p.1 

OAR Box 1147 

Prepped by Ollie Stewart 

Document Number: 

138) IV-E-11 

Docket Number: 

A-87-11 

Printed 4/22/2008 2:42:55 PM Sheet 138 of 242 
P.2 

Note to Docket A-87-11 

On October 26, 1987, representatives of the Motor Vehicles Manufacturers 

Association (MVMA) met with scientists at the Environmental Protection Agency 

to present the results of research sponsored by the MVMA, most of which is 

unpublished but has been submitted for publication. Presentations were made 

by John Shi Her , B. D. Garg, and Michael Olson concerning their research. 

A list of the meeting attendees and a copy of the papers distributed 

before and during the meeting is attached. 

Submitted by 

Robert E. McGaughy ' 

October 26, 1987 

Attachment 
P.3 

MVMA GASOLINE RESEARCH MEETING 

OCTOBER 26, 1987 

Participant 

Karl Baetcke 

Michael J. Olson 

Robert McGaughy 

Graham Chatburn 

Kathy Steilen 

B. 0. Garg 

C. C. Green 

R. T. Paul 

J. Shi 1ler 

J. Vostal 

Arthur Chiu 

William Pepelko 

Barbara Mandula 

Affiliation 

EPA 

General Motors 

EPA 

Ford 

EPA 

General Motors 

General Motors 

Motor Vehicles Manufacturers 

Association 

Ford 

General Motors 

EPA 

EPA 

EPA 
P.4 

87-31.3 

GASOLINE VAPOR EXPOSURE PARAMETERS 

DURING REFUELING OPERATIONS 

JOHN W. SHILLER 

FORD MOTOR COMPANY 

DEARBORN, MICHIGAN 

FOR PRESENTATION AT THE 80TH ANNUAL MEETING AND EXHIBITION OF APCA 

New York, New York 

June 21-26, 1987 

— — ' ' — —*—* "i^T-i — -rmX*-
P.5 

GASOLINE VAPOR EXPOSURE PARAMETERS DURING REFUELING OPERATIONS, J. W. Shiller. 

Ford Motor Company, Dearborn, MI 48121. 

In order to accurately characterize the potential health effects of exposure to gasoline 

vapors during refueling operations, a determination of the concentration, time duration 

and chemical characteristics of exposure patterns must be made. Because available field 

data are limited, theoretical modeling of fuel properties became necessary to generate a 

more complete picture of exposure. Available exposure data indicate that the most 

significant parameters affecting gasoline vapor concentration are distance from the fuel 

tank opening, wind direction, and orientation of the person filling the tank. Small 

changes in these parameters produce a large scatter in experimental field data. 

Theoretical considerations indicate that exposure to gasoline vapors should decrease in 

the winter compared to summer levels even with the greater volatility of winter fuels. 

The composition of refueling vapors consists mainly of the lighter components of 

gasoline, CS and lower. Exposure duration is typically 1/2 to 1-1/2 minutes per 

refueling event adding up to a total of about 70 minutes per year per vehicle on average. 

It is found that the chemical composition of refueling vapor is so different from the 

actual fuel composition that this must be considered in any evaluation of potential 

health effects. 
P.6 

- 2 - 87-31.3 

I. INTRODUCTION 

Gasoline Refueling Vapors 

Regulations that are currently In place Co control hydrocarbon 

emissions, in general, focus on reducing non-methane hydrocarbons that 

participate in ozone-generating reactions in che atmosphere. The U.S. 

Environmental Protection Agency (EPA) has estimated that if che emissions 

of gasoline vapors during motor vehicle refueling operations ac service 

stations are conerolled, ic would resule In about a 2 percent reduccion in 

total hydrocarbon emissions^-. A number of strategies have been identified 

to control vapor emissions during motor vehicle refueling at gasoline 

service stations. One option is a vapor recovery systen ac service 

stations which returns the vapor to the underground tank (Stage II vapor 

control). Another option is to store and dispose of the vapor In the 

vehicle (onboard vapor control). The Environmental Protection Agency is 

expected to propose rulemaking on controlling gasoline refueling emissions 

in 19871. One important question in that rulemaking process is to 

determine whether the direct emissions of gasoline vapor or specific 

constituents of gasoline vapor warrant control to protect public health. 

If the best, available answer to that question indicates chat direct 

control is not warranted, then control of gasoline vapor may be required 

only in areas needing additional ozone control. If the best available 

answer indicates chac direct control is necessary, then a nationwide 

control program may be Implied. 

Gasoline Vapor Health Effects 

Motor vehicle manufacturers and EPA requested that the Health Effects 

Institute (HEI) review the issues concerning the potential adverse health 

effects of gasoline refueling vapors. These issues arose from a two-year 

animal study which found increased kidney tumors in male rats and 

increased liver tumors in female mice, following chronic exposure to 

wholly vaporized unleaded gasoline^. In response, the Institute sponsored 

a nine month study of the existing data and issued a final report in 

September 1985^. Some of the facts brought out in the HEI report are as 

follows: 

The bioassay study* has shown wholly vaporized gasoline 

to be an animal carcinogen and in the absence of 

evidence Co the contrary, lc might also be prudent to 

assume that vapors from wholly vaporized gasoline are 

human carcinogens. 

Yet because people rarely, if ever, encounter wholly 

vaporized gasoline vapors during the course of their 

daily activities, findings from the bioassay study do 

not necessarily meet the informational requirements for 

the first step in quantitative risk assessment, hazard 

identification. 
P.7 

- 3 - 87-31.3 

A possible mechanism for the toxic response observed in 

. male rats was identified and suggests that the male rat 

may be uniquely susceptible to effects from gasoline 

vapor, thus diminishing its relevance to humans. 

Benzene, a constituenC of gasoline vapor and a 

carcinogen in humans and rodents, is not believed to 

have contributed to the increased incidence of tumors 

found in the rodent bioassay studies. 

