in new or renovated buildings. EPA has established an intramural research program to address this question. The current report details the results of the initial study in this program. Objectives of this study were to confirm and extend the pioneering work of Molhave et al (1986) and to identify sensitive measures for use in subsequent studies of sick-building syndrome. Molhave et al. demonstrated sensory irritant and neurobehavioral effects of controlled human exposure to a complex VOC mixture at concentrations of 5 and 25 mg/m3, representing the usual and maximal levels found in new homes. The design of the Danish experiment was problematic, however. A repeated-measures design was used in which subjects completed clean air and exposure conditions in the morning and afternoon of the same day. Three types of confounding may have occurred in this design—practice effects, VOC exposure carry-over effects from morning to afternoon, and time-of-day differences. The present study was designed to minimize or eliminate these problems by scheduling a separate training day to minimize practice effects, testing all subjects at the same time of day (afternoon), and separating clean air and exposure runs by a minimum interval of one week. Other important differences between studies included the subject population ------- and tests used. The Danish study included both males and females aged 18-64, all with documented histories of chemical sensitivity. The present study was limited to young adult males aged 18-39 with no history of chemical sensitivity. Only two neurobehavioral performance tests were used in the Danish study, while fourteen tests were used in the present study to fully characterize the nature of the possible neurobehavioral effects of VOC exposure. Results of this study confirm the adverse subjective reactions of subjects to a 25 mg/m3 concentration of volatile organic compounds reported by Molhave et al. Ratings of general discomfort (defined as irritation of the eyes, nose and throat), symptom questionnaire responses on odor intensity, air quality, eye and throat irritation, headache and drowsiness, and mood scale measures of fatigue and confusion all differed in predicted directions between clean air and exposure conditions. However, no convincing evidence was found of any neurobehavioral impairment associated with exposure to the VOC mixture. That is, we were unable to confirm the Molhave et al report that VOCs impair short term memory as measured by auditory digit span. Nor were other tests of memory, sensorimotor performance, or more complex cognitive functions affected by exposure to this concentration of VOCs. In summary, clear adverse subjective reactions to VOC exposure, but no functional (neurobehavioral) impairments, were found. Confirmation of sensory irritation in a normal, healthy adult male population is quite important. This finding demonstrates that subjective reactions to VOCs at levels found in new buildings are not limited to "complainers" or chemically sensitive subgroups in the general population. In fact, subjects in this study probably represent the subgroup least likely to be affected by VOC exposure. Sensory irritation, headache and drowsiness are also of considerable importance in the community and workplace. Perceived dissatisfaction with air quality in the home, office, market place, or factory can lead to reduced productivity, decreased quality of life among home-owners and workers, and could result in mass exodus from buildings with air quality problems. This is an important factor in the sick-building syndrome. Further consideration should be given to the relevance of subjective reactions in ------- evaluating the adverse health effects of indoor pollutants. Subjects in this study and previous Danish studies found the odor of VOCs to be very strong and unpleasant. The unmistakable odor of VOCs confounds efforts to conduct a double-blind experiment. Subjective reactions to the VOC mixture can be attributed, at least in part, to the unpleasant odor. If this were the only objectionable characteristic of VOCs, we would probably conclude that VOCs at concentrations found in the indoor environment do not constitute any significant health risk. The time course of responses to odor and symptoms of irritation differ, however, suggesting that odor accounts for only part of the subjective reaction to VOCs. Further study is needed to differentiate olfactory and trigeminal components of the VOC response. Failure to find any functional consequences of exposure to the VOC mixture however, raises important questions in assessing health risks and planning subsequent indoor air studies. For instance, we cannot conclude that a 25 mg/m3 concentration of VOCs poses no neurotoxic risk to susceptible subgroups of the general population, or even to the general population. Further study with a chemically sensitive group of subjects is needed and the relationship of age and gender to VOC response needs to be explored. It is also possible that the neurobehavioral tests used were not sensitive (or difficult) enough to detect effects. If subjects find that VOCs irritate the eyes and throat and induce headaches, one would expect performance to be impaired, at least indirectly, by distraction or impaired attention. Another possibility to consider is physiological endpoints. If VOCs irritate the eyes, nose and throat, we should examine secretions of these tissues for evidence of inflammation. Kjaergaard et al (1989) recently reported evidence of inflammatory response in eye fluids following VOC exposure. However, no evidence of inflammatory response in nasal secretions was found, consistent with symptom response in the present study. Further study of inflammatory effects of VOC exposure in eye fluid and other mucosal tissues are needed. ------- B. BACKGROUND AND RATIONALE. "Sick Building Syndrome" costs industry and the government considerable time and dollars in lost vorktime, worker dissatisfaction, and temporary abandonment of facilities (Melius et al, 1984). Beyond the economic impact is the question of possible adverse health effects of exposure to indoor air pollutants found in new and renovated homes and commercial buildings. Most people spend the majority of their lives indoors (NRC, 1981). Therefore, agencies and institutions responsible for protecting human health therefore need to carefully study the potential adverse effects of chemicals commonly found in indoor environments. Molhave and his colleagues (1986) have studied the health effects of controlled exposure to mixtures of volatile organic compounds found in new- construction Danish homes. Subjects were healthy adults selected by means of a questionnaire survey to identify individuals who have experienced discomfort associated with indoor air quality. Subjects were exposed to three concentrations (0, 5, 25 mg/m3) of a mixture containing 22 VOCs in the climate chamber at the Institute of Hygiene, Environmental and Occupational Medicine, University of Aarhus. The composition of this mixture was based on VOCs commonly found in newly constructed Danish homes (Molhave and Holler, 1979) and is similar to the levels and types of VOCs found in U.S. homes (White, 1987). Several measures of performance and sensory irritation were obtained. Subjective ratings of sensory irritation and air quality varied significantly with VOC exposure. Results also indicated impaired memory as measured by the Digit Span Test. The implications of these results are that exposure to low level mixtures of VOCs commonly found in new or renovated buildings, levels hitherto considered to be harmless to human health, may impair-the performance, productivity, comfort and well-being of workers and home-owners. These findings provide preliminary evidence of neurotoxic effects of ------- exposure to complex low-level mixtures of VOCs, although further study is needed to determine the precise nature of the observed effects. For instance, are these findings limited to a particular subset of the population who dislike the odor of VOCs? Is the response psychosomatic or does low-level mixed VOC exposure produce measurable decrements in objective health endpoints? Which health endpoints are most sensitive to VOC exposure—e.g., sensory, memory pulmonary, or immunological measures? Are there other vulnerable subsets of the general population such as young children, elderly, asthmatics or persons with allergies? Are VOC exposure effects due to single constituents of the mixture or the combination of several constituents? Systematic study of these questions under controlled laboratory conditions is necessary to clarify the human health risks of exposure to indoor air pollutants. Very little evidence is currently available concerning the neurotoxic effects of low-level human exposure to complex VOC mixtures. Controlled exposure studies are also necessary before undertaking related field studies. The type of symptoms affected by VOC exposure in the Molhave et al study has been identified as the Sick Building Syndrome (SBS) by WHO (1982). This syndrome is characterized by subjective and physiological symptoms of sensory irritation of mucosal tissue and other symptoms as shown in Table 1. These symptoms are acute, nonspecific, and cannot be ascribed to any specific cheraical(s) found in the problem environment. In the Molhave et al study, individual VOCs in the complex mixture were present at concentrations well below levels that would be expected to produce irritant effects for any single chemical in the mixture. Molhave (1986) has hypothecized that the summation of effects of exposure to the mixture, rather than any single con- stituent chemical, may be the cause of the effects observed in the study. Molhave (1986) has further hypothecized that the observed effects are mediated by trigeminal nerve function which is generally considered to be the neuro- physiological substrate of the chemical sense. the The objective of the present study was to confirm the principal findings of Aarhus study—i.e., increased perception of sensory irritation and ------- discomfort associated with exposure to a low-level complex mixture of VOCs. Molhave et al also reported an effect on digit span performance suggestive of a VOC-related impairment of short-term memory. The functional significance of the digit span finding, however, requires clarification. The observed effect could be a primary neurotoxic effect of VOC exposure or a secondary effect of sensory irritation (e.g., distraction). Digit span performance also reflects both attentional and memory processes. The present study included a battery of neurobehavioral tests designed to clarify the nature and validity of the Danish findings. Response to low-level VOC exposure was studied in normal healthy adults. ------- C. METHODS C.I Subject Selection and Characterization. Normal healthy adult volunteers were recruited by the UNC Subject Recruitment Office (operated under EPA Contract 68-02-3800). Advertisements posted on campus and published in local newspapers were used to recruit subjects. Selection criteria based on those used in the Danish study are specified in Table 2. In brief, healthy, non- smoking Caucasion males aged 18 to 40 with no history of asthma, allergic rhinitis, cigarette use or other medical conditions were referred for physical examination and allergy testing. It should be noted that these requirements were more stringent than the Danish study which included smokers and persons up to 64 years of age. Smokers and persons over 40 years were excluded from the present study in order to minimize the variability in performance and sensory irritation that might be associated with these variables. Exposure history was assessed by a questionnaire adapted from Molhave et al (1986). Exposure history was used to exclude individuals with significant occupational or recreational exposures that could influence neurobehavioral measures and to exclude individuals with histories of chemical sensitivity. Volunteers who met selection criteria were referred for physical examination and allergy skin testing. A standard scratch test for 18 common allergens including airborne pollens, cat and dog danders, molds and house dust was administered. Subjects with positive skin allergy tests were not included in the study in accordance with the selection criteria of Molhave et al (1986). Payment for participation conformed to guidelines established by the EPA Clinical Research Branch. A base rate of $7 per hour and bonus for completion of the study was paid to subjects. $30 was paid to potential subjects who completed the physical examination. Subjects who qualified for participation in th* study earned $84 more, depending on the number of hours devoted to training ------- and running in the control and exposure sessions. Subjects also received a bonus of $30 for completion of the experiment. Subjects completing all phases of the study earned a maximum of $144. Payments were made at the end of the experiment or upon termination of participation. Although a subject could elect to discontinue the experiment at any time due to discomfort, concern or other reasons, if the subject chose to leave for non-medical reasons, only fees earned to that point were paid. On the other hand, if the investigators terminated an individual on medical or technical grounds once an exposure session had commenced, the subject(s) received full payment for that session. Subjects commuting from Durham and Raleigh were paid an additional $6 and $11, respectively, and all parking fees were paid. To conform to the Federal Privacy Act, each subject was assigned a number at the time of physical examination. Subject privacy was maintained thereafter by exclusive use of the subject number for subject identification. Seventy-six subjects completed the VOC study. Data from ten subjects vere discarded for technical reasons including the addition of the auditory digit span test after seven subjects had been run and the accidental termination of exposure during one session with three subjects. Therefore, data from sixty-six subjects vere used in statistical analyses. The mean age of subjects was 25.0 yrs. (s.d. 5.6). C.2 Physical Facilities. The study vas carried out in the exposure facilities of the