Available human epidemiologic evidence neither negates 

nor confirms the interpretation that gasoline vapors are 

a potenCial human carcinogen. Thus, Che actual 

magnitude of associated health hazards, if any, cannot 

be estimated accurately without additional data. 

Purpose of Paper 

As discussed above, the Health Effects Institute pointed out the need 

for additional data before an accurate estimate of the public health risk 

due to exposure to gasoline vapors can be, made. This paper deals with one 

Important element of the health assessment issue, exposure-dose 

parameters. Such parameters include chemical characteristics as well as 

concentration levels and time duration of typical exposure patterns. Such 

information is believed essential to medical professionals in making their 

determination of public health risk. 

2. EXPOSURE PARAMETERS 

Variables 

There are many factors that influence potential exposure to gasoline 

vapor constituents during refueling operations. Some of the more 

important factors are: 

Fuel chemical components and relative abundance. 

Ambient and fuel temperatures. 

Wind"'speed and direction. 

Refueling time duration and fill rate. 

Distance from pump nozzle and orientation of person. 

The field sampling data so far available cover only a small subset of 

the range of these variables. Therefore, field exposure data were 

combined in this paper with fuel modeling and other data to help generate 

a more complete picture of potential exposure. 

Field Exposure Data 

Data taken to describe exposure to gasoline vapors during actual 

motor vehicle refueling operations are available from only a few studies. 

In one study, short term self-service exposure concentrations were 

measured by Clayton Environmental Consultants (CEC) at 13 gasoline 

stations using a portable sampling apparatus . Shown in the first two 

major sections of Table I are the CEC data for unleaded, leaded and 
- 4 - 87-31.3 

premium unleaded fuel, first averaged by fuel type and second by the 

geographic location of che service stations chosen in that study. Also 

shown in Table I are data from similar studies by employees of Shell Oil 

Co.5, Baeeella6»

7 EPA8, Mobil Oil Corp.9, and General Motors10 (GM). All 

of these daea have been converted Co che same basis for comparison in 

Table I. The following observations are made regarding these six field 

studies. 

(1) The measured average concentrations of gasoline vapor listed in 

Table I cover a large range from a low of 0.01 to a high of 218 ppm. 

Although there are many factors that influence the measured concentration 

level, the EPA study8 appears to have clearly identified some of the more 

significant variables; wind direction and distance fro© pump nozzle. 

Highest concentrations were observed when che wind first passed over che 

gasoline tank and pump nozzle, blowing emitted vapors directly toward the 

hydrocarbon detector or the person filling the tank (called parallel wind 

assuming the wind is also parallel to the axles of the car). Lowest 

concentrations were observed when the wind was blowing vapors directly 

away from the hydrocarbon detector or the person filling the tank (called 

reverse parallel wind). The significant effect of wind direction and 

distance from pump nozzle is illustrated in Figure 1. Both Che parallel 

and reverse parallel wind conditions are illustrated, for vapor 

concentration (logarithmic scale) as a function of the horizontal distance 

from the fuel tank filling neck (linear scale). ' The 2 foot distance was 

selecced by che authors" as the approximate location of an individual 

during a refueling operation ("breathing zone"). As the distance from che 

fuel tank filler neck increased, the measured hydrocarbon vapor 

concentration rapidly decreased as indicated. The data in Figure 2 have 

background levels subtracted out. At the 2 foot location, hydrocarbon 

concentration for the reverse parallel wind was more than a factor of 2000 

times less than the parallel wind case. The GM study*" also found widely 

varying results with changes in the position of the person and wind 

direction. In fact, the dominant reason why their winter results were 

higher than their summer values, contrary to expectations, was that the 

person filling the tank was closer to the fuel nozzle in the winter 

program. 

(2)' The'average ambient temperature data listed in Table I for the 

CEC, EPA, and GM studies were taken directly from their reports. The 

temperature data listed for the other studies are 30 year normal 

temperatures for the location and month used during their sampling 

prograe^. Most of the data in Table I were obtained for ambient 

temperatures above 60°F and are not completely representative of all 

seasons and geographic locations. Furthermore, the large scatter in the 

daea due to the dominant effect of differences in sampling distance, 

orientation, and.wind directions make it difficult to discern CemperaCure 

effeces based on available experimenCal daea. Figure 2 is an illuscracion 

of che expecCed CemperaCure dependence of refueling losses, and hence 

ambient vapor concentration, based on theoretical considerations^>13 

assuming similar tank and delivered fuel temperature for various fuel 

volatility levels indicated by Reid Vapor Pressure (RVP, psi). The effect 

of fuel weathering also is approximated in the figure represented by 

vehicle tank fuel level at the beginning of the refueling event. 
P,9 

- 5 - 87-31.3 

Based on che expected temperature effect (Figure 2), exposure to 

gasoline vapors, other things held constant, is expected to decrease from 

summer to winter if there is no change in the fuel specifications. 

However, as "indicated in Table II (columns 4,5,7 and 8) , fuel properties 

are adjusted by suppliers for season and geographical location. This 

adjustment, in part, helps to Insure good driveability and engine starting 

throughout the year. Average normal teaperatures on record*-* for each 

state also are listed In Table II along with average fuel volatility 

data,*^ RVP. The ratio of the average change in fuel RVP in going fro® 

summer (July) to winter (January) divided by the change in average normal 

temperature over the same period can be used as an indication of both fuel 

and seasonal changes. This ratio for each state is listed in the far 

right column of Table II. Based on Figure 2, however, if the ^P/<^>T 

ratio is less than about 0.18 psi/°F, then winter refueling emissions and 

exposure would be less than in the summer. The 0.18 psi/°F value was 

calculated by drawing a horizontal line (no change in emissions) between 

the 8.9 and 12.2 psi fuel curves and dividing this pressure difference 

(3.3 psi) by the temperature difference along the horizontal axis. The 

ratio will vary slightly with starting position. The ^ P/^>T ratios in 

Table II for each state are less than 0.18 psi/°F with the single 

exception of Los Angeles, California which has a relatively large change 

in summer to winter fuel volatility compared to a rather small change in 

temperature (Los Angeles already has stage II). Therefore, almost without 

exception, winter refueling emissions and exposure concentrations are 

expected to be smaller than during the summer. 