EPA Human Studies Division in Chapel Hill (Strong, 1978). The environment including temperature, humidity, light levels and pollutant concentration vas continuously controlled and monitored by computer. Personnel operating this facility included EPA psychologists and technicians and a team of technical and engineering personnel from CE, Inc. vho operate and maintain the facility under contract to EPA. These personnel vere highly trained individuals vho routinely 8 ------- administer other similar protocols. A licensed physician was immediately available to the operating team during all experiments. C.3 VOC Generation and Monitoring. The complex VOC mixture described by Molhave et al (1986) was used in the present study with the substitution of one compound (1,1-dichloroethane for 1,2 dichlorethane). The Molhave mixture of 22 volatile organic compounds is shown in Table 3. The compounds were selected on the basis of previous Danish surveys (Molhave and Holler, 1979) including the 10 most commonly found and the 10 found in greatest concentration in indoor nonindustrial environments, but ex- eluding known carcinogens. That is, this mixture is characteristic of the chem- icals and concentrations to which residents in new Danish and American homes are often exposed. Any compounds classified by the International Agency for Research on Cancer (IARC monographs) as Group 1 (chemicals for which there is "sufficient evidence from epidemiological studies to support a causal associa- tion between the exposure and cancer") or Group 2 (chemicals that are probably carcinogenic to humans—i.e., "exposures for which there [is] at least limited evidence of carcinogenic!ty to humans" or "sufficient evidence in animals") were excluded. The concentration of individual constituents of the mixture shown in Table 3 is specified in ug/m3, each of which is below the threshold limit value (TLV) for occupational exposure. TLV is the concentration of a chemical to which workers may be continuously exposed for 8 hours without adverse health effect as defined by OSHA. A system for generating and monitoring the VOC mixture equivalent to that used in the Danish study was constructed by CE, Inc. A des- scription is contained in Appendix A. Subjects were exposed to a 25 mg/m3 concentration of VOCs measured as a toluene equivalent concentration with a flame ionization detector (FID). The duration of exposure was 2.75 hours. C.4 Equipment Readiness and Pilot Study. Prior to initiating the main study, a pilot study was conducted to test equipment, to train the operating staff, and to check-out the protocol to insure ------- that the testing schedule ran smoothly. Prior to exposing subjects to the VOC mixture, extensive testing of the gas generating and monitoring system was conducted by CE to ensure that the system operated reliably and to ensure that gas concentrations did not vary by more than +3.0% once the target level (25 mg/m3) was reached. When CE completed testing of the gas generating and monitoring equipment, two groups of three subjects (total N=6) were run through the complete protocol in order to work out any scheduling problems and to train the operating staff. Both groups were run under control (clean air) and exposure conditions as specified for the main protocol in the following sections. C.5 Experimental Design. Molhave et al (1986) used a repeated measures design in which each sub- ject completed both clean air control and VOC exposure (either 5 or 25 mg/m3) conditions on the same day. Half the subjects received clean air in the morn- ing and half in the afternoon in a counterbalanced design. There are several problems with this design including possible confounding from carry-over effects (i.e., carry-over effects on afternoon control performance from morning exposure), from practice or fatigue effects (session 1 to session 2), and from time-of-day effects (morning vs. afternoon). Carry-over effects were prevented by separating control and exposure sessions by a week or more. This separation also reduced, but did not eliminate practice effects. It also permitted each subject to complete exposure and control sessions at the same time of day, eliminating time-of-day confounding. A disadvantage was that subjects were required to return for an extra day, introducing the possibility of subject drop-out as well as other possible confounding factors such as different weather conditions or subject mood and preparedness. To simplify the design further, only one exposure level—25 mg/m3—was run. Control and exposure sessions were administered in, a double-blind manner counter-balanced across subjects similar to the design shown in Table AA. Although the chamber could accommodate four subjects per run, subject availability limited testing on many days to less than four as shown on Table 10 ------- 4B. C.6 Behavioral Battery A battery of behavioral tests was used to assess which behavioral processes are affected by VOC exposure. The battery included the digit span test reported by Molhave et al (1986) to be impaired by VOC exposure. This test was administered in the same manner as Molhave et al—i.e., the paper-and-pencil form derived from tfechsler (1955) with auditory stimuli presented by tape recorder. Instructions for the administration and scoring of this test are contained in Appendix B. Since performance of the digit span test includes components of attention as well as memory, other behavioral tests were included to evaluate which processes are affected by VOC exposure. A main set of eight computerized behavioral tests developed by Baker and Letz (1985) and described in Appendix C was used. The tests included motor speed, memory, attention and mood. These tests were administered twice during each testing session—at the beginning and end. A secondary set of five other sensorimotor, memory and cognitive tests were administered once midway through each testing session. These tests are also described in Appendix C. C.7 Subjective Reactions A 25-item questionnaire related to sensory irritation, discomfort, and indoor air quality was used to obtain subjective ratings as described by Molhave et al (1986). This questionnaire is presented in Appendix D. The feeling of irritation in the eyes, nose and throat was also measured at 15-30 minute intervals using a linear potentiometer described in Appendix E. In the questionnaire subjects were asked to indicate the degree of perceived discomfort by adjusting the position of a vertical marker on a video screen by means of a joystick. The range of response varied between two well-defined extremes of items such as dryness of the nose or skin, irritation of the throat, and room noise, temperature or humidity. The linear potentiometer is a device that reduces the questionnaire to a single scale representing the collective irritation of eyes, nose and throat. The potentiometer consisted of an aluminum 11 ------- box with a sliding lever which electronically and continuously recorded the comfort rating of a particular subject during the experiment. C.8 Confirmatory and Exploratory Hypotheses. Confirmatory and exploratory hypotheses specified a priori are stated below. Measurement variables from individual tests and questionnaires are listed in Table 5. a. Confirmatory Hypotheses. (1) Exposure to complex VOC mixtures will result in higher ratings of sensory irritation as measured by the linear analog rating scale (potentiometer). (2) Exposure to complex VOC mixtures will result in impaired cognitive performance relative to clean air as follows: i. Short-term memory as measured by span length (sum of forward and backward span lengths) in the auditory digit span test will be reduced; ii. Coding time (msec) for symbol-digit substitution items will be longer; iii. Selective attention, as measured by mean time/item in the switching attention test (incompatible trials) will be longer. b. Exploratory Hypotheses. Exposure to complex VOC mixtures will: (3) Increase sensory irritation as indexed by questionnaire ratings; (4) Not influence ratings of controlled climate variables—i.e., air temperature, humidity, light level and sound level; (5) Not affect motor performance on the finger tapping test; (6) Alter selected dimensions of mood—i.e., tension, confusion and 12 ------- fatigue—as indexed by pre-post mood scale difference scores; (7) Impair other measures of cognitive performance as indexed by continuous performance, serial digit learning and pattern memory tests. C.9 Data Analysis Plan. The main analyses of the experiment were done to determine if there were any VOC effects. Secondary analyses of most variables were done to determine if learning or practice effects were present and, if so, to determine the nature of those effects. In the main or confirmatory analysis were: (1) auditory digit span (forward plus backward), (2) potentiometer (comfort meter) measurements, (3) symbol-digit substitution (mean response time) and (4) switching attention test (reaction time in alternating condition). All other variables and analyses are considered as secondary or exploratory in nature. Variables measured only once during each testing session were analyzed by a paired t-test on the two measurements for each subject. Variables with pre and post measurements were analyzed by a paired t-test on the air and VOC change (post-pre) for each subject. This analysis is equivalent to a two-way analysis of variance with subjects as one factor and treatment changes as the other factor. The two response profiles of each of the 25 questions in the comfort questionnaire and the response profiles of the comfort meter measurements were tested for parallelism using multivariate analysis of variance of repeated measures methods. If the parallelism hypothesis for a question was rejected, the three additional hypotheses were tested in order to explain the nature of the lack of parallelism. The first hypothesis compared the mid-exposure changes on air and VOC, the second hypothesis compared the post-exposure changes and the third hypothesis compared the changes from mid to post-exposure. The first three questions required either a yes or no response and were analyzed in the above manner by coding "1" for yes and "0" for no. In other words, the profile analyzed was the proportion of "yes" responses at each measurement point. 13 ------- Pretest questionnaire responses were analyzed by the nonparametric McNemar test for the significance of changes. This test was used in order to answer the question of whether or not potential covariates vere changing from one exposure to the other. All variables except questionnaire and comfort meter responses vere analyzed to determine if learning or practice effects vere present and, if so, as far as possible, to determine the nature of those effects. For variables vith pre- and post-measurements during each exposure, the exposure vas ignored and the actual order of the four measurements vas analyzed by fitting a first, second and third degree polynomial to the means. For variables vith only one measurement during each exposure, the exposure vas ignored and the first measurement vas compared to the second to determine if learning effects vere present. C.10 Risks and Safeguards. The concentrations of all volatile organic compounds used in this study vere belov the TLVs for occupational exposure by orders of 10 to 1000 (ACGIH, 1982). Although mucosal irritation and discomfort vere reported by subjects in the study conducted by Molhave et al (1986), these effects vere gone vithin 24 hours as indicated by follow-up questionnaires (Molhave, personal communication). Reported effects on digit span performance vere marginal and transient as veil. There is no evidence of any permanent adverse effect of acute VOC exposure at these levels. There is no known risk involved in the performance of the computerized behavioral battery or discomfort rating instru- ments. In the unlikely event of medical emergency, a licensed physician vas be present in the EPA facility at all times during the experiment. Comprehensive emergency procedures used as general practice in the operation of the facility could have been activated vithin seconds of the occurrence of an emergency. Emergency equipment vas kept at the site of the exposure chambers at all times. Furthermore, subjects vere kept under constant surveillance by trained 14 ------- biomedical personnel via closed circuit TV monitoring, direct observation, and voice (intercom) communication. D. RESULTS D.I Comfort Meter (Potentiometer) Comfort meter response profiles for exposure and clean air were significantly different (p < .001) as shown in figure 2. That is, subjects expressed much more discomfort (irritation of mucous membranes) during VOC exposure than clean air. An abrupt divergence of comfort levels occurred when VOC exposure commenced at the 75 min. point in testing. The maximum difference in comfort levels occurred about 75 min. after exposure onset. Mean potentiometer readings (and standard errors) at each sampling point are shown in Table 6. D.2 Symptom Questionnaire A 25-item symptom questionnaire was administered three times during each testing session—during pre-exposure baseline, immediately after VOC build-up, and at the end of the 2.75 hr. exposure period. Mean response levels at the three measurement points for continuous items 1-22 are shown in Table 7. Significant main effects of VOC exposure (25 mg/m3) compared to clean air were found for six items including headache, odor level, eye and throat irritation, drowsiness and air quality. Figures 3 and 4 illustrate these findings. Five environmental variables (light and noise levels, temperature, humidity and air movement) were held constant during testing. Responses to control variables did not vary with exposure (Figure 5). Different patterns of change over time in response levels were apparent for different symptoms. Further analysis of temporal patterns (Table 8) indicated that the perceived odor level increased dramatically after VOC exposure onset, but then decreased significantly with continued exposure at a constant level 15 ------- (Figure 3). A similar (but inverse) pattern vas observed for air quality. Headache, eye and throat irritation also increased significantly after VOC onset, but remained