(3) Reported averages of all available yearly average surface wind 

speed data through 1984 for 43 U.S. cities** was found to have a mean 

value of 9.2 mph plus or minus one standard deviation of l.S mph. To a 

first approximation, one would expect gasoline vapor concentration to be 

proportional to the tank fill rate and inversely proportional to wind 

speed. Because mechanical turbulence in wind velocity is likely to be 

large around physical entities in the fuel service area, no attempt to 

correct for wind speed or wind direction was made. 

(4) Average reported benzene concentration data also are listed in 

Table I along with the computed ratio of gasoline vapor to benzene. The 

ratio of gasoline vapor to benzene depends upon a number of factors 

including fuel benzene level, gasoline volatility, and temperature. The 

ratio tends to be higher at lower temperatures and can change by as much 

as a factor of 2 or more over the ambient temperature range of the data in 

Table I for typical gasolines. The GM data*0 are an example of this. The. 

ratio is lowest at high temperature, low volatility, and high fuel benzene 

content. As an example, a mathematical fuel model*^ predicts a gasoline 

vapor to benzene ratio of 90 for 100°F, indolene gasoline (9 psi RVP), and 

2 volume percent benzene. Because the data in Table I represent lower 

temperatures, generally higher volatility fuels, and in many cases lower 

volume percent fuel benzene than the example given above, the ratios would 

most likely exceed 90. 

Except for the unleaded samples taken in Philadelphia and premium 

unleaded samples in Houston, the gasoline vapor to benzene ratios for the 

CEC data appear to be too low. Therefore, unless the CEC study was 

conducted for gasoline stations selling significantly higher than expected 
-

 6 - 87-31.3 

benzene content (not very likely), the data appear questionable. An 

inquiry with CEC*^ revealed that the reported total HC and hence the 

gasoline* vapor data, may be somewhat low due possibly to some missing 

light hydrocarbon species but ehae ehe benzene daea were considered 

reliable for the sampling conditions used. This explanation appears 

correcto The daea from some of ehe oehee studies in Table I also may have 

failed to measure all hydrocarbon vapors but this appears eo be much less 

of a problem due to their generally higher ratios. Samples in the CEC and 

Shell Oil program were originally collected using charcoal tubes and 

processed in the laboratory using carbon disulfide extraction. This 

method is known to lose very light hydrocarbon species and requires a 

relatively large sample for adequate detection limits. 

(5) The EPA study took the most care in defining the "breathing 

zone" sampling location. However, the selected placement of the sampling 

probe in the other studies, although not well defined, still must be 

considered because there remains some question as to what location besc 

represents average exposure. Because of these uncertainties, one might 

base an exposure estimate on the simple average of all data In Table I 

(about 60 ppm) However, when referring to such an exposure estimate one 

must keep in mind that the missing hydrocarbon species may have caused the 

estimate to be too low. On the other hand, an overrepresentation of 

reverse parallel wind, low wind speed, above average temperatures, and 

untypically short exposure distances may have biased such an exposure 

estimate on the high side. 

3. EXPOSURE TIME 

The duration of exposure is dependent upon the number of gallons of 

gasoline to be transferred to the vehicle and the pumping rate assumed for 

each self-service fillup. Gasoline pumps typically operate between 8 to 

12 gallons per minute. The average yearly fuel consumption for all motor 

vehicles (cars and trucks) is almost 700 gallons per year*&. At a 10 

gallon per minute fill rate, total average exposure duration would be 

about 70 minutes per year, of course not all at one time. Assuming 1 to 2 

trips to the service station per week per vehicle, an exposure duration 

per fill would range from about 1/2 to 1-1/2 minute. The EPA selected the 

1-1/2 minute time period for their sampling program8. 

4. COMPOSITION OF REFUELING VAPOR 

Both theoretica l computations*^»* ' and refuelin g vapor 

measurements*'s*8»*9 demonstrate that the composition of refueling vapor 

is strongly dependent on the composition of the liquid gasoline, but, as 

expected, the vapor is composed primarily of the more volatile components 

of gasoline. 

The chemical components found in gasoline encompass a wide-boiling 

range (-47 to above 400°F) and represent a large number of homologous 

hydrocarbons derived from petroleum. Such mixtures are nearly ideal in 

the absence of oxygenates (alcohols). An ideal mixture, by definition, 

obeys Raoult's law which states that the partial pressure of each 

component in the vapor phase Is directly proportional to its molar 

composition or to the number density of component molecules in the liquid 

fuel. Raoult's law may be described by the equation: 
P.11 

- 7 - 87-31.3 

Pi ° Pi .

Ei 

where p^ ° the partial pressure of component i 

?l ° the vapor pressure of pure component i 

m^ <=> the molar or number fraction of component i in the 

liquid fuel 

If we divide both sides of the above equation by the total fuel vapor 

pressure and multiply by 100, then we have the vapor mole percent or vapor 

concentration percent VC^ which is given by: 

VCA o 100 (Pi m t / ( 2 Pi taj.)) 

This equation can be used to estimate the percent concentration of 

any particular component in gasoline vapor if its fuel molar concentration 

and vapor pressure are known. Of course non-ideal mixtures, such as an 

alcohol and gasoline blend, are more complex, but Raoult's law can still 

provide useful preliminary concentration estimates in such cases for 

initial health evaluations, until more accurate data become available. 