elevated for the duration of VOC exposure (Figure 4). Increased drowsiness, relative to clean air, was significant only at the end of exposure. Three other questions solicited binary (yes-no) responses concerning air quality as follows: (1) Do you feel now that staying in the chamber is comfortable? (2) Would you be satisfied with this air quality in your own home? (3) Would you ventilate your home more? VOC exposure in each case elicited significant change in perceived comfort as shown in Figure 6 and Table 9. Sixty-four percent more of the subjects, for instance, indicated that they would not be satisfied with the air quality perceived after VOC exposure build-up (mid) than at the same point during clean air. A small (14%) but significant reduction in dissatisfaction occurred by the end of exposure. D.3 Auditory Digit Span (ADS) Six scores were derived from the ADS test including the standard measures of the longest spans recalled in forward and backward conditions and the total (forward + backward) span. Three variant measures, designated as "weighted" scores, were also obtained as described in the methods section. A problem was encountered in the administration of ADS to some subjects who failed to reverse numbers in the backward condition. That is, numbers were written down from right-to-left on the answer sheets, enabling these subjects to complete the backward condition without mentally reversing numbers. The problem was observed in 12 subjects, despite explicit instructions and training to the contrary. Therefore, ADS results were analyzed both with and without the 12 16 ------- aberrant subjects. Hean pre- and post-exposure scores are shown in Tables 10 (without 12 subjects) and 11 (all subjects). VOC exposure did not affect auditory digit span in the predicted manner. One measure (standard backward span length) varied significantly with VOC exposure, but in the wrong direction—i.e., that is, auditory digit span (backwards) was slightly longer during exposure than clean air. ADS results are shown in Figure 7. D.4 NES Main Test Results D.4a Finger Tapping As predicted, finger tapping speed was not altered by VOC exposure. The mean numbers of finger taps obtained with preferred, non-preferred and alternating hands are shown in Table 12. D.4b Visual Digit Span (VDS) Three scores were derived from the VDS test including the longest spans recalled in forward and backward directions and the total (forward + backward) span. These scores are comparable to the standard scores obtained form the auditory digit span test. Mean pre- and post-exposure scores are shown in Table 13 and Figure 8. Forward span length increased slightly but significantly more under clean air than VOC exposure. Backward and total span lengths did not vary with exposure. D.4c Continuous Performance Test (CPT) Three CPT measures were analyzed—mean reaction time (RT), errors of omission, and false positives. The primary measure (RT) did not vary with VOC exposure, nor did omission errors. A marginal trend (p = .061) toward increased false positives was observed during VOC exposure. Mean scores are provided in Table 14 and illustrated in Figure 9. 17 ------- D.4d Symbol Digit Substitution (SDS) Two scores were obtained from the SDS test—the mean number of incorrect responses per trial, and the mean time to complete a trial (requiring nine digit substitutions). Hean scores and results are shown in Table 15. Neither score was affected by VOC exposure. D.4e Serial Digit Learning (SDL) Two measures were analyzed from the SDL test, a performance score described in the methods section, and a variant of the performance score in which the total number of trials to reach criterion was added to the performance score. Unlike related auditory and visual digit span tests, smaller scores indicate better performance in the SDL test. Mean scores and results are detailed in Table 16. SDL performance was not affected by VOC exposure. D.4f Pattern Memory (PM) Three measures were analyzed including the mean response latency of all trials, mean response latency for correct trials only, and the mean number of correct trials. Results and means are shown in Table 17. VOC exposure did not affect pattern memory. D.4g Switching Attention (SWATT) Seven response-time measures were derived from the SWATT test including mean response times for three basic conditions—side, direction and the alternation of side and direction (switching). The switching condition was then broken down into four subsets of trials—side compatible, side incompatible, 18 ------- direction compatible, and direction incompatible. Mean response times for each of these measures are presented in Table 18. Response times improved over the testing session (pre - post difference) for all measures during clean air. The pre - post difference was less or in the opposite direction for six of seven measures during VOC exposure, although none of the differences were statistically significant. Response times also increased markedly relative to the complexity of the type of trial—the side direction is easiest, the direction condition is more difficult, and the alternating (switching) direction condition is most difficult as shown in Figure 10. D.4h Mood Scales Mean scores obtained for the five mood scales during clean air and VOC exposure are shown in Table 19. Post - pre change scores differed significantly during VOC exposure on two scales (confusion and fatigue) as predicted. That is, increases in fatigue and confusion over the 4-hour testing session were greater during VOC exposure than during clean air as shown in Figure 11. D.5 NES Secondary Test Results D.5a Associate Learning/Recall Two measures were obtained for this test—the total number of correct pairings over 3 trials and the number of correct pairings made during the subsequent recall trial. Means and results are shown in Table 20. Associate learning and recall were not affected by VOC exposure. D.5b Pattern Comparison Three measures were analyzed including the number of correct trials, mean latency for all trials, and mean latency for correct trials only. Mean scores and results are presented in Table 21. VOC exposure did not affect any pattern 19 ------- comparison measures. D.5c Simple Reaction Time (SRT) Mean reaction-times were examined for preferred and non-preferred hands. Means and results are shown in Table 22. SRT was not altered by VOC exposure. D.5d Grammatical Reasoning Three measures were obtained from this test including the mean response time for individual trials, the total number of errors, and a composite score of the total number of correct responses minus the total number of errors. None of these measures vas affected by VOC exposure as shown in Table 23. D.5e Horizontal Arithmetic Three measures were obtained including the number correct, mean response time for all trials and the mean response time for correct trials only. Slightly more problems were answered correctly during VOC exposure (p = .031) than during clean air as shown in Table 24. D.6 Water Consumption Subjects consumed slightly more water during VOC exposure (2.60 cm) than during clean air (2.36 cm), but this difference was not significant. D.7 Learning/Practice Effects First, Second and third degree polynomials were fitted to the four serial order means of measures from each of the NES main tests to determine whether learning or practice effects occurred beyond testing. A significant linear contrast implies that there are learning or practice effects across the two training sessions (or four serial test administrations). A significant 20 ------- quadratic effect implies that the learning or practice effect is approaching or has reached asymptote. A significant cubic effect within the present design suggests that learning or practice effects occur within each testing session (pre-post comparison). The absence of linear, quadratic or cubic effects implies no learning or practice effects. Most measures showed significant serial order effects within or across testing sessions. The auditory digit span test illustrates significant linear trends for all measures (Figure 12, Table 25). The total span length measure on the Visual Digit Span Test (Figure 13, Table 26) demonstrates a clear quadratic pattern. An example of a cubic effect in the absence of linear or quadratic trends occurred on the Confusion Mood Scale (Figure 14). Response latencies on the pattern memory test exhibited a complex serial order pattern in which all three contrasts were significant (Table 27, Figure 15). Table 28 summarizes the results of polynomial contrasts for all computerized NES tests used in this study. 21 ------- E. DISCUSSION E.I Confirmatory Hypotheses. Four confirmatory hypothese were specified, only one of which was confirmed. As hypothesized, subjects expressed considerably more general discomfort on the linear analog rating scale of eyes, nose and throat irritation during VOC exposure than clean air. This finding is consistent with previous Danish reports (Molhave et al., 1986; Kjaergaard et al, 1989). Other hypotheses that VOC exposure would impair cognitive performance of short-term memory on the auditory digit span test, coding time on the symbol digit substitution test, and selective attention on the switching attention test were not confirmed. Contrary to Molhave et al's (1986) report, short-term memory was not affected by VOC exposure. E.2 Exploratory Hypotheses Five exploratory hypotheses were specified, four of which were confirmed. E.2a Sensory Irritation. Symptoms of sick building syndrome, listed in Table 1, include irritation of the eyes, nose and throat; mental fatigue, and headache. Subjects expressed more irritation of eyes and throat (but not nose) during exposure to VOCs than clean air. Subjects also reported more headache and drowsiness during VOC exposure, consistent with Danish studies (Molhave et al, 1986; Kjaergaard et al, 1989). Symptom questionnaire results provide a more detailed picture of the irritant effects of VOC exposure than the general measure of discomfort provided by the linear analog rating scale. Although the potentiometer rating was defined as an index of "eye, nose and throat irritation", responses to specific symptom questions suggest that VOCs irritated the nose less than eyes and throat. This finding has implications for future VOC studies—i.e., that measures of inflammatory process in eye fluids and throat secretions may yield more useful information than in nose secretions. Failure to find a significant 22 ------- increase in nose irritation is not in accord with the SBS profile, although it is consistent with Kjaergaard et al's (1989) report that VOC exposure did not produce any signs of inflammation in nasal secretions. The most dramatic reactions to VOC exposure in the present study and in previous Danish studies were obtained on ratings of odor intensity and air quality. Subjects exposed to the 25 mg/m3 VOC mixture found the odor very strong and unpleasant. Subjects similarly reported that VOCs degrade air quality very much. Molhave et al (1988), moreover, found a systematic dose-dependent relationship of perceived odor intensity and air quality at VOC exposure levels of 3, 8, and 25mg/m3. The strong and unmistakable odor of the VOC mixture poses a methodologic challenge—i.e., VOCs and clean air cannot be administered to subjects in a double-blind manner. Subjects know unequivocally when exposure begins. This problem was reduced by using a counterbalanced design. The rapid habituation of olfactory sensation also helps to mitigate this problem. Results of the present study, for instance, indicate that perceived odor intensity decreased at the end of the testing period, while eye, throat, headache, fatigue and confusion scores either increased or remained elevated during the 2.75 hour exposure period. The irritant effects of VOC exposure thought to be mediated by the trigeminal nerve (Molhave, 1986), can thus be distinguished from olfactory response over time. Further study is needed to characterize in detail the time course of olfactory and trigeminal response to VOCs. E.2b Controlled Climate Variables It was further hypothesized that ratings of five climate variables (air temperature, humidity, light and noise levels, and air flow), which were held constant throughout testing, would not vary with VOC exposure. This hypothesis was confirmed, providing construct validity to the symptom questionnaire. E.2c Motor Performance. The finger tapping test provides a simple, straightforward measure of motor 23 ------- performance. Since motor dysfunction has not been associated with VOC exposure, we predicted no effects on finger tapping. This hypothesis was confirmed. E.2d Mood Scale Alterations. Pre-post differences on three mood scales—tension, confusion and fatigue— associated with VOC exposure were hypothesized. This hypothesis was confirmed for confusion and fatigue, but not tension. These findings are consistent with SBS syndrome as veil as the drowsiness item on the symptom questionnaire. The lack of effect on the tension and anger scales is of some interest, particularly in view of the perceived increase in discomfort and sensory irritation. Many constituents of the mixture are organic solvents with known anesthetic or CNS depressant properties—e.g., n-butylacetate, 1-1-dichloroethane, n-hexane, and p-xylene (Anger and Johnson, 1985). The anesthetic or narcotic effects of the solvents may account for the absence of tension or anger in subjects during VOC exposure. E.2e Cognitive Performance. The final exploratory hypothesis concerning the impairment of performance on three different tests of cognitive performance (continuous performance test, serial digit learning and pattern memory) was not confirmed. The results of all memory, sensoriraotor and other types of cognitive tests were very consistent. None of these tests showed any convincing evidence that VOC exposure impaired performance, despite marked subjective reactions on the symptom questionnaire and comfort rating scale. Nor is this finding particularly surprising in view of the low aggregate exposure level (7 ppm toluene equivalent) of the VOC mixture. Neurobehavioral effects have seldom been observed below 300 ppm toluene (Johnson et al, 1987). Concentrations of individual