Figure 3 illustrates the difference in composition between refueling 

vapor and liquid fuel based on calculations using Raoult's law for an 

unleaded gasoline fuel (#8532, 80°F, Reference 12). For convenience, the 

fuel components along the X-axis are ordered right to left in terms of 

increasing component volatility. The figure illustrates the expected 

higher concentration of the more volatile components in the vapor compared 

to the liquid phase. In this particular case, more than 94% of the gas 

vapor molecules are C5 and lighter hydrocarbons whereas these same 

components were only 34 volume percent of the liquid gasoline. At lower 

temperatures, the percentage (but not the absolute quantity) of light 

hydrocarbons in gasoline vapor increases. Experimental data*°>*' are 

consistent with the results shown in Figure 3. Therefore, the composition 

of gasoline vapor differs significantly from the wholly vaporized gasoline 

used in animal studies.* 

5. NATIONAL AVERAGE VAPOR CONCENTRATION EXPOSURE ESTIMATE 

Several years ago, EPA made an estimation of the public health risk 

from exposure to gasoline vapors during vehicle refueling.*0 In that 

estimate, EPA took the arithmetic average of the reported values in the 

CEC study and derived exposure concentration levels very close to those 

listed as "CEC Overall Averages" in Table I (small differences were due to 

rounding and conversion factor assumptions). However, the CEC study did 

not attempt to select a random sample of service stations representative 

of the geographic, population, or seasonal temperature distributions, 

biasing the exposure estimate high. On the other hand, some missing light 

hydrocarbons may have biased the estimate low as discussed previously. 

Until better data become available, the 60 ppm hydrocarbon estimate, 

discussed in Section 2 (5), can be used with caution and with the 

understanding that variations from the average can be large for each 

individual refueling event. 

t "-^yg .'"a l •r-.^AA irArr^n^jm.-r^mrmm .r..^-. 
- 8 - 87-31.3 

6. CONCLUSIONS 

Based on the study contained in this paper, the following conclusions 

are drawn: 

The orientation of the person to wind direction and his 

distance from the pump nozzle are the most significant 

parameters affecting potential exposure to gasoline 

vapors. 

Other things being equal, such as wind direction and 

distance, winter refueling exposure, on average, is 

expected to be smaller than summer exposure with few 

exceptions despite higher volatility winter fuel. 

For most situations, the ratio of gasoline vapor to 

benzene concentration will be greater than 90. This 

indicates that benzene concentration usually will be 

about IZ (ratio of 100) or less of the hydrocarbon 

vapors. Exposure to benzene during refueling operations 

in the winter is expected to be lower not only because 

the concentration of total hydrocarbons is lower but 

also because the percentage of benzene can be half that 

during summer refueling operations due to temperature 

effects. Refueling vapor contains mainly light 

hydrocarbons, in many cases 90+2 C5 and lighter. At 

lower temperatures, the percentage of light hydrocarbons 

increases. 

There still remains some question as to what location 

best represents average exposure. Until better data 

become available, an average hydrocarbon concentration 

of about 60 ppm can be used with caution. Variations 

from the average can be large for each individual 

refueling event. 

Exposure duration per fill ranges from about 1/2 to 1 

and 1/2 minutes. This would amount to about 70 minutes 

exposure per year. 

Raoult's law is a useful tool to estimate the 

concentration of individual components in gasoline 

vapor. 

Because the composition of refueling vapor is vastly 

different from wholly vaporized gasoline, one should not 

extrapolate potential health effects from one situation 

to the other without taking this difference into 

consideration. So far, this recommendation has not been 

adequately addressed. 

JWS.20 
P.13 

- 9 - 87-31.3 

REFERENCES 

1. Statement of Administrator Lee Thomas of the Environmental Protection 

Agency Before The Subcommittee on Health and the Environment, 

Committee on Energy and Commerce, U.S. House of Representaclves, on 

Nationwide Ozone Nonattainment, Washington, D.C, February 19, 1987. 

2. A Chronic Inhalation Study with Unleaded Gasoline Vapor, 

H. N. MacFarland, et al., Journal of the American College of 

Toxicology, Vol. 3, Number 4, 1984. 

3. Gasoline Vapor Exposure and Human Cancer: Evaluation of Existing 

Scientific Information and Recommendations for Future Research, 

Report of the Institute's Health Review Committee, Health Effects 

Institute, September, 1985. 

4. Clayton Environmental Consultants, Report on a Gasoline Exposure 

Study for the American Petroleum Institute, CEC Job No. 18629-15, 

draft copy, August 25, 1983. 

5. Service Station Attendants' Exposure to Benzene and Gasoline Vapors, 

McDermott and Vos. J., Shell Oil Company, Am. Ind. Hyg. Assoc. J. 40, 

315-321, April, 1979. 

6. Battelle-Columbus Laboratories (1977), as cited in p. 115, of 

Reference 7. 

7. Mara and Lee, Assessment of Human Exposures to Atmospheric Benzene, 

EPA-450/3-78-031, 1978. 

8. Self Service Station Vehicle Refueling Exposure Study, 

Andrew E. Bond, Vinson L. Thompson, and Gordon C. Ortman, EPA 

Environmental Monitoring Systems Laboratory, and Francis M. Black and 

John E. Sigsby, Jr., EPA Atmospheric Sciences Research Laboratory, 

June 1985. 

9. Gasoline Vapor Exposures at a High Volume Service Station, 

C A. Kearney and 0. B. Dunham, Mobil Oil Corporation, Am. Ind. Hyg. 

Assoc. J. 47 (8), 535-539, 1986. 

10. Measurement of Vapor Exposure During Gasoline Refueling, SAE paper 

#860087, G. Tironi, G. Nebel and R. Williams, presented at the 

Society of Automotive Engineers 1986 International Congress and 

Exposition, Detroit, Feb. 24-28, 1986. 