chemicals in the mixture were below TLVs by factors of 10 to 100 or more. Molhave et al (1986) reported a small reduction in digit span during VOC exposure. The consistent negative results on fourteen neurobehavioral tests argue rather persuasively against any functional impairment in healthy young adult males exposed to this concentration (25 mg/m3) of volatile organic 24 ------- compounds. Exposure and clean air sessions were run in the morning and and afternoon of the same day in the original Danish study, giving rise to possible confounding from exposure carry-over effects, practice or fatigue effects, and time-of-day effects. Modifications in the experimental design, to mitigate these confounding factors render the present results more reliable than the original study. On the other hand, several important differences in the subject samples used in the two studies could possibly account for the discrepant digit span findings. (1) Molhave et al carefully selected chemically sensitive subjects with documented histories of indoor climate problems. Chemically sensitive subjects were excluded from the present study. (2) The range of ages in the Danish study (18-60 years) was much wider than the present study (19-39 years). Older subjects could be more sensitive than younger subjects to VOC exposure. (3) Finally, the Danish study included equal numbers of males and females. Maizlich et al (1987) have shown that females perform more poorly than males on many NES tests. Whether cognitive performance is more susceptible to impairment by VOC exposure in women then men, in older than younger subjects, and in chemically sensitive compared to normal healthy subjects are questions that require further study. E.3 Effects £f Practice and Learning The question of practice or learning effects is an important concern in repeated-measures studies employing neurobehavioral tests. Such effects could exaggerate or obscure effects of chemical exposure if not controlled properly in the experimental design. It is also of considerable interest to the research community to characterize the effects of practice and learning in a widely used neurotoxicity testing system. For the present discussion the terms "practice" and "learning" will be used synonymously. Although the study was not specifically designed to examine practice effects, the study provided a convenient and extensive dataset to evaluate this question. Nine tests were administered twice on each of two test days, yielding four 25 ------- replications. Test days were separated by at least one week. Practice effects were evaluated by testing for linear, quadratic and cubic trends in the data arrayed in the serial order of presentation. As noted previously, a significant linear trend implies extended practice effects across the four test administrations, quadratic effects imply that practice effects approach or reach asymptote levels, and cubic effects imply transient practice effects (within a testing session). Results of polynomial tests for serial order effects are summarized in Table 28 including data on nine tests repeated four times. Evidence of extended practice effects (significant linear trends) were found on five tests—auditory and visual digit span, continuous performance, symbol digit substitution, and switching attention tests. Results of three secondary NES tests (associate learning, pattern comparison, and grammatical reasoning tests), administered once during each testing session, also suggest extended practice effects. Only one of five mood scales (tension) exhibited a significant linear trend. Tests which do not appear to have extended practice effects include finger tapping, serial digit learning, other mood scales, simple reaction time, and horizontal addition. Finger tapping and two mood scales (fatigue and confusion), however, showed significant cubic trends which imply within-session effects, but no between-session effect. Mood scales, in particular, are designed to assess short-term subjective reactions to experimental variables. Fatigue and confusion scales, if valid, should vary over a demanding task and then return to previous values after resting or at the beginning of another testing session. Significant quadratic effects were observed in two tests—visual digit span and pattern memory—and marginal quadratic trends were found for some variables in serial digit learning, switching attention, and the tension scale. All three trends occurred in the pattern memory test. The implication of the serial order analysis is that practice or learning effects occur in the performance of most neurobehavioral tests commonly used in neurotoxicity testing of humans. It should be noted that these effects were observed despite an initial training session. Data from the training session were not included in the present analysis because the length and complexity of 26 ------- some tests varied on training and testing days. However, a single training session does not appear to be adequate to eliminate practice/learning effects on most NES tests. Further parametric studies need to be done to systematically evaluate the practice/learning characteristics of each NES test. Task complexity, length of task and interest trial interval must be carefully assessed in order to determine optimal parameters for the use of NES tests in repeated-measures studies in the laboratory as well as in the field. 27 ------- 14. REFERENCES American Conference of Government Industrial Hygienists. Threshold Limit Values for Chemical Substances and Physical Agents in the Workroom Environ- ment with Intended Changes for 1982. ACGIH Publ. Office, Cincinnati, 1982. Bach B, Molhave L, Pedersen OF. Human reactions during controlled exposures to low concentrations of organic gases and vapors known as normal indoor air pollutants: Performance tests. In Berglund et al (eds) Indoor Air, Vol 3, Sensory and Hyperreactivity Reactions to Sick Buildings. Swedish Council for Building Research, Stockholm, 1984, pp397-401. Hathaway SR, McKinley JC. The Minnesota Multiphasic Personality Inventory. The Psychological Corporation, 304 East 45th St, New York, NY 10017, 1966. Hollingshead AB. Two-factor index of social position. Unpublished ms., Yale University, 1957 (Available from the author, 1965 Yale Station, New Haven, CT 06520). IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Volumes 1-41, Lyon, 1971-1986. Johnson BL (ed). Prevention of Neurotoxic Illness in Working Populations. Wiley, New York, 1987. Kjaergaard S, Molhave L, Pedersen O.F. Human reactions to a mixture of indoor air volatile organic compounds. Environment Int., 1989 (in press). Letz R, Baker EL. Computer-administered neurobehavioral testing: On defining its limitatins. In: Environmental Health, Doc 3, World Health Organization, Copenhagen, 1985:158-162. Maizlich N, Schenker M, Weisskopf C, Seiber J, Samuels S. A behavioral evaluation of pest control workers with short-term, low-level exposure to the organophosphate Diazinon. Am J Indus Med, 1987, 12:153-172. Melius J, Wallingford K, Carpenter J, Keenlyside R. Indoor air quality: the NIOSH experience. Am Conf Indus Hyg, Rep 10, 1984: 3-7. Molhave L. Indoor air quality in relation to sensory irritation due to volatile organic compounds. ASHRAE Transactions, Vol 92(1), paper 2954, Dec 1986. Molhave L, Bach B, Pedersen OF. Human reactions during controlled exposures to low concentrations of organic gases and vapors known as normal indoor air pollutants. In Berglund et al (eds) Indoor Air, Vol 3, Sensory and Hyperreac- tivity Reactions to Sick Buildings. Swedish Council for Building Research, Stockholm, 1984, pp431-436. 28 ------- Molhave L, Bach B, Pedersen OF. Human reactions to low concentrations of volatile organic compounds. Environment Int., 1986, 12: in press. Molhave L, Jensen JG, Larsen S. Acute afnd subacute subjective reactions to volatile organic air pollutants. Unpublished Report, Inst. Environ. Occup. Med., Univ. of Aarhus, Denmark, Dec. 1988. Molhave L, Holler J. The atmospheric environment in modern Danish dwell- ings-Measurements in 39m flats. in Indoor Climate, P Fanger and 0 Valbjorn (eds), SBI, Horsholm, Denmark, 1979, pp!71-186. National Research Council, Committee on Indoor Pollutants. Indoor Pollutants. National Academy Press, Washington, DC, 1981. Strong AA. Description of the CLEANS Human Exposure System. EPA-600/1-78-064, Health Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC, Nov 1978. Wechsler D. Wechsler Adult Intelligence Scale Manual. Psychological Corpora- tion, New York, 1955. White JR. A Review of Indoor Air Contaminants and Recommendations for a "Typical" Indoor Air Mixture. Internal Report, Air and Energy Engineering Laboratory, U.S. Environmental Protection Agency, Research Triangle, 1987. World Health Organization. Indoor Air Quality Research. EURO Reports 103, Stockholm, 1986. 29 ------- TABLE 1. SYMPTOMS OF SICK BUILDING SYNDROME (WHO, 1982) Irritation of eye, nose and throat Dry mucous membrane and skin Erythema Mental fatigue, headache Airvay infections, cough Hoarseness of voice, wheezing Nonspecific hyperreactivity reactions Nausea, dizziness TABLE 2. SUBJECT SELECTION CRITERIA Age: 18-40 years Race: Caucasion Gender: Male Non smokers No pre-existing diseases or abnormal conditions such as: *chronic airway disease *cardiac disease *Asthma *Chronic bronchitis *tuberculosis *Psychiatric disorder *Neurological disorder *Skin disease *Chronic eye disease *0besity *Cancer or other severe disease *Alcohol or Drug Addiction *IgE Atopy (negative skin test to common agents) Native language must be English Subjects must be able to perform tasks without difficulty Normal psychological profile as determined by MMPI 30 ------- TABLE 3. MOLHAVE et al (1986) VOC MIXTURE No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Compound n-Hexane n-Nonane n-Decane n-Undecane 1-Octene 1-Decene Cyclohexane 3-Xylene Ethylbenzene 1,2, 4-Trimethylbenzene n-Propylbenzene a-Pinene n-Pentanal n-Hexanal Iso-propanol n-Butanol 2-Butanone (MEK) 3-Methyl-3-Butanone 4-Methyl-2-Pentanone n-Butylacetate Ethoxyethylacetate 1,1-Dichloroe thane Ratio 1 1 1 0.1 0.01 1 0.1 10 1 0.1 0.1 1 0.1 1 0.1 1 0.1 0.1 0.1 10 1 1 cone. (ug/m3) 825 825 825 75 8 825 75 8250 825 75 75 825 75 825 75 825 75 75 75 8250 825 825 *Based on a mixture with a total concentration of 25 mg/m3 TABLE 4. EXPERIMENTAL DESIGN A. TREATMENT SESSION 1 SESSION 2 A 0 25 B 25 0 B. No. of Subjects in Chamber 1 2 3 4 Frequency of Occurrence during Testing 20 23 15 10 31 ------- TABLE 5. MEASUREMENT VARIABLES A. MAIN NES TESTS TEST MEASURE C/E* Finger Tapping Visual Digit Span Continuous Performance Test Symbol Digit Substitution Serial Digit Learning Pattern Memory Switching Attention Mood Scales flaps for preferred, non preferred, and alternating condition Total span length (forward+backward) Forward span length Backward span length Mean Reaction Time tMisses #False Alarms Mean msec/digit terrors ^trials attempted + score Mean msec/trial #correct Mean msec/side condition Mean msec/direction condition Mean msec/switching condition Tension Depression Anger Fatigue Confusion E E E E E E C E E E E E C E E E E E 32 ------- TABLE 5 (cont.) B. SECONDARY NES TESTS TEST MEASURE C/E Associate Learning Associate Recall Pattern Comparison Simple Reaction Time Grammatical Reasoning Horizontal Arithmetic # Correct (trials 1-3) # pairs correctly recalled mean latency of trials 2-25, excluding incorrect trials # trials correct mean RT for preferred hand (P2-P5) and non preferred hand (N6-N9), excluding block 1 mean response time # errors # correct mean response time E E E E E E E E C. OTHER TESTS Auditory Digit Span Comfort Scale (Potentiometer) Symptom Questionaire total span length (Fwd + Bwd) forward span length backward span length difference profile between VOC exposure and clean air difference between VOC exposure and clean air for individual items C E E E *C=Conformity, E=Exploratory Measures 33 ------- TABLE 6 MEAN POTENTIOMETER READING BY EXPOSURE AND TIME EXPOSURE AIR VOC TIME (MINUTES) 0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 MEAN* .048 .059 .234 .235 .235 .310 .321 .361 .362 .438 .461 .531 .467 .476 .586 .572 .570 .103 .106 .373 .382 .380 .570 .921 1.084 1.136 1.277 1.303 1.131 1.166 1.178 1.223 1.222 1.223 S.E. .017 .020 .045 .045 .045 .056 .055 .056 .056 .069 .070 .081 .077 .078 .097 .093 .094 .033 .033 .055 .054 .054 .074 .096 .100 .106 .117 .123 .115 .116 .118 .123 .131 .131 * N=63 Ho: F(16,47) = 3.98 (P<.001) ------- TABLE 7 QUESTIONNAIRE QUESTION 1. BODY/SKIN IRRITATION 2. NOISE LEVEL 3. EYES DRY / WATERY 4 . HEADACHE 5. FACIAL SKIN TEMPERATURE 6. SLUGGISHNESS 7. GDOR LEVEL 8. EYE IRRITATION 9. BODY TEMPERATURE 10. -I5HT INTENSITY 11. ZOUGHING TIME PRE MID POST PRE MID POST PRE MID POST PRE MID POST PRE MID POST PRE MID POST PRE MID POST PRE MID POST PRE MID POST PRE MID POST PRE MID : POST ft MEAN(tt) 1.77 3.35 5. 18 11.48 12.91 13.80 12.71 12.23 11.30 2.00 2.12 3.41 14.00 12.30 11.33 6.33 8.27 9.83 8.73 6.53 0.68 2.56 5.21 7.59 14.03 11.05 10.05 17.11 17.35 18.08 1.38 3.94 1.05 tIR S.E. 0.51 0.73 0.80 0.76 0.82 0.89 0.36 0.45 0.56 0.61 0.53 0.76 0.37 0.55 0.63 0.85 0.95 1.09 0.95 0.89 0.91 0.57 0.87 0.97 0.46 0.56 0.66. 0.44 0.48 3. 55 3.41 0.32 0.37 V MEAN ( * ) 1.73 5.26 6.39 10.88 13.45 14.09 13.30 12.39 11.24 1.64 5.05 6.97 14.53 12.82 13.21 5.70 8.70 11.71 B. 96 21.68 17.77 3.70 9.68 10.71 13.86 11.58 11.55 17.44 17.68 18.42 1.1* 2.06 1.82 OC S.E. 0.48 0.90 0.92 0.75 0.81 0.88 0.34 0.59 0.60 0.48 0.82 1.04 0.39 0.63 0.60 0.84 0.94 1.10 0.99 0.80 1.09 0.76 1.04 1.13 0.49 0.62 0.64 0.50 a. 52 : 0.53 : 13.41 : 3.57 : 0.55 F« (P) 1.71 ( .190) 1.37 ( .261) 0.47 ( .626) 9.52 ( < . 001 ) 2.05 (.138) 2.42 C . 098 ) 77.90 «.001) 4.13 (.021) 1.84 ( .167) 0.00 ( . 999 ) : 2.31 ( . 108 ) ; * N « •66 & 64 Degre< es of Freedom 35 ------- TABLE 7 (Can t) QUESTIONNAIRE QUESTION 12. FACE IRRITATION 13. HUMIDITY 14. AIR MOVEMENT 15. NOSE IRRITATION 16. TEMPERATURE 17. TIRED SLEEPY IB. SKIN. BODY no IST /DRY 19. THROAT IRRITATION 20. AIR QUALITY 21. ^ACIAL SKIN MOIST/DRY 22. CONCENTRATION TIME PRE MID POST PRE MID POST PRE MID POST PRE MID POST PRE MID POST PRE MID POST PRE MID POST PRE MID POST PRE MID POST PRE MID POST PRE MID POST f. MEAN(*») ! 1.70 3.35 4.55 11.62 11.38 11.44 17.86 19.79 20.09 4.33 5.26 6.85 10.82 8.61 7.70 6.97 8.79 10.45 12. 