11. The World Almanac and Book of Facts, 1986, Newspaper Enterprise 

Association, Inc., Published for the Detroit News, 1986. 
P.14 

- 10 - 87-31.3 

12. Mathematical Predictions of the Effects of Gasoline Composition on 

Reid Vapor Pressure, Refueling Emissions and Their Reactivity, 

J. W. Shiller, SAE paper #860532, presented at the Society of 

Automotive Engineers' 1986 International Congress and Exposition, 

Detroit, Feb. 24-28, 1986. 

13. Technical Report, Refueling Emissions from Uncontrolled Vehicles, 

D. Rothman and R. Johnson, EPA-AA-SDSB-85-6, August 1985. 

14. MVMA 1985 winter and summer Gasoline Surveys. 

15. Phone communication, E. C. Su, Ford, and Mr. Walcott, Clayton 

Environmental Consultants, February, 1985. 

16. MVMA Motor Vehicle Facts and Figures '86. 

17. Composition of Vapor Emitted From a Vehicle Gasoline Tank during 

Refueling, R. L. Furey and B. E. Nagel, SAE paper #860086, presented 

at the Society of Automotive Engineers' 1986 International Congress 

and Exposition, Detroit, February 24-18, 1986. 

18. Quest for a Gasoline TLV, H. J. McDermott and S. E. Killiany, Jr., 

Am. Ind. Hyg. Assoc. J. (39), February, 1978. 

19. Gasoline Vapor Exposure During Bulk Handling Operations, 

C. F. Phillilps and R. K. Jones, Am. Ind. Hyg. Assoc. J. (39), 

February, 1978. 

20. Estimation of the Public Health Risk from Exposure to Gasoline Vapor 

Via the Gasoline Marketing System, EPA Draft Staff Paper Submitted 

for review to the Science Advisory Board by OHEA and OAQPS, EPA, 

June, 1984. 

21. Motor Vehicle Manufacturers Association U.S. Gasoline Survey, Summer 

1983. 

JWS.20C 
P.15 

87-31.3 

Table I - Refueling Studies of Short Term (Few Minutes) Exposure to "Breathing 

Zone" Gasoline Vapor 

Study/Date/Fuel/Location 

*CEC Overall Averages (Aug.,'83)(1) 

Unleaded 

Leaded 

Premium Unleaded 

Philadelphia (8l°F, 10.5 mph) 

Mariecca, Ga. (9l°F, 10 mph) 

AClanca (81°F, 5.7 mph) 

Monterey, Ca. (75°F, 5 mph) 

Carmel, Ca. (66°F, 8 mph) 

Salinas, Ca. (70°F, 7 mph) 

Bellaire, Tx. (92°F, 7 mph) 

Houston (87°F, 10.3 mph) 

*Shell Oil Company (2) 

Houston (May, '77) (75°F) 

N. Britain, Ct.(Jul,'77) (73°F) 

N. Orleans (Jun, '77) (80°F) 

*Battelle (3) 

*EPA Raleigh, N.C. (4) 

(Feb., '85) 

Average 

Gasoline 

Vapor/ppm 

1 

56.5* 

70.5* 

66.2* 

ied/Premium i 

89/129/-* 

36/ 51/-* 

61/ 70/73* 

20/ 19/-* 

34/-/51* 

62/ 47/-* 

80/ 16/55* 

48/ 82/68* 

54.4 

40.0 

77.3 

Average 

Benzene 

(ppm) 

0.96 

1.42 

0.81 

(D 

0.63/1.9/-

0.61/0.97/-

1.12/1.28/0 

0.57/0.94/-

1.2/-/1.4/-

1.8/1.1/-

1.6/0.57/0.', 

0.71/1.94/0 

0.11 

0.46 

0.96 

Ratio of 

G. Vapor 

to Benzene 

59 

50 

82 

141/68/-

59/53/-

.84 54/55/87 

35/20/-

28/-/36 

34/43/-

74 50/28/74 

.61 .„ 68/42/111 

495 • 

87 

81 

Benzene in 

Gasoline/% 

-

-

(7) 

1.1/.93 

1.8/.63 

1.5/.96 

1.3/1.3 

.97/.88 

.43/.38 

(2)' 

(2) 

(2) 

E. 

Ga 

N. 

s. 

S. 

N. 

Pa. 

m 

Ca. 

Ca. 

Tx. 

Tx. 

0.27 

Parallel Wind (57°F, 4.5 mph) 24* 

(13.1 RVP) 

Reverse Parallel (55°, 6.5 mph) 0.01* 

(12.9 RVP) 

0.23 104 2.02 of Carbon 

1.9% of Carbon 

*Mobil Oil Corporation (5) 

E. Pennsylvania 

(May, '83) (63°F) ~ 29 

•General Motors Detroit Area (6) 

(Aug./Sept. '85, 64'F, 10.4 RVP) 57* 

(Feb.'85, 19°F, 13.7 RVP) 218* 

0.50 

0.97 

114 

224 

3.2Z of Carbon 

3.2Z of Carbon 

(1) Computed from the data in Reference 4. 

(2) Taken from Reference 5. 90Z of gasoline samples had benzene content 

< - IZ (Leaded), < - 1.3Z (Unleaded), and < - 1.5Z (Unleaded Premium). 

(3) Taken from Table IX-4 of Reference 7. 

(4) Taken from Reference 8. Parallel and Reverse Parallel refer to the wind direction, 

either toward (parallel) or away from (reverse parallel) the person filling the tank 

with reference to the vapor source. 

(5) Taken from Reference 9, corrected to 2 minute exposure time. 

(6) Taken from Reference 10. 

(7) Average benzene content in 1983-summer gasoline, MVMA gasoline survey, vol Z, Ref. 21. 

* The conversion to ppm was made assuming 4.6 carbons per molecule, an average molecular 

weight of 66 and a hydrogen/carbon ratio of 2.4 for gasoline vapor. 
FIGURE 1 

Hydrocarbon Vapor Concentration Measured During 

Vehicle Unleaded Gasoline Refueling Operations 

•=^ & 

Parallel Wind 

4.5 mph Velocity 

O . 

Reverse Parallel 

Wind. 6.5 mph 

—O— 

Wind blowing vapors 

toward detector 

Wind blowing vapors 

away from detector 

012345678. 9 

Horizontal distance from fuel tank opening, feet 

Note: The 2 foot horizontal distance (5 feet 

above ground) was selected as the approximate 

location of the breathing zone during refueling. 

10 

TJ 
FIGURE 2 

Calculated Refueling Loss Versus Temperature 

Assuming Tank And Added Fuel At Same Temperature 

Refueling Loss. Grans Per'Gallon 

9r 

8 

7 

6 

5 

4 

3 

2 

1 

RVP 12.2 — 40X Full 

,QRVP 12.2 — 10% Full 

•~G -

^r*—2&r« •*"-"' o^iV' RVP 10.5 — 10% Full 

RVP 8.9 — 40% Full 

m^mWm-mm^mmm^m * ^MM » 

RVP 8.9 — 10% Full 

—£ r— 

.... i... . i... . i. . i . i... . i... . i... . i... . i....i... . i....i....i....i....i....i.... i 

$ *$> *$> 4> & &<$>4>&4><p4>#4><fm.4>tt$ 

Fuel Temperature. Degrees F 

Reference 12. 

P 
FIGURE 3 

Relative Abundance Of Gasoline Refueling Vapor 

Components Compared To Liquid Fuel Composition 

Vapor/Fuel 

40 

35 

30 

25 

20 

15 

10 

5 

0 

volume percent 

1 

1 Ix 

1 '^K^ 

1 fl K 

' • H X m-TrH B S B ( EL & 

<> r<> m<> vO -vO V ^ , C V<> , 0 V ^ rS> VO 

K

V

«AV<AV A v

 t O \ ^ A% A % \\>\0\0\v>\\>\0'\0'\K v

^

v

' -

V 

oV^oO^ <$> 0 % VJ *v VJ % <-."• CP 

r?> r°> K<> ° * 

Increasing volatility 

Vapor Volume % 

Fuel Volume % 

Fuel #8532. 80 degrees F, Reference 12. 

TJ 

co 
Sft C 

Alabama 

Alaska 

Arizona 

Arkansas 

California 

Colorado 

Connecticut 

Delaware 

Florida 

Georgia 

Hawaii 

Idaho 

Illinois 

Indiana 

Iowa 

Kansas 

Kentucky 

Louisiana 

Maine 

Maryland 

Massachusetts 

Michigan 

Minnesota 

Mississippi 

Missouri 

Montana 

Nebraska 

Nevada 

New Hampshire 

New Jersey 

New Mexico 

New York 

North Carolina 

North Dakota 

Ohio 

Oklahoma 

Oregon 

Pennsylvania 

Rhode Island 

South Carolina 

South Dakota 

Representative 

Cities 

Mobile 

Juneau 

Phoenix 

Littl e Rock 

Los Angeles, 

San Francisco 

Denver 

Hartford 

Ui Islington 

Jacksonville, 

Miami 

Atlanta 

Honolulu 

Boise 

Chicago 

Indianapolis 

Des Moines 

Wichita, 

Kansas City 

Louisville 

New Orleans 

Portland 

Baltisnre 

Boston 

Detroit, Sault 

Ste. Marie 

Him-St. Paul 

Jackson 

St. Louis 

Helena, Billings 

Oaaha 

Reno, Las Vegas 

Concord 

Atlantic City 

Albuquerque, 

New York, Albany 

Charlotte, Raleigh 

Bismark 

Cincinnati, 

Cleveland 

Oklahoma City 

Portland 

Philadelphia, 

Harrisburg 

Block Island 

Charleston 

Huron, Rapid City 

t 

State 1980 

Population 

Fraction 

1.72 

0.18 

1.20 

1.01 

10.45 

1.28 

1.37 

0.26 

4.30 

2.41 . 

0.43 

0.42 

5.04 

2.42 

1.29 

1.04 

1.62 

1.86 

0.50 

1.86 

2.53 

4.09 

1.80 

1.11 

2.17 

0.35 

0.69 

0.35 

0.41 

3.25 

0.58 

7.75 

2.60 

0.29 

4.77 

1.34 

1.16 

5.24 

0.42 

1.38 

0.30 

TABLE 11 

'/Winter unleaded Regular Gasoline and Jeapprature Data by State 

Suaaer 

(July) 

Gasoline 

Class 

ASTN 0439 

C/B 

0 

s: A 

B 

B/A 

B/A 

C 

C 

C 

C/B 

C 

B 

C/B 

C 

B/C 

B 

C 

C/B 

C 

c 

c 

c 

c C/B 

B 

B 

B 

B/A 

C 

C 

A 

C 

C/B 

B 

C 

8 

C/B 

C 

C 

C/B 

B 

Unleaded • 

SUM - C1985) 

<Juty> 

Fuel Volatility 

(osi. RVP) 

11.1 

12.7(u) 

8.3* 

10.7 

8.9* 

10.2* 

11.5 

11.1 

11.4* 

10.3* 

11.6 

10.3 

12.6* 

12.6 

12.6 

10.7* 

11.6 

11.1* 

11.5 

11.1 

11.5* 

12.0* 

11.3* 

11.1 

10.4* 

10.3* 

10.7 

8.6* 

11.5 

11.1 

9.5* 

11.5* 

11.4 

10.3 

12.1* 

10.7 

11.4 

11.1* 

11.5 

10.3 

10.3 

Suaaer 

(July) 