88 12.03 11.76 2.48 3.92 4.45 11.00 11.59 11.52 12.65 12.23 11.86 2.42 2.55 3.20 IIR S.E. 0.46 0.76 0.84 0.56 0.61 0.65 0.67 0.59 0.62 0.80 0.89 1.03 0.42 0.47 0.53 0.89 1.02 1.02 0.37 13.60 13.56 0.60 0.79 0.92 0.74 0.80 0.87 0.51 0.56 0.59 0.59 0.55 0.66 V, MEAN ( * ) 2.36 4.33 5.17 12.24 12.50 12.24 17.88 19.41 20.23 5.12 7.77 9.41 10.47 8.36 7.95 5.98 8.08 12.15 13.02 12.00 12.24 1.71 5.65 6.18 10.74 19.98 18.36 12.45 12.17 11.53 2.26 3.42 4.03 'DC S.E. 0.56 0.79 0.86 0.54 0.62 0.60 0.65 0.62 0.57 0.85 0.97 1.11 0.35 0.46 0.44 0.90 0.91 1.05 0.42 0.42 0.47 0.46 0.94 1.00 0.77 0.70 0.80 0.49 0.52 (3.56 0.56 0.69 0.75 F* (P) 0.08 ( . 924 ) 0.24 ( .784) 0.50 ( .612) 1.16 ( .319) 0.60 ( .550) 4.38 ( .017) 3.36 i .696) 4.41 ( .016) 32.86 ( <.001) 0.08 ( . 922 5 1.75 ( .183) « 2 4 e>4 Degrees of 36 ------- TABLE 8 ANALYSIS OF QUESTIONS 4, 7, 8, 17, 19, AND 20 QUESTION 4 . HEADACHE 7. ODOR LEVEL 8. EYE IRRITATION 17. TIRED / SLEEPY 19. THROAT IRRITATION 20. AIR QUALITY CONTRAST (*) 1 2 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 CONTRAST MEAN 3.29 3.92 0.63 15.02 10.96 -4.06 3.33 1.98 -1.35 0.28 2.69 2.41 2.50 2.50 0.00 8.65 7.10 -1.55 F(*«) 16.04 15.99 0.70 153.16 79.04 12.79 8.24 2.34 1.01 0.05 4.65 7.80 6.73 6.84 0.00 66.47 39.92 5.30 P <.001 <.001 0.406 <.001 <.001 <.001 0.006 0.131 0.318 0.815 0.035 0.007 0.012 0.011 0.999 <.001 <.001 0.024 *1: Change to mid on VOC - Change to mid on AIR 2: Change to post on VOC - Change to post on AIR 3: Contrast 2 - Contrast 1 ** 1 and 65 Degrees of Freedom 37 ------- TABLE 9 QUESTIONNAIRE (YES-NO QUESTIONS) QUESTION 23 24 25 TIME PRE MID POST PRE MID POST PRE MID POST (• PROP. "YES" 0.95 0.92 0.80 0.79 0.73 0.74 0.35 0.3B 0.36 >IR 5.E. 0.03 0.03 0.05 0.05 0.06 0.05 0.06 0.06 0.06 V PROP. "YES" 0.97 0.62 0.59 0.79 0.09 0.23 0.33 0.83 0.73 /DC S.E. 0.02 0.06 0.06 0.05 0.04 0.05 0.06 0.05 0.06 F* (P) 15.19 «.001) 44.68 «.001) 22.35 ( <.001 ) # N=66 t 2 & 64 Degrees of Freedom TABLE 9A QUESTIONNAIRE (YES-NO QUESTIONS) : QUEST I ON i : 23 1 : 24 1 25 CONTRAST (*) 1 2 3 1 2 3 1 2 3 CONTRAST MEAN -0.32 -0.23 0.09 -0.64 -0.51 0.13 0.47 0.39 -0.08 F(«> 26.62 12.60 1.51 90.14 50.63 4.84 42.18 28.59 2.03 P <.001 <.001 0.223 <.001 <.001 0.031 <.001 < .001 0.159 *1: Change to mid on VOC - Change to mid on AIR 2: Change to post on VOC - Change to cost on AIR 3: Contrast 2 - Contrast 1 ** 1 and 65 Degrees of Freedom 38 ------- TABLE 10 AUDITORY DIGIT SPAN (WITHOUT 12 SUBJECTS) TEST FORWARD (STANDARD) BACKWARD (STANDARD) TOTAL (STANDARD) FORWARD (WEIGHTED) BACKWARD (WEIGHTED) TOTAL (WEIGHTED) TRT AIR VOC AIR VOC 5 S5SS^SS AIR VOC AIR VOC AIR VOC AIR VOC TIME PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST MEAN* 7.85 8.09 7.89 8.09 7.43 7.24 7.22 7.54 15.28 15.33 15.11 15.63 60.61 62.74 60.96 61.91 53.43 54.43 53.81 58.59 114.04 117.17 114.80 120.50 S.E. 0.14 0.13 0.15 0.14 0.11 0.15 0.13 0.11 0.22 0.22 0.24 0.21 1.99 1.71 1.95 2.04 1.61 1.73 1.74 1.69 3.31 2.89 3.32 3.14 CHANGE (POST -PRE) 0.24 0.20 -0.19 0.31 0.06 0.52 2.13 0.94 1.00 4.78 3.13 5.70 P-value ** 0.869 0.028 0.132 0.638 3.096 0.462 * N=54 for all means ** Ho: Change on VOC - Change on AIR = 0 39 ------- TABLE 11 AUDITORY DIBIT SPAN (ALL SUBJECTS) TEST FORWARD ( STANDARD ) BACKWARD (STANDARD) TOTAL ( STANDARD ) FORWARD (WEIGHTED) BACKWARD (WEIGHTED) TOTAL (WEIGHTED) TRT AIR VOC AIR voc AIR VOC AIR VOC AIR VOC AIR voc TIME PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST MEAN* 7.80 8.11 7.86 8.03 7.17 7.17 7.14 7.38 14.97 15.27 15.00 15.41 59.95 62.47 60.62 61.58 50.95 50.20 52.52 54.47 110.91 115.67 113.15 119.05 S.E. 0.13 0.12 0.13 0.13 0.15 0.13 0.13 0.14 0.24 0.20 0.22 0.22 1.72 1.54 1.76 1.90 1.82 1.68 1.58 1.65 3.20 2.69 3.02 3.00 CHANGE (POST -PRE) 0.30 0.17 0.00 0.24 0.30 0.41 2.52 0.95 2.24 4.95 4.76 5.89 P-value ** 0.503 0.259 0.721 0.481 0.208 0.715 * N=66 for all means *» Ho: Change on VOC - Change on AIR = 0 40 ------- TABLE 12 'INGER TAPPING CHANGE NUMBER TAPS 'REFERRE HAND) ; NUMBER ITAPS (NON-: I PREFERRED HAND) NUMBER : TAPE : (ALTER- : NAT ING) TRT , AIR 1 : voc ; : AIR ! : VOC TIME PRE POST PRE POST PRE POST PRE POET MEAN* 116.6 113.3 114. 1 113.5 106.3 102.7 104.7 102.1 5 . E • 1.9 1 .9 1.8 1.9 1.7 1.4 1.5 1 e (POST -PRE ) -3.3 -0.6 -3.6 -2.6 P— vai ue x* 0.213 0.396 AIR VOC PRE POET PRE POST 101.0 99.9 102.4 98. 9 * . c 1.9 -1 .1 -3.5 * N=66 for all means *» Ho: Change on VOC - Change on AIR = 0 TABLE 13 VISUAL DIGIT SPAN TEST LONGEST FORWARD SPAN LENGTH g=^^»^sg?~ •— -• LONGEST BACKWARD SPAN : LENGTH TOTAL TRT AIR VOC B*^^B«v^e^*e ^V^^^E^Bfi AIR voc AIR TIME PRE POST PRE POST PRE POST PRE POST PRE POST MEAN* 7.68 8.20 7.97 8.06 7.53 7.62 7.59 7.83 15.21 15.62 S.E. 0.17 0.16 0.17 0.16 0.21 0.20 0.21 0.21 0.34 0.32 CHANGE (POST -PRE) 0.52 0.09 0.0" • 0.24 0 . 61 P-value XX 0.030 0.512 VOC PRE POS" 15.56 15.8* E.34 0.41e « N=do *or all means ** no: Change on VOC - Change on .'SIR = C ------- TABLE 14 CONTINUOUS PERFORMANCE TASK TEST MEAN REACTION TIME TOTAL OMISSION ERRORS ============ TOTAL FALSE POSITIVES TRT AIR VOC AIR VOC ======= AIR VOC TIME PRE POST PRE POST PRE POST PRE POST EtEssrsass PRE POST PRE POST MEAN* 376.3 380.4 375.5 383.7 0.26 0.48 0.26 0.35 ========== 0.91 0.88 0.74 1.20 5 . E . 4.23 4.08 4.14 4.37 0.11 0.14 0.08 0.09 ======== 0.14 0.19 0.14 0.15 CHANGE (POST -PRE) 4.1 8.2 0.22 0.09 ========== -0.03 0.46 P- value ** 0.156 0.406 ========= 0.061 * N=66 for all means »> Ho: Change on VOC - Change on AIR = 0 TABLE 15 SYMBOL-DIGIT SUBSTITUTION TEST MEAN NUMBER CORRECT as • SB • ' ' I sssssssa MEAN TIME ; TRT AIR VOC AIR VOC TIME ESSSSSSS& PRE POST PRE POST PRE POST PRE POST MEAN* .028 .027 .034 .029 15.05 14.61 14.94 14.50 S.E. '35^5 XS SS ^E ^5 S .008 .009 .007 .009 0.27 0.27 0.24 0.25 CHANGE (POST -PRE) -0.001 -0.005 -0.44 " -0.44 P-value ** 0.812 0.954 * N=66 for all means ** Ho: Change on VOC - Change on AIR = 0 42 ------- 10 TABLE 16 SERIAL DIGIT LEARNING TEST SCORE SCORE -»• NUMBER OF TRIALS TRT AIR VOC AIR VOC TIME PRE POST PRE POST PRE POST PRE POST MEAN* 2.55 2.48 2.70 2.89 6.45 6.30 6.80 7.06 S.E. 0.2B 0.34 0.27 0.34 0.44 0.53 0.45 0.52 CHANGE (POST -PRE) -0.07 0.19 -0.15 0.26 ========== P-value X* 0.54B 0.539 * N=66 for all means ** Ho: Change on VOC - Change on AIR = 0 TABLE 17 PATTERN MEMORY TEST LATENCY (ALL TRIALS) LATENCY ( CORRECT TRIALS ONLY) NUMBER CORRECT TRT AIR VOC AIR VOC AIR VOC TIME PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST MEAN* 5.21 4.29 5.08 4.31 4. 98 4.02 4. 82 4.08 23.3 22.7 23.2 22.7 S.E. 0.1B 0.14 0.16 0.14 0.17 0.13 0.15 0.14 0.20 0.23 0.17 0.24 CHANGE (POST -PRE) -0.92 -0.77 -0.96 -0.74 -0.6 -0. 5 P-value ** 0.359 0.157 0.B56 * N=66 for all means ** Ho: Change on VOC Change on AIR = 0 43 ------- 11 TABLE 18 SWITCHING ATTENTION TEST TEST SIDE CONDITION DIRECTION CONDITION SWITCHING CONDITION SWITCHING- SIDE COMPAT . SWITCHING- SIDE INCOMPAT. SWITCHING DIRECTION COMPAT . SWITCHING DIRECTION INCOMPAT. TRT .AIR VOC AIR VOC AIR VOC AIR VOC AIR VOC AIR VOC AIR VOC TIME PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST MEAN* 319.5 315.7 320.3 321.1 432.2 428. 0 430.9 419.9 489.3 474.9 485.9 483.4 426.4 416.4 410.6 414.6 444.1 427.5 446.0 438.7 550.4 537.0 551.5 553.7 536.1 518.8 535.6 526.8 S.E. 6.2 7.1 6.6 6.7 6.9 8.4 7.7 7.7 13. B 13.8 16.6 15.4 14.7 14.6 14.5 15.0 15.4 17.0 22.1 18.8 14.0 13.6 17.6 16.9 13.8 13.2 15.6 14.4 CHANGE (POST -PRE) -3.B 0.8 -4.2 -11.0 -14.4 -2.5 -10.0 4.0 -16.6 -7.3 -13.4 2.2 -17.3 -8.8 P-value 0.611 0.473 0.341 0.337 0.561 0.321 0.532 * N=66 for all means ** Ho: Change on VOC - Change on AIR = 0 ------- 12 TABLE 19 MOOD SCALE ============ TEST TENSION SCORE DEPRESSION SCORE ANGER SCORE FATIGUE SCORE CONFUSION SCORE TRT AIR VOC AIR VOC AIR VOC AIR VOC AIR VOC TIME PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST PRE POST MEAN* 1.97 1.89 1.94 1.98 1.57 1.58 1.50 1.57 1.18 1.20 1.23 1.22 2.68 2.75 2.58 2.82 1.94 1.97 1.93 2.10 5 .E . 0.07 0.06 0.08 0.08 0.05 0.06 0.05 0.05 0.04 0.05 0.06 0.04 0.09 0.09 0.09 0.08 0.06 0.06 0.07 0.08 CHANGE (POST -PRE) -0.08 0.04 0.01 0.07 0.02 -0.01 0.07 0.24 0.03 0.17 P-vai ue 0 . 1 53 0.197 0.722 0.045 0.026 * N=66 for all means *» Ho: Change on VOC - Change on AIR 0 ------- 13 TABLE 20 ASSOCIATIVE LEARNING TEST TOTAL CORRECT RECALL TRT AIR VOC AIR VOC MEAN* 21.15 22.06 7.94 8.02 S.E. 0.62 0.55 0.17 0.21 DIFF- ERENCE: VOC-AIR 0.91 0.0B V ^HB ^ ^ ^^ •« P-value X* 0.132 0.747 * N=66 for all means «» Ho: VOC - AIR = 0 TABLE 21 PATTERN COMPARISON TEST NUMBER TRIALS CORRECT LATENCY (ALL TRIALS) LATENCY ( CORRECT : ONLY ) TRT AIR VOC AIR VOC AIR VOC MEAN* 24.80 24.85 3.07 3.07 3.07 3.07 S.E. 0.06 0.05 0.09 0.08 0.09 0.08 DIFF- ; ERENCE: IP-value VOC-AIR I »* 0.05 ! 0.581 t 0*00 : 0.968 0.00 : 0.969 * N=fa6 for all means ** Ho: VOC - AIR = 0 46 ------- 14 TABLE 22 SIMPLE REACTION TIME TEST REACTION TIME (PREFERRED HAND) REACTION TIME (NON- PREFERRED HAND) TRT AIR VOC AIR VDC MEANX 244.3 246.4 253.7 254.3 S . E . 3.38 3.41 3.91 3.54 : DIFF- ERENCE: VOC-AIR 2.1 p-value x» 0.455 0.6 0.862 * N=66 for all means *« Ho: VOC - AIR = 0 TABLE 23 GRAMMATICAL REASONING TEST MEAN RESPONSE TIME SUM OF ERRORS SCORE TRT AIR VOC AIR VOC AIR VOC MEAN* 3396 3375 6.7 6.2 : 54.5 : 54 . o : DIFF- S.E. 1ERENCE: ! VOC-AIR 96 I 101 : . -21 0.9 : 0.9 : -0.5 1 t ; : l.o. 0.4 ._______. P-value IX 0.737 0.4B2 : 0.694 * N=66 for all means xx HO: VOC - AIR = 0 47 ------- 15 TABLE 24 HORIZONTAL ADDITION TEST NUMBER CORRECT RESPONSE TIME (ALL TRIALS) =======:=== RESPONSE TIME (COR- RECT ONLY) TRT AIR voc AIR VOC AIR voc EAN* 58.5 56.9 3.26 3.25 3.25 3.24 S.E. 0.18 0.11 0.09 0.0B 0.09 0.08 DIFF- ERENCE: VOC-AIR 0.4 -0.01 -0.01 P— va lue ** 0.031 0.766 0.806 * N=66 for all means ** Ho: VOC - AIR = 0 ------- 16 TABLE 25 AUDITORY DIGIT SPAN (ALL SUBJECTS) TEST FORWARD ( STANDARD ) BACKWARD ( STANDARD ) TOTAL ( STANDARD ) FORWARD (WEIGHTED) BACKWARD (WEIGHTED) TOTAL (WEIGHTED) ORDER 1 2 3 4 1 1 ^ 3 4 i 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 MEAN* 7.74 8.06 7.92 8.08 7.03 7.11 7.27 7.44 14.77 15.17 15.20 15.52 58.76 61.64 61.82 62.41 50.73 54.32 52.74 56.35 109.50 115.95 114.56 118.76 S.E. i CONTRAST i 0.13 ! 0.13 ILINEAR 0.13 ! QUADRAT 1C 0.12 JCUBIC 0.15 ! 0.14 iLINEAR 0.13 ! QUADRAT 1C 0.13 ICUBIC 0.24 ! 0.22 ILINEAR 0.21 ! QUADRAT 1C 0.19 ICUBIC 1.68 ! 1.82 ILINEAR 1.77 I QUADRAT 1C 1.64 1 CUBIC 1.80 ! 1.64 ILINEAR 1.60 1 QUADRAT 1C 1.72 ICUBIC 3.22 ; 2.94 ILINEAR 2.96 [QUADRATIC 2.75 ICUBIC P-value 0.042 0.379 0.081 0.002 0.644 0.836 <.001 0.788 0.301 0.022 0.291 0.521 0.002 0.994 0.036 <.001 0.465 0.053 N=66 for all means 49 ------- 17 TABLE 26 VISUAL DIGIT SPAN TEST LONGEST FORWARD SPAN LENGTH LONGEST BACKWARD SPAN LENGTH TOTAL 1 ORDER : 1 > 4U • 3 : 4 : 1 : 2. • 3 : 4 1 <^ : 3 : 4 MEAN* 7.55 8.00 B.li 6.26 7.26 7.71 7.B6 7.74 i4.ee 15.71 15.97 16.00 5 .£ . 0.17 0.15 0.17 0.17 0.21 0.20 0.20 0.21 0.34 0.31 0.32 0.33 ; CONTRAST ! LINEAR ! QUADRAT I C ! CUBIC J I LI NEAR ! QUADRAT 1C i CUBIC I LINEAR ! QUADRAT 1C .'CUBIC P-vaiue <.001 0.129 0.377 0.005 0.024 0.957 <.001 0.007 0.556 N=66 for all means TABLE 27 PATTERN MEMORY TEST LATENCY (ALL TRIALS) LATENCY ( CORRECT TRIALS ONLY) NUMBER CORRECT ORDER 1 2 3 4 1 2 3 4 i _j" T ', ** MEAN* 5.73 4. SB 4.56 4.02 5.47 4.25 4.33 3.84 23.1 22.2 23.4 23.1 S.E. 0.17 0.16 0.14 0.12 0.16 0.14 0.14 0.12 0.20 0.23 0.17 : 0.23 CONTRAST LINEAR QUADRATIC CUBIC LINEAR QUADRATIC CUBIC LINEAR QUADRAT I C CUBIC P— value : <.00l : <.001 : <.00l : <.001 : <.001 : <.00i ; 0.10B • 0.053 X .001 for ail means 50 ------- TABLE 28. SERIAL ORDER/PRACTICE EFFECTS (p-Values) A. NES MAIN TESTS 1. 2. 3. 4. 5. 7. 8. TEST Finger Tapping Visual Digit Span Continuous Performance Test Symbol Digit Substitution Serial Digit Learning Pattern Memory Switching Attention Mood Scales MEASURE Preferred Hand Nonpreferred Alternating Fvd Span Bvd Span Total React ion- time Omissions False Positives Correct Response Time Score Score + tt trials Latency (all trls) Latency (corr. trls) Side Direction Switching Sw. Side Compat. Sw. Side Incomp. Sw. Dir. Compat. Sw. Dir. Incomp. Tension Depression Anger Fatigue Confusion LINEAR NS NS NS <.001 0.005 <.001 0.024 0.087 0.012 0.090 <.001 NS NS <.001 <.001 (0.085) NS <.001 <.001 0.004 <-001 <.001 0.002 NS NS NS NS QUADRATIC NS NS NS NS 0.024 0.007 NS NS NS NS NS (0.102) NS <.001 <.001 NS NS NS (0.065) NS NS (0.072) (0.089) NS NS NS NS CUBIC 0.014 <.001 0.009 NS NS NS 0.003 NS NS NS NS NS NS <.001 <-001 NS NS NS (0.097) NS NS NS NS (0.062) NS 0.004 0.02 51 ------- Table 28 (cont.) B. AUDITORY DIGIT SPAN TEST Forward (Std) Backward (Std) Total (Std) Forward (Wtd) Backward (Wtd) Total (Wtd) 0.042 0.002 <.001 0.022 0.002 <.001 NS NS NS NS NS NS (0.081) NS NS NS 0.036 (0.053) C. NES SECONDARY TESTS DIFFERENCE (ORDER 2 - ORDER 1) 1. Associate # Correct 0.022 Learning Recall NS 2. Pattern # Correct (0.095) Comparison Latency (All) <.001 Latency (Corr. trls) <.001 3. Simple Preferred Hand NS Reaction-Time Nonpreferred NS 4. Syntactic Mean Response Time <.001 Reasoning f Errors <.001 Score 0.001 5. Horizontal # Correct NS Arithmetic Response Time (All) NS Response Time (Corr.) NS 52 ------- FIGURE 1 SCHEMATIC OF TEST PROTOCOL COMFORT RATING SCALE SYMPTOM QUESTIONNAIRE NES MAIN BATTERY AUDITORY DIGIT SPAN NES FILLER TESTS PRE-EXPOSURE BUILDUP CONSTANT EXPOSURE I I I I I I I I I I I I I 0 60 120 180 Time Expired (Minutes) 240 ------- FIGURE 2 COMFORT RATING SCALE RESULTS 1.4 r Ln AIR VOC 60 90 120 150 TIME (minutes) 180 210 240 ------- FIGURE 3 HKADACHE, ODOR & AIR QUALITY Ln 24 20 ^D ID II 16 12 8 O o CO < LJ 0 '— AIR VOC HEADACHE AIR VOC ODOR AIR VOC AIR QUALITY ------- FIGURE 4 ODOR, EYE & THROAT IRRITATION cr» AIR VOC THROAT VOC EYE AIR VOC ODOR ------- FIGURE 5 CONTROL QUESTION RESPONSES Ln 0 AIR VOC NOISE AIR VOC LIGHT AIR VOC TEMPERATURE ------- FIGURE 6 YES-NO QUESTION RESPONSES "DO YOU FEEL NOW THAT STAYING to IN THE CHAMBER IS COMFORTABLE?" | ,i . . y I ,f !"^~ &J t £UJ- O Mr- O r O 014. § ' t &3»- O g - fe oL K PRE MID POST EXPOSURE PERIOD "WOULD YOU BE SATISFIED WITH THIS « AIR QUALITY IN YOUR OWN HOME?" «f -^ _ . JS. f -^— . • , a. : ~ _ i PRE MID POST EXPOSURE PERIOD "WOULD YOU VENTILATE YOUR HOME MORE?" to W 1, . « 1 ! < OJf r *.