Average 

82 

56 

92 

82 

68 

73 

73 

76 

82 

79 

80 

75 

73 

75 

76 

82 

78 

82 

68 

77 

74 

68 

73 

82 

79 

68 

76 

70 

70 

74 

79 

74 

78 

70 

74 

82 

68 

76 

70 

82 

74 " 

Winter 

(January) 

Gasoline 

Class 

ASTND439 

0 

E 

D 

E/D 

E/D 

E 

E 

E 

D 

D 

C 

E 

E 

E 

E 

E 

E 

0 

E 

E 

E 

E 

E 

D 

E 

E 

E 

E/D 

E 

E 

E/D 

E 

E/D 

E 

E 

E/D 

E 

E 

E 

D 

E 

Unleaded • 

Winter (1985) 

(January) 

Fuel Volatility 

(osi. RVP) 

12.7 

14.6 

12.5* 

12.7 

12.2* 

13.4* 

14.0 

14.6 

12.8* 

13.0* 

11.6(8) 

14.1 

14.9* 

14.9 

14.9 

14.7* 

14.5 

12.7* 

14.2 

14.6 

14.2* 

15.3* 

14.8* 

12.7 

14.4* 

14.1* 

14.7 

12.6* 

14.2 

14.6 

13.3* 

14.0* 

12.7 

14.1 

15.2* 

12.7 

14.6 

14.6* 

14.2 

13.0 

14.1 

Winter 

(January) 

Average 

Teaperature 

51 

22 

52 

40 

53 

30 

25 

31 

60 

42 

73 

30 

21 

26 

19 

30 

33 

52 

22 

33 

30 

18 

11 

46 

29 

18 

19 

32 

20 

34 

35 

27 

40 

7 

27 

36 

39 

31 

31 

49 

16 

A? 

A*i 

mSmmmi-tk 

0.052 

0.056 

0.105 

0.048 

0.220 

0.074 

0.052 

0.078 

0.064 

0.073 

0.000 

0.084 

0.044 

0.047 

0.040 

0.077 

0.064 

0.053 

0.059 

0.080 

0.061 

0.066 

0.056 

0.044 

0.080 

0.076 

0.070 

0.105 

0.054 

0.088 

0.086 

0.053 

0.034 

0.060 

0.066 

0.043 

0.110 

0.078 

0.069 

0.082 

0.066 
TABLE 11 (Continued) 

State 

Tennessee 

Texas 

Utah 

Vermont 

Virginia 

Wash ington 

West Virginia 

Wisconsin 

Wyoming 

Nat. Capitol 

Representative 

Cities 

Nashville 

Amarilo, Galveston 

Houston, San 

: Antonio, Dallas 

Salt Lake City 

Burli ngton 

Norfolk 

Seattte-Tacoma, 

Spokane 

Huntington 

Madison, Milwaukee 

Cheyenne 

Washington, D.C. 

i 

State 1980 

Population 

Fraction 

2.03 

, 6.28 

0.64 

0.23 

2.36 

1.82 

0.86 

2.08 

0.21 

0.25 

Stmner 

(Juty) 

Gasoline 

Class 

ASTM D439 

C/B 

B/A 

i 

B/A 

C 

C 

C/B 

C 

C 

B 

C 

Unleaded • 

Sterner (1985) 

(July) 

Fuel Volatility 

(esi. RVP) 

10.3 

10.7* 

10.2 

11.5 

11.1 

11.4* 

11.1 

12.6 

10.2 

11.1* 

* 

Summer 

(July) 

Average 

Te»ger*,tyre 

79 

82 

78 

70 

78 

67 

76 

71 

69 

76 

Winter 

(January) 

Gasoline 

Class 

ASTRD439 

E/D 

0 

E 

E 

E 

E 

E 

E 

E 

E 

Unleaded • 

Winter (1985) 

(January) 

Fuel Volatility 

fosi. RVP) 

13.0 

13.0* 

13.4 

14.2 

13.8 

14.6* 

13.8 

14.9 

13.4 

13.8* 

Winter 

(January) 

Aver mat 

Temperature 

37 

47 

29 

17 

40 

32 

33 

17 

26 

31 

4: 

Smil^l 

0.064 

0.066 

0.065 

0.051 

0.071 

0.091 

0.063 

0.043 

0.046 

0.060 

Total 100.00 

• Reference 14. States with an asterisk (*) are covered in the fuel survey. Values given for states not surveyed represent the a nearby survey 

result. The average temperature data are normal temperatures on record for the thirty-year period 1951 to 1980 inclusive for the months, and 

for the representative cities indicated, reference 11. 

JUS.200 
P.21 

The Engineering 

Resource For 

Advancing Mobility 400 COMMONWEALTH ORIVE WARRENOALE. PA 15096 

SAE Technical 

Paper Series 

860532 

Mathematical Prediction of Effects of 

Gasoline Composition on Reid Vapor Pressure. 

Refueling Emissions and their Reactivity 

J. W. Shiller 

Automotive Emissions and Fuel 

Economy Office 

Ford Motor Co. 

International Congress and Exposition 

Detroit, Michigan 

February 24-28, 1986 
P.22 

860532 

Mathematical Prediction of Effects of 

Gasoline Composition on Reid Vapor Pressure, 

Refueling Emissions and their Reactivity 

J. W. Shiller 

Automotive Emissions and Fuel 

Economy Office 

Ford Motor Co. 

ABSTRACT 

Concern ove r the Impacts of change s ln 

gasolin e composition on refueling , emission s 

stimulated a search for a simple technique to 

provide estimate s of related fuel parameters. 