__ I VDC PRE MID POST EXPOSURE^gERIOD ------- FIGURE 7 AUDITORY DIGIT SPAN Ln vo AIR VOC PRE POST FORWARD PRE POST BACKWARD PRE POST TOTAL ------- FIGURE 8 VISUAL DIGIT SPAN PRE POST FORWARD PRE POST BACKWARD PRE POST TOTAL ------- FIGURE 9 CONTINUOUS PERFORMANCE TASK 400 MEAN REACTION TIME voc I o.u TOTAL OMISSION ERRORS VOC TOTAL FALSE POSITIVES ------- FIGURE 10 SWITCHING ATTENTION TEST UJ 600 550 500 450 UJ en z CL 400 cn UJ CE 350 300 -G -A PRE POST L_ PRE -• . J_-_ POST 600 550 500 u Q) UJ 450 £ UJ en 400 a. in UJ tr 350 300 SIDE DIRECTION SNITCHING SHITCHING-SIDE COMPATIBLE SHITCHING-SIDE INCOMPATIBLE SWITCHING-DIRECTION COMPATIBLE SWITCHING-DIRECTION INCOMPATIBLE AIR VOC ------- FIGURE 11 MOOD SCALE DIFFERENCE SCORES ON U> LiJ O ^ X O bJ OH Q- Z> H- CO O Q. 0.30 0.25 0.20 0.15 0.10 0.05 0.00 -0.05 r\ 1 n - - - - . AIR VOC TENSION DEPRESSION ANGER (p=.153) (p=-197) (p=.722) EATIGUE CONEUSION (p=.045) (p=.026) ------- 12 16.5 16 15.5 TOTAL FORWARD BACKWARD 15 (O CO FIGURE 12 SERIAL ORDER EFFECTS AUDITORY DIGIT SPAN b: o Q 7.5 6.5 -j- _i_ 2 3 ORDER ------- 13 16.5 16 - 15.5 15 CO co 2 H.5X '*^ fc O o 7.5 6,5 FIGURE 13 SERIAL ORDER EFFECTS VISUAL DIGIT SPAN ORDER 65 ------- UJ UJ 2.06 2.04 2.02 2.00 CL 1.98 (/I LU ^ 1.96 1.94 1.92 1.90 CONFUSION FIGURE 14 SERIAL ORDER EFFECTS CONFUSION SCALE CONTRAST p-VALUE LINEAR QUADRATIC CUBIC 0.639 0.761 0.027 L- 3 4 SERIAL ORDER ------- FIGURE 15 SERIAL ORDER EFFECTS PATTERN MEMORY 6.00 Ld O CL CO 5.50 - 5.00 4.50 4.00 3.50 CONTRAST LINEAR QUADRATIC CUBIC p-VALUE < 0.001 < 0.001 < 0.001 SERIAL ORDER ------- APPENDIX A DESIGN AND PERFORMANCE OF A SYSTEM TO CONTROL VOC CONCENTRATIONS WITHIN AN ENVIRONMENTAL LABORATORY USED FOR HUMAN EXPOSURE Jon Berntsen C-E Environmental, Inc. Chapel Hill, NC 27514 INTRODUCTION The Environmental Protection Agency's Clinical Environmental Laboratory (CEL) on the campus of the University of North Carolina at Chapel Hill, is used to provide human exposure data which can withstand vigorous technical and legal 3 3 challenge. This facility consists of of two 84 m (3000 ft ) environmental laboratories which are used to expose human volunteer subjects to constant or varying concentrations of 03> N02, S02, CO and water soluble aerosols. The pollutant delivery system and the aerosol delivery system are under computer control. The computers are used to monitor the concentration of the pollutants, control the pollutant delivery systems, validate the data as it is taken, alarm the operator of unsafe conditions and record the data for later use. These chambers have been in continuous use since 1977 exposing subjects to pollutants and measuring responses with a computerized Physiological Data Acquisition System. The EPA's interest in indoor air pollution and the effects of these pollutants on humans has expanded the requirements of the facility. To enable the exposure of humans to carefully controlled concentrations of Volatile Organic Compounds (VOCs), the pollutant delivery system was modified to allow injection and monitoring of the VOC compounds in one of the chambers. The following sections discuss the design and operation of the VOC generation and monitoring system. 68 ------- SYSTEM DESIGN Existing System Constraints The VOC monitoring and delivery system was designed to be compatible with the existing pollutant control system and chambers. In contrast to other chambers, these chambers recirculate air with a flow of 3.8 mVsec (8000 ftVmin) through the main loop. Fresh (make-up) air is provided at a rate of 0.56 m3/sec (1200 ftVmin) to replace the same amount of recirculating air exhausted through a purge damper, leaks or the chamber door when opened. The total volume of the system, chamber and associated duct work, has been calculated to be 175 m3 (6180 ft3) with a time constant of about 5 minutes for exchanging 67K of the chamber air. For the purpose of controlling a pollutant gas concentration, the chambers behave as a one pass chamber with a total volume of 175 m3 and a flow rate of 0.56 m3/sec except for the amount of pollutant needed to bring the concentration to the desired value. Due to space constraints, the VOC generator was connected to the aerosol generator. The aerosol generator uses .38 mVsec (800 ftVmin) for sweep air to dry and remove the aerosol particles from the generator. This air also moves aerosols from the generator into the main air duct where the aerosols are mixed with chamber air. This sweep air and the aerosol delivery ducts are used by the VOC generator. The chamber design is shown in Figure 1. This figure shows recirculated air through the main chamber air loop, the fan and bypass to force air through the VOC generator, the location of the makeup air injection point and the location of the exhaust air outlet. VOC Generator Design Considerations The VOC generator was designed to meet a variety of stringent requirements. 69 ------- The generator had to deliver precisely controlled amounts of a vaporized mixture of highly flammable VOC's to the chamber. The amount of vapor had to be automatically controlled by an existing computer and associated system. Since the mixture contains compounds with boiling points between 69 °C and 181 °C, the generator must vaporize all constituents without changing the relative concentrations of any components. The generator must also vaporize the components with a minimum of chemical reactions so that only the components in the mixture are in the chamber without generating impurities. Since VOCs are flammable, the generator must minimize the danger of explosion or fire. The other major factor is the chamber design. Since the chamber is multipass with large internal volume and a relatively low air exchange rate, the generator uses an on-off type of control instead of proportional control. Since the design of the existing system has a 30 second control cycle, the VOC generator also incorporates this 30 second controlling period. Thus the VOC concentration can be controlled at a steady concentration by turning the solenoid valve on at the start of the 30 second period and off sometime in the middle of the period. Since the computer reports 2 minute averages, the effects of this on and off control are further minimized. VOC Generator Design The VOC generator (Figure 2) is a modification of a design by Molhave, which was scaled up and changed to meet increased flow requirements. The mixture of hydrocarbons is placed in an Erlemeyer flask. The top of the flask has been modified to accept a screw type fitting. This fitting allows the connection of a N» pressure line and a mixture delivery line. A Fairchild Model 81 precision pressure regulator is used to control the pressure over the liquid mixture. This pressure was held at 3 psig for these tests but can be varied to control the liquid feed rate. A dip tube which almost touches the bottom of the flask removes liquid from the flask. The flask is placed on a magnetic stirrer and agitated throughout the run to keep the mixture homogeneous. The mixture delivery tube was connected through a T-connection to a Nupro 70 ------- SS-SS2-KZ fine metering valve. The metering valve vas fitted with Kalrez and Teflon seals. The other end of the T-connection was connected to a tube several feet long. This tube was uncapped after the mixture was pressurized to allow the mixture to fill the delivery tube. This tube was securely capped after the delivery tube was filled. The outlet of the metering valve was connected to the inlet of a Sierra Model 840VO-020 solenoid valve. The valve has a 0.02 inch orifice with Kalrez seals. The outlet of the solenoid valve was connected to the (evaporator) through a sprayer. This sprayer was constructed of two concentric tubes. The inner capillary tube allowed the mixture to enter the evaporation container. About 0.05 liters/sec (0.1 CFM) of nitrogen was passed through the outer tube. The flow of nitrogen helped spray the mixture on the bottom of the evaporation container and also forced the vapor out of the container. The evaporator is made of glass and during operation is placed in a large brass block (15 cm in diameter and 15 cm high). The evaporator is about 8 cm in diameter and 10 cm high. A fritted glass disk separates the evaporator into two. sections. The fritted glass keeps any liquid from moving from the bottom section to the top section. An arm on one side of the bottom section allows access for the sprayer. The angle of this arm allows the VOC mixture to be sprayed on the bottom of the container. Another arm, sealed at the bottom and almost touching the bottom of the container, contains a thermocouple (Omega Model C03-E) probe. The large brass block contains six 250 V cartridge heaters (McMaster-Carr P/N 3618K615) which were controlled by an Omega Model CN5001E1 proportional controller. For this mixture of compounds, the block was heated to 250 °C. After the VOC mixture has evaporated, the nitrogen forces it through the fritted glass into the top section. Here the vapor is diluted with another 0.5 liters/sec (1 CFM) of nitrogen. The flow of nitrogen was measured and controlled with an Omega Model FM064-63ST flowmeter. The concentrated VOC vapor is then diluted with about .02 m3/s (40 CFM) of air from the chamber. This air cools the mixture and dilutes the VOC concentration below the lower explosive limit of the compounds. A vertical 4 inch glass pipe 5 feet long allows any liquid to condense and run down the glass 71 ------- into a trap. After passing through this condenser, the VOC mixture is then diluted with about .36 m3/s (760 CFM) of the chamber air. This air is then center injected into the main air duct where it is further diluted and mixed with main recirculated air and passed through the chamber. The use of nitrogen to dilute the VOC vapor and to force it out of the evaporator reduces the explosion and fire risk, prevents the oxidation of VOCs and minimizes the time for reactions caused by the heat required to evaporate the compounds. VOC Monitoring and Control System The VOC concentration in the chambers is monitored by two GowMac 23-500R total hydrocarbon analyzers. These analyzers use a flame ionization detector to monitor the hydrocarbon concentration in the chamber. The analyzers sample from a 3 inch glass sample manifold used by all of the gaseous pollutant analyzers in the facility. This manifold draws a sample through an inverted glass funnel. The opening of this funnel is 3 meters from the floor and in the center of the chamber ceiling. The manifold exits the chamber near the ceiling of the chamber and passes over the back top of the instrument racks containing the gas analyzers. Chamber air is pushed through the sample manifold by the 0.1 inch of water positive pressure in the chamber and two sample manifold exhaust blowers. The flow though the sample manifold is 0.2 m3/s. Each analyzer has a tap consisting of a I/A inch teflon line inserted into the glass pipe so the opening is in the middle of the glass pipe. For the hydrocarbon analyzers a Mace Model 800-2334-2-0 solenoid valve is used to allow the analyzers to sample from the sample manifold or a calibration manifold. The sample is pushed into the analyzer with a Thomas Model 107CA14TFEL pump with teflon coated piston, valves and cylinder. A PDF 11/35 computer measures the output of each analyzer once per second. These samples are used to generate 2 minute averages which are reported to 72 ------- investigators as the chamber concentration. For control purposes, the computer samples the concentration in the chamber every 30 seconds and determines the percent on time for the VOC valve accordingly. This value is converted to a voltage and sent to the VOC control card. Depending on the state of the system, this card uses a voltage from a potentiometer in the manual mode or the voltage from the computer in the automatic mode, to control the duty cycle of the valve. This allows either automatic or manual control of the generator. In the automatic mode, another signal from the computer forces the card to synchronize with the computer. System Calibration The VOC concentration in the Molhave study was 25 mg/m3 toluene equivalent. Molhave calibrated FID analyzers with toluene and controlled the VOC concentration with instrument output at the same level obtained with the toluene calibration standard. The 25 mg/m3 was converted to 7 ppm toluene equivalent for these studies. The VOC analyzers were calibrated with toluene and the concentrations reported in terms of toluene. A standard of toluene diluted with nitrogen was diluted to the various concentrations with ultra high purity zero air with the dilution calibration system which has been used for 12 years. This system is automatically controlled by the computer which after the calibration has been specified by the operator, automatically calibrates the analyzers. The standard calibration consisted of three span points (5, 7 and 9 ppm) and a zero point. The zero was taken first, then the highest point was taken twice with the first one thrown out. System Operation The following procedure was used to run a session of the protocol. 1. The instruments were calibrated the morning of the protocol using the method given above. 2. A new solution was mixed up. Several safeguards were followed to assure that the mixture was made correctly. The operator used a check sheet to remove a bottle of each of the chemicals from a storage cabinet. Each chemical was pipetted using its own premarked pipette 73 ------- (both with chemical name and the volumne) from the bottle to the flask. As this was done, the chemical was checked off on the check sheet. The bottle was then placed back in storage. 3. After the mixture was prepared, it vas connected to the system. A. The generation system vas turned on and heated to the proper temperature during instrument calibration and chemical mixing. 5. The results of the calibration were then checked and the new calibration constants loaded into the computer. 6. The computer vas instructed to monitor the VOC concentration in the chamber. 7. Just before VOC generation was initiated, the ambient chamber VOC concentration was measured and the exposure level (7 ppm) was incremented by the ambient VOC level. For example, if the ambient VOC level was 0.37 ppm, the exposure level vas set at 7.37 ppm. 8. Seventy-five minutes after commencement of testing, the operator initiated exposure by activating the automatic control of the VOC concentration in the chamber. SYSTEM PERFORMANCE This system has been used in two different studies for a total of 110 hours of VOC control during a protocol. The two studies used a mixture very similar to the tventy-tvo compounds Molhave used. Figure 3 shovs a plot of the VOC concentration in the chamber over the period of the protocol. For this study, the subjects were in the chamber while the VOC control was established and the system equilibrated to the required valve. ------- MAKEUP AIR COMPUTER SVI CTRL FID ANALYZER PURGE r MIXER A CHAMBER t voc GENERATOR FAN Figure 1 Overall Chamber Design ------- Spil NITROGEN o\ VOC MIXTURE Figure 2 VOC Generator Design METERING SOLENOID VALVE VALVE NITROGEN 2 l/m SPRAYER GLASS EVAPORATOR BRASS BLOCK DAMPER HEATERS ------- H« IN OHMM* ft I'll/il IHUlll I . 4 . 3 - i . I . I . — lit HI 9M 911 IN (41 I I I I I I I t»M Itl* 1IM »•<• UN lilt I1M 1141 II »CtU»l MIIWD TIM Figure 3 Typical Plot of VOC Control ------- REFERENCES Molhave L, Bach B and Pedersen 0. Klimakammerundersogelse af geneforekomster hos personer, der udsaettes for organiske gasser og dampe fra byggematerialer [Human reactions to volatile organis gases]. Project Report ROO-52(22411), Institute of Hygiene, Universitetsparken, Aarhus, Denmark, 1985. Strong AA; "Description of the CLEANS Human Exposure System". U.S. EPA. EPA-600/1-78-064, 1978. Crider VL, Landis DA, Weaver FH. In "Generation of Aerosolos and Facilities for Exposure Experiments". Villeke K, (ed.) Ann Arbor, Michigan, 1980:493-515. Glover DE, Berntsen JH, Crider, VL, Strong AA. "Design and Performance of a System to Control Concentrations of Common Gaseous Air Pollutants within Environmental Laboratories Used for Human Exposure". J. Environ. Sci. Health, A16(5), 1981, pp501-522. 78 ------- APPENDIX B AUDITORY DIGIT SPAN TEST DESCRIPTION AND INSTRUCTIONS H. Kenneth Hudnell Neurotoxicology Division, Health Effects Research Laboratory U.S. Environmental Protection Agency, RTF, NC The following tape-recorded instructions were given to subjects. "Attention Subjects: you will now be given another version of the digit span test. Unlike the Digit Span Test you took earlier on the computer, this test will be administered by a cassette recording played over your speakers. Please write your responses on the answer sheet attached to the clipboard. Please pick up the clipboard at this time and assume a comfortable writing position. The answer sheet should say "auditory digit span - pretest" at the top. The test will have two parts, Digit Forward and Digit Backward. Please find the section labeled Digit Forward. As you can see on the answer sheet, blank spaces are provided for you to write down each of the digits in the string. The first string will contain 3 digits. Please listen carefully as the digits are called out. Be sure not to repeat the digits verbally, but you may rehearse them in your mind. After the string has been called out, there will be a pause during which you are to write down the digits as you remember them and in the same order as they were presented. Please do not begin writing until all digits in the string have been called out. After the pause, a different string of 3 digits will be presented and then followed by a pause during which you can write your answers. This process will be repeated until 2 trials with strings of up to 9 digits have been presented. After the digit forward test has been completed there will be a 1 min. pause. Then, the digit backwards test will begin. This test is exactly like the digit forward test except that you are to write down the digits in the reverse order of their presentation. That is, the last digit you hear should be 79 ------- the first digit you write on the answer line. The digit backwards test will begin with a 2 digit string and end with an 8 digit string. Please remember, do not repeat the digits out loud and do not begin to write your answers until the pause begins after all the digits in the string have been called out. Please prepare to begin the digit forward test. Write the digits in the same order as they are presented. The test will begin now. Trial 1 of 3 digits (say 1-4-7 and pause 10 sec.) Trial 2 of 3 digits (say 3-7-2 and pause 10 sec.) " 1 " 4 " ( » 2-9-5-6 and " 12 sec.) " 2 " 4 " < " 2-6-5-4 and " 12 " ) " 1 " 5 " ( " 2-8-7-4-5 and " 14 " ) " 2 " 5 " ( " 2-9-6-7-5 and " 14 ") " 1 " 6 " ( n 1-6-9-2-4-1 and M 16 " ) " 2 " 6 " ( " 4-7-6-1-2-8 and " 16 " ) M 1 " 7 " ( " 5-7-1-6-2-4-9 and M 18 " ) " 2 " 7 " ( " 1-5-2-8-9-4-6 and " 18 " ) " 1 " 8 " ( " 7-3-9-8-4-5-2-7 and " 20 " ) " 2 " 8 " < " 8-7-4-2-5-4-3-1 and " 20 " ) •" 1 " 9 " ( " 3-7-4-9-7-3-8-4-2 and " 22 " ) " 2 " 9 " ( " 1-3-2-5-2-7-8-4-8 and " 22 ") The digit forward test is now completed. We will pause for 1 min. before beginning the digit backwards test, (pause 1 min.) Please prepare to begin the digit backwards test. Please write the digits in the order opposite that in which they are presented. The test will begin now". Trial 1 of 2 digits (say 3-5 and pause 10 sec.) Trial 2 of 2 digits (say 4-2 and pause 10 sec.) " 1 " 3 " ( " 2-9-2 and " 12 " ) " 2 " 3 " ( " 3-2-5 and " 12 " ) " 1 " 4 " ( " 2-7-9-4 and " 14 " ) " 2 " 4 " ( " 2-8-9-4 and " 14 " ) " 1 " 5 " ( " 3-6-7-5-1 and " 16 " ) " 2 " 5 " ( " 2-6-9-7-3 and " 16 " ) M 1 " 6 " ( " 1-4-8-3-9-3 and " 18 " ) " 2 " 6 " ( " 7-3-6-2-4-1 and " 18 " ) " 1 " 7 " . ( " 4-1-6-7-9-5-1 and " 20 " ) " 2 " 7 " ( " 2-9-6-7-2-1-4 and " 20 " ) " 1 " 8 " ( " 1-4-2-6-7-5-6-9 and " 22 " ) M 2 " 8 " ( " 1-6-1-3-9-8-3-7 and " 22 " ) Similar instructions were given for the post test. Digits presented in the post test were: 80 ------- FORWARD 2-8-6 3-2-6 1-5-7-3 9-3-5-1 6-8-1-9-2 8-2-5-7-3 2-8-6-1-3-7 2-4-8-7-5-1 9-5-1-3-1-7-2 6-3-5-7-2-8-5 1_5_9_5_7_3_9_2 1-6-9-3-4-8-6-2 2-8-1-5-2-6-1-7-3 4-1-4-8-2-6-9-4-1 Digit Sequences used on day 2 vere as follows: FORWARD PRE-TEST 6-1-3 3-5-1-7 8-9-4-2 5-9-8-1-6 2-8-9-4-6 3-1-4-2-4-7 3-7-9-2-6-1 2-9-3-7-9-4-6 8-5-1-7-2-4-3 7-1-4-6-8-3-2-9 9-4-7-3-2-9-5-6 1-6-2-5-1-8-2-7-3 7-3-9-3-8-4-5-7-2 BACKWARD TT~ 2-8 5-2-7 1-3-8 3-7-9-5 6-1-4-7 2-8-9-5-6 2-9-5-2-1 1-3-4-8-2-1 5-9-3-8-7-2 4-5-7-4-1-6-7 3_9_5_9_1_5_7 1-7-4-3-2-9-1-6 9-4-8-2-7-1-3-2 BACKWARD PRE-TEST T-3 2-6 1-4-2 1-8-7 5-2-4-1 2-4-6-5 3-1-5-4-6 6-3-5-1-7 2-9-1-3-8-4 3-6-7-5-2-8 7-3-7-1-8-1-4 6-1-3-7-9-8-2 2-9-4-7-5-1-4-9 4-2-4-1-7-3-6-8 POST TEST-FORWARD 7-4-1 2-8-9 1-6-8-7 5-2-7-1 2-7-3-1-4 l_5_8-3-9 9-3-4-7-6-1 4-6-2-8-1-3 3-7-8-9-1-3-5 3-2-4-5-7-9-6 2-7-8-6-9-3-4-8 9-4-3-5-8-2-1-5 8-3-7-4-6-9-2-3-1 5-2-3-1-4-9-2-6-8 POST TEST-BACKWARD 539 3-1 2-9-5 2-6-9 1-4-5-8 3-2-8-6 7-3-2-4-1 5-6-1-4-3 7-3-5-1-2-6 8-5-2-7-3-1 3-2-3-5-4-2-1 4-3-6-5-7-8-2 1-5-3-9-4-8-2-6 1-6-9-3-8-2-7-1 81 ------- Digit Span Answer Sheet Subject ID Number : Date : Session # (Circle one): 147 Day (Circle one) : 0 1 2 A. Digit Forward 3. (1) (2) 4. (1) (2) 5. (1) (2) 6. (1) (2) 7. (1) (2) 8. (1) (2) 9- (1) (2) B. Digit Backward 2. (1) (2) 3. (1) (2) 4. (1) (2) 5. (1) (2) 6. (1) (2) 7. (1) (2) 8- (1) (2) 82 ------- INSTRUCTIONS FOR SCORING AUDITORY DIGIT SPAN DATA ADS data will be scored in two ways, the standard method (std) and the weigh ted method (wtd). Procedure. Both Methods. Start with the 3 digit forward string, trial 1. Use the answer key to see if all 3 digits are correctly recorded on the answer sheet. If so, put a checkmark at the right end of the string. If no, put an X at the right end. Repeat this process for each string in the digit forward test. Then score the digit backward test. Std Method. At the bottom of the answer sheet under the forward responses, write the number of the string that corresponds to the longest string in which at least 1 of the 2 trials was answered correctly for that string and all previous strings. For example, if at least 1 trial is correctly answered on strings 3, 4, 5 and 6 but neither trial is answered correctly on the string of 7 digit, the score is "std = 6". Repeat this procedure for the digit backwards test. At the center, record the total score. For example, if the forward score is 6 and the backwards score is 5, write "Tstd = 11". Wtd Method. For the digit forward test, multiply each string number (3-9) by the number of trials answered correctly (0,1 or 2). Add all products together. For example, if the scores are: string # trials correct the products are 3 2 3 x 2 = 6 4 2 4x2 = 8 5 2 5 x 2 = 10 6 1 6x1 = 6 7 0 7x0 = 0 8 1 8x1=8 9 0 9x0 = 0 The product sum = 38. Write "Wtd = 31" under the forward responses. Repeat this procedure for the backwards test. Record the total Wtd score at the bottom of the answer sheet in the center. For example, if the forward score is 38 and the backwards score is 26, write "Twtd = 64". Note. For the backwards test, remember that the answer must be in the reverse order of presentation to be correct. 83 ------- APPENDIX C NEUROTOXICITY TESTING OF VOC MIXTURES IN HUMANS In order to characterize the neurobehavioral effects of VOC exposure, a microprocessor-based test battery was used. The neurobehavioral evaluation system (NES) developed by Baker et al (1985) for neurotoxicity field testing contains a menu-driven battery of tests selected on the basis of proven sensitivity to environmental toxicity and WHO recommendations (Johnson et al., 1987). A variety of sensorimotor and cognitive functions including reaction time, motor coordination, attention, memory and spatial integration were assessed. The NES battery has been used previously in the study of nitrous dioxide (Greenberg et al, 1985), styrene, alcohol, is currently being used by CDC in the AGENT ORANGE project, and in NHANES III (National Health and Nutrition Survey). Specific tests to be used in the VOC study included finger tapping, continuous performance, digit span, symbol-digit substitution, visual memory and switching attention. Individual tests are described below. NES TESTS USED IN VOC STUDY (1) Finger tapping (mean no.) 3 min. (2) Digit span (no. correct fwd, bvd) 7 min. (3) Continuous Performance Test (msec, errors) 7 min. (A) Symbol-digit substitution (sec/digit, errors) 5 min. (5) Serial digit learning (no. correct, no. trials) 7 min. (6) Pattern memory (no. correct, msec) 5 min. (7) Switching attention ((msec/no, correct) 7 min. (8) Mood scales (5 scale avs.) 5 min. 84 ------- Total time: 46 min. Descriptions of Individual Tests (1) Finger-Tapping. This test evaluates motor response speed. The subject is instructed to press a button as many times as possible within a 1-second interval. In the first trial, the subject presses one button with the left hand, The second trial tests the right hand, and in the third trial, the subject alternately taps two buttons with the preferred hand. This test provided a straightforward measure of VOC effect on motor response. It was a negative control measure since VOCs were not expected to affect motor response per se. (2) Digit Span. The auditory version of this widely used clinical test has been adapted from the Vechsler Adult Intelligence Scale (Wechsler, 1955). Subjects must enter into the computer progressively longer series of digits which are presented visually at a rate of one per sec by the computer. New digit sequences are created at each span length. After incorrectly responding to two trials at a span length, the task changes such that the digits must be entered in reverse order. This test has been used as a measure of short-term memory and attention in studies of solvent and lead toxicity (Hanninen, 1979). Molhave et al (1986) reported that digit span performance was impaired by exposure to the complex VOC mixture, a finding that the present study attempted to replicate. (3) Continuous Performance Test (CPT). This test measures sustained visual attention by having the subject press a button upon seeing a large letter "S" when it is projected on a video display. Letters flash briefly (for about 50 msec) on the screen at a rate of one per second for five minutes. Recording and storage of individual response latencies permits the computation mean (and standard deviation) of reaction times. Omission and commission errors are also recorded. Molhave et al (1986) failed to observe any effect of VOC exposure on "graphic" CPT performance, but the present test is very 85 ------- different and has proven to be sensitive to toxicant exposure in previous studies (Baker et al, 1988; Erstin et al, 1988; Mahoney et al, 1988). (4) Symbol-Digit (SD) Substitution Test. This test is similar to the Digit-Symbol Substitution Test from the WAIS. SD evaluates speed and coding ability and has been found useful in previous studies of lead, carbon disulfide and solvent mixtures (Anger and Johnson, 1985). In this adaptation, nine symbols and digits are paired at the top of the screen and the subject has to press the digit keys corresponding to a re-ordered test set of the nine symbols. The time required to complete each SD set and the number of digits incorrectly matched are recorded. Five sets of nine SD pairs are presented in succession. The pairing of symbols vith digits is varied between sets to avoid learning. (5) Serial Digit Learning (SDL). This is a test of short-term memory and attention in which subjects are presented a series of 10 digits on the display screen at a rate of one per second. Upon completion they are asked to enter that series into the computer. The series is presented until two successive trials are performed correctly, or at most 8 times. The number of correct digits entered on each trial is recorded. The series is changed for each test session to avoid learning effects. This test is a variant of the digit-span test found to be sensitive to solvent and lead exposure (Anger and Johnson, 1985) and is similar to Supraspan in the neurotoxicity test battery developed by Eckerman et al (1985). Based on the digit span findings of Molhave et al (1986), we predicted that SDL performance would be impaired by VOC exposure. (6) Pattern Memory (PM) Test. This test of short term visual memory involves the brief presentation of a block-like pattern followed by three similar figures, one of which is identical with the original pattern. The degree of correspondence of the two incorrect patterns to the correct one varies. The number of correct responses and the response time are recorded. Vhile digit span and serial digit learning assessed verbal memory, PH examined if VOC exposure impaired visual memory. 86 ------- (7) Switching Attention (SVAT). This test evaluated the effect of VOC exposure on selective attention with little memory demands. During two initial sets the subject is trained to respond differentially to target stimuli on the left or right side of the screen, and then to stimuli in the center of the screen pointing to the left or right. The target stimuli are large rectangles with inset arrows pointing left or right which may appear on the right or left side of the screen. In the third set of trials, these two response conditions are mixed. When the word on the screen is "SIDE", the subject must press the left button if the target stimulus is on the left and vice versa. When the word on the screen is "DIRECTION", then the subject must press the right button if the arrow points to the right and vice versa. The word on the screen is randomly assigned each trial, the numbers of incorrect responses and mean react ion-times are recorded. This test was chosen to clarify whether the Molhave et al digit span finding was due to memory or attentional impairment. (8) Mood Scales. This test consists of a series of adjectives or phrases with which subjects rate themselves on their feelings. Similar instruments have been used to evaluate the efficacy of psychotherapeutic drugs and to classify individuals with various neurobehavioral disorders (McNair et al, 1971) The present test contains 25 items and yields a five-dimensional mood profile (tension, depression, anger, fatigue, confusion) by combining ratings on individual items. Prior studies have shown that this approach is sensitive in the evaluation of central nervous system effects of occupational lead exposure (Baker et al, 1984). Molhave et al (1986) reported that subjective ratings of sensory irritation (comfort) and air quality were altered by VOC exposure. The mood scale was chosen to clarify these findings by assessing which dimension(s) of mood were affected by VOC exposure. (9) Associate Learning/Recall. This test is similar to the paired associate learning test in the Wechsler Memory Scale (Wechsler, 1945). Three letter names (e.g. Jan, Pat, Tom) are paired with occupations (e.g. florist, lawyer, plumber). Nine pairs were presented on the video monitor for two sees. each with one sec. intervals between pairs. After all pairs were presented, each name was displayed and subjects were required to select the appropriate 87 ------- alternative from a list of occupations. Three trials were presented with the same pairings, but in different order. The score consisted of the total number of correct pairings in trials 1-3. A single recall trial vith the same pairings vas presented about 20 mins. later. This test provided an alternative type of memory challenge for testing VOC exposure effects. (10) Pattern Comparison. This test is similar to the Pattern Memory Test described above and utilizes the same 10x10 character arrays. Three patterns are presented simultaneously on the video screen. Two patterns are identical and the third is slightly different. The task is to identify the odd array. 25 trials were presented. Two measures were recorded—the mean latency of correct trials 2-25 and the number of correct trials. Schmedtje et al (1988) found that performance on this test improved after administration of dextroamphetamine and worsened after scopalomine. (11) Simple Reaction Time (SRT). SRT is the oldest tool in the psychologist's armamentarium. This visual task requires subjects to button-press as fast as possible when a large square is displayed on the screen. Nine blocks (five with the preferred hand and four with the nonpreferred hand) containing 12 trials each and variable intertrial intervals of 2-4 sees were used. Mean reaction times, excluding block one, were calculated for each hand. SRT tests have been used extensively in occupational field studies including workers exposed to solvents (Anger and Johnson, 1985). (12) Grammatical Reasoning. This test was adapted from Baddeley (1968). Each trial consists of a logical sequence such as "A FOLLOWS B: AB" to which the subject must respond "true" or "false". Sequences vary along four dimensions: active-passive, positive-negative, true-false, and whether the first letter is A or B. Two blocks of 32 trials each were presented. Mean response time for all correct trials and the number of correct trials were recorded. This was the most difficult NES test used. Little evidence of the utility of this test in neurotoxicity testing is available. (13) Horizontal Addition Test. Subjects were presented with 60 simple 88 ------- addition problems in which three single digits were presented in a row. Subjects were instructed to sum the digits as rapidly and accurately as possible. Responses had to be entered on the keyboard. Mean response times and number of correct trials were recorded. Subjects made very few errors. This test served primarily as a filler. REFERENCES Anger WK, Johnson BL. Chemicals affecting behavior. In: O'Donoghue JL (ed) Neurotoxicity of Industrial and Commercial Chemicals. CRC Press, Baton Rouge, 1985. Baddeley, AD. A 3 minute reasoning test based on grammatical transformation. Psychonom Sci 1968, 10: 341-342. Baker E, Letz R, Fidler A, etal. A computer-based neurobehavioral evaluation system for occupational and environmental epidemiology: Methodology and validation studies. Neurobehav Toxicol Teratol 1985, 7:369-377. Baker E, Letz R, Eisen EA et al. Neurobehavioral effects of solvents in construction painters. J Occup Med, 1988, 30:116-123. Eckerman D, Carroll J, Force D, et al. An approach to brief field testing for neurotoxicity. ibid:387-393. Estrin WJ, Moore P, Letz R, Wasch HH. The P300 event-related potential in potential in experimental nitrous oxide exposure. Clin Pharmacol Ther, 1988, 43:86-90. Greenberg B, Moore P, Letz R, Baker E. Computerized assessment of human neurotoxicity: Senitivity to nitrous oxide exposure. Clin Pharmacol Ther- apeutics, 1985, 38:656-660. Hanninen H. Psychological test methods: Sensitivity to long term chemical exposure at work. Neurobehav Toxicol Teratol, 1979, l(supp 1):157-161. Mahoney P, Moore PA, Baker EL, Letz R. Experimental nitrous oxide exposure as a model system for evaluating neurobehavioral tests. Toxicol., 1988, 49:449-457. McNair DM, Lorr M, Drpopleman LF. BITS Manual—Profile of Mood States. San Diego: Educational and Testing Service, 1975. Molhave L, Bach B, Pedersen OF. Human reactions to low concentrations of volatile organic compounds. Environ Internal, 1986, 12:167-175. 89 ------- Schmedtje, J.F., Oman C., Letz, R., Baker, E.L. Effects on scopalomine and dextroamphetamine on human performance. Aviat Space Environ Med, 1988, 59:407-410. Vechsler D. A standardized memory scale for clinical use. J Psychol 1945, 19:87-95. Vechsler D. Vechsler Adult Intelligence Scale Manual. Psychological Corp- oration, Nev York, 1955. 90 ------- APPENDIX D SYMPTOM QUESTIONNAIRE SUBJECT ID: DO YOU FEEL NOW THAT STAYING IN THE CHAMBER IS COMFORTABLE? (Y/N) WOULD YOU BE SATISFIED WITH THIS AIR QUALITY IN YOUR OWN HOME? (Y/N) WOULD YOU VENTILATE YOUR HOME MORE? (Y/N) (1) DOES THE SKIN OF YOUR BODY FEEL IRRITATED OR ITCHY? NOT AT ALL | 1 1 VERY MUCH (2) HOW IS THE NOISE LEVEL IN THE ROOM? VERY QUIET | 1 1 VERY NOISY (3) DO YOUR EYES FEEL DRY OR WATERY? VERY DRY | 1 1 VERY WATERY (4) DO YOU HAVE A HEADACHE RIGHT NOW? NOT AT ALL | 1 1 VERY STRONG (5) DOES THE SKIN ON YOUR FACE FEEL WARM OR COLD? VERY COLD | 1 1 VERY WARM (6) DO YOU FEEL SLUGGISH? NOT AT ALL | 1 1 VERY MUCH (7) HOW IS THE ODOR LEVEL IN THE ROOM? NO ODOR | 1 1 VERY STRONG (8) DO YOU FEEL ANY IRRITATION IN YOUR EYES? NOT AT ALL | 1 1 VERY STRONG (9) DOES THE SKIN OF YOUR BODY FEEL WARM OR COLD? VERY COLD | 1 1 VERY WARM (10) HOW IS THE LIGHT LEVEL IN THE ROOM? VERY DARK | 1 1 VERY BRIGHT (11) HAVE YOU BEEN COUGHING? NOT AT ALL | | 1 A LOT (12) DOES YOUR FACE FEEL IRRITATED OR ITCHY? NOT AT ALL | 1 1 VERY MUCH 91 ------- (13) HOW IS THE AIR HUMIDITY IN THE ROOM? VERY DRY | 1 1 VERY HUMID (14) HOW IS THE MOVEMENT OF AIR IN THE ROOM? VERY STILL | 1 1 VERY DRAFTY (15) DOES YOUR NOSE FEEL DRY OR IRRITATED? NOT AT ALL | 1 1 VERY MUCH (16) HOW IS THE AIR TEMPERATURE IN THE ROOM? VERY COLD | 1 1 VERY HOT (17) ARE YOU TIRED OR SLEEPY? NOT AT ALL | 1 1 VERY MUCH (18) DOES THE SKIN OF YOUR BODY FEEL MOIST OR DRY? NOT AT ALL | 1 1 VERY MUCH (19) DOES YOUR THROAT FEEL IRRITATED? NOT AT ALL | 1 1 VERY MUCH (20) HOW IS THE QUALITY OF AIR IN THE ROOM? VERY GOOD | 1 1 VERY BAD (21) DOES THE SKIN ON YOUR FACE FEEL DRY OR MOIST? VERY DRY | 1 1 VERY MOIST (22) HOW HARD DID YOU HAVE TO CONCENTRATE ON THESE QUESTIONS? VERY LITTLE | 1 1 VERY MUCH 92 ------- APPENDIX E THE USE OF LINEAR POTENTIOMETERS FOR SUBJECTIVE ASSESSMENTS OF INDOOR AIR QUALITY 12 3 Lars Molhave , David Otto , Gregory Rose Institute of Environmental and Occupational Medicine University of Aarhus Aarhus, Denmark 2 Human Effects Research Laboratory U.S. Environmenatal Protection Agency Chapel Hill, NC 27599-7315 3 NSI Environmental, Inc. Research Triangle Park, NC 27711 93 ------- Introduction The principle of potentiometer assessment is called modified magnitude estimation and is based on a linear analog rating scale (Bond and Lader, 1974). Subjects may continuously indicate individual or collective ratings of any specified indoor climate factor presented in the form of questions with defined continuous answers between two well defined extremes. Measuring Principles Each subject has his own rating box (Fig. 1) with a sliding pointer that can be positioned along a 60 mm track. The device can be used in two modes. Ratings can be signalled by external prompt (e.g., a visual prompt on the subject's video screen or verbal instruction from the investigator) or ratings can be initated spontaneously by the subject whenever a change in indoor air quality is perceived. In this study comfort ratings were signalled every 15-30 minutes by video prompts or verbal instruction. The extremes were defined as "no irritation" and "unacceptably strong irritation". Prior to commencement of testing, the potentiometer pointer was positioned at the "no irritation" extreme. Instructions to the Subject should include: a) Definition of the endpoints and rating scale. b) The nature of the indoor climate factor (s) to be rated. c) That the measurement scale must be established by the subject himself. d) That only the pointer should move and that nobody else should see the setting of the pointer. 94 ------- e) That the scale includes a neutral range (e.g., at the "no irritation" end). f) That the initial position of the pointer has no particular relationship to the present IAQ. g) Operation of the box and pointer must be demonstrated and explained to subjects prior to its use. h) The time intervals at which ratings are to be made should be specified. i) If external timing is used, the subjects should be instructed to go to the box and make a simulated rating even if they don't feel any change. They may delay their rating if they are occupied with other tests when the sound signal appears. j) The pointer must be set in a neutral position (i.e., no irritation) at the start of the tests. k) Subjects should not move the pointer back to neutral and then to a new position when a rating is required. That is, if a change in rating is desired, the pointer should be moved from its previous position directly to the new position. The Equipment The rating box is shown in Figure 1. The box contained a linear potentiometer with sliding pointer which could be positioned along a one-dimensional 60 mm axis. A 5 volt power source in the breakout box supplied the voltage across the resistive arm to give a O to 5 volt output range. Due to a non-linear response in the 4 to 5 volt range, the slide was limited to a maximum output of 4 volts. Thus 0 volts corresponded to "no irritation" and 4 volts corresponded to "unacceptabley strong irritation". Each of the four potentiometer outputs was directed to an input channel of an analog-to-digital 95 ------- realtime module mounted in the backplane of the Compaq Plus microcomputer control station. A BASIC algorithm automatically sampled each of the four channels simultanelusly every 15 minutes throughout the test day. Sampling was initiated manually two minutes after testing commenced to permit subjects to complete the initial comfort rating. Subjects vere prompted on video monitors while testing and by operators over loudspeakers during break periods to set their comfort level prior to the 15 minute auto sampling. The algorithm converted the sampled digital units back to voltage units to store and report irritation in a range of 0.0 to 4.0. 96 ------- 4 VOLTS 0 RATING BOX 1 RATING BOX 2 RATING BOX 3 RATING BOX 4 i BREAKOUT BOX (WITH 5 VOLT POWER SUPPLY) FIGURE 1 COMPAQ PLUS WITH A/D CONVERTER ------- The calibration of the potentiometers with the analog-to-digital converter was checked prior to each test day and stored by the computer in 0, 25%, 50%, 75%, 100% increments of the slide of the potentiometer which corresponded to measures of 0.0, 1.0, 2.0, 3.0, and 4.0. Instructions to the Subjects "An aluminum Comfort Meter is located to the right of your computer. The lid should be open to show a sliding brass knob with a pointer that can be moved from "No irritation" to "Strong irritation". You will use this comfort meter to rate the degree of comfort or irritation that you feel in your eyes, nose or throat during the experiment. There are no scale markings on the box, so you will have to establish your own scale when you start". "Remember, we want to monitor the level of comfort or irritation that you feel in your eyes, nose and throat during the experiment. You will be instructed at regular intervals, both by computer prompts and over the intercom, to indicate your comfort level. Do not interrupt an ongoing test to reset the comfort meter unless instructed to do so. During rest periods between tests, however, you are encouraged to indicate any change in comfort level that you feel. Please close the lid after each rating in order to prevent others from seeing your comfort rating". REFERENCE Bond A., and Lader H. The use of analoque scales in rating subjective feelings. Br. J. Med. Psycholo. 1974, 47:211-218. 98 ------- |