Accordingly, a mathematical computer model was 

developed based on the assumption that gasoline 

liquid-vapor equilibri a can be predicted with 

reasonable accuracy from Raoult's law for Ideal 

mixtures. Gasoline- fue l parameters such as 

vapor pressure (Including Reid Vapor Pressure , 

RVP), refueling loss, OH reactivit y in forming 

atmospheric ozone pollution , and othe r fue l 

characteristic s wer e addressed . The 

assumption s made and mode l result s wer e 

validate d by compariso n t o measured fue l 

parameters from 9 unleaded gasolin e fuels . 

Extrapolations were made to Illustrat e fue l 

temperature and vapor/liqui d rati o effects . 

Calculated RVP levels were found to be within 

5% of measured values for the fuels evaluated. 

A complete- listin g of the model's source code 

written,speciflcall y fo r a Fortun e 32:1 6 

microcomputer Is provided. 

OVERVIEW - Befor e emissio n controls on 

automobile s were established , the amount of 

gasoline vapor lost during refueling operations 

was only a small fraction (less than 3Z) of the 

tota l vehicl e hydrocarbon s emitted . (*•' 

However, as the control requirements on exhaust 

and evaporative emissions were progressivel y 

made more stringent, refueling emissions as a 

calculate d percentag e o f tota l vehicl e 

emissions became large r in comparison although 

not In magnitude . Due t o the difficult y 

experienced by a number of locations ln the 

United State s in the l Q70's and early 1980's In 

trying to achieve the ambient ozone ai r qualit y 

standar d throug h hydrocarbo n control s by 

mandated Clean Air Act deadlines, the potentia l 

for any additiona l hydrocarbon reduction became 

of major concern for some states. Because of 

this , al l hydrocarbo n sources , includin g 

refueling emissions, have come under scrutiny . 

An importan t facto r t o conside r i n 

evaluatin g refuelin g emission s Is th e 

composition of commercial gasoline . Gasoline 

composition has been changing over time in 

response to a number of factors. For example, 

the addition of butane (a relativel y volatil e 

hydrocarbon ) t o gasolin e ha s becom e 

economicall y attractiv e as butane's market 

price has been low ln relationship to the pric e 

of gasoline . Addition of butane Increase s 

octane , decreases anergy content and Increases 

fuel volatility . Figure I Is art illustratio n 

of the historica l Increas e In the averag e 

volatilit y of summer unleaded regular gasoline 

FIGURE 1 

SUM P Gasoline Volatility Trend. MVMA Survey 

Average RVP Of Unleaded Regular Gasolines 

Reid Vipar Pressure. 

11 r 

10 

psi 

9 -

•Numbers in parentheses designate references at end ot paper. 

1978 1971 19M 19 

Ci lender Veer 

1984 

0148-7191 /86/0224-0532S02.50 

Copyright 1986 Society of Automotive Engineers, Inc. 
860532 

P.23 

fina l equilibrium vapor pressure depends on the 

temperature and on the vapor/liquid ratio . 

The above simplifying assumptions regarding 

the gasolin e vapor-liqui d system allo w the 

vapor and liquid composition, such as In an RVP 

bomb, to be calculated from a knowledge of the 

fuel composition and the vapor pressures of the 

major constituents in the gasoline . Similarly , 

for a given fuel leve l in a vehicl e fuel tank, 

the vapor composition In equilibrium with the 

gasoline in the tank and hence the refueling 

losse s may he estimate d fo r a specifie d 

temperature . For suc h computation s an 

Iterativ e procedur e is require d to adjust 

component concentrations unti l the vapor/liquid 

equilibrium is found. The iterattv e procedure 

Incorporated In the model ensures convergence, 

usually within 10 iteration s for a vapor/liquid 

rati o of 4 . The smalle r the vapor/liqui d 

ratio , the faste r the convergence. 

The general vapor pressure and temoerature 

relationshi p used ln the model is based on 

published work by van Kranen and van Nes 

Their equation relate s the vapor pressure of a 

pure hydrocarbon t o Its boilin g poin t as 

follows : 

logio Pt 

where 

6 07918-1 io(n7 * ( 232+B)«(U20-t) 6.07918-3.19837* (U20 _ B ) * ( 232+c ) 

Pt • vapor pressure of the hydrocarbon in 

ram Hg at temperature t in degrees C. 

B « boiling point of the hydrocarbon at 

atmospheric pressure , in degrees C. 

Although thi s equatio n Is quit e good , tt 

represents a generalizatio n applie d to al l 

hydrocarbons. However, when a generalization 

is not essentia l as a simplifying assumption, 

othe r equations can be used to more closel y 

match the behavio r of individua l gasolin e 

components . A numbe r o f functiona l 

relationship s are availabl e for this purpose 

(e.g. , Riedel, Antolne", Frost-Kalkwart-Thodos, 

etc.^Vapo r pressure equatlons)^1-^). r n this 

study specia l equations were generated for some 

of the more volatil e gasoline components based 

on published vapor pressur e and temperature 

data(">^ ) • Published data were related to a 

second orde r La Grange polynomia l fo r the 

logarithm of pressur e as the form for the 

selecte d functiona l relationship . Other 

functional relationships als o are possible . A 

separat e equatio n was generate d fo r each 

componen t o f interest . Suc h specifi c 

modifications to the generalized van Kranen and 

van Nes approach are desirable , particularl y 

for the more volatil e hydrocarbons. The La 

Grange polynomial technique assures perfec t fit 

to published data points with interpolatio n 

between points. The order of the polynomial 

plus one represents the number of points of 

perfec t fit . Three dat a points fo r each 

constituent were used in this study, selected 

to include the expected range of pressure s 

Important for that constituent In calculating 

In-use automotive gasoline tank vapor pressure. 

HYDROCARBON REACTIVITY - Hydrocarbon 

emissions are controlled because of the role 

the y pla y In formin g atmospheri c ozon e 

pollution . Individual hydrocarbon compounds, 

however, do not al l contribut e equally to the 

production of photochemical smog and diffe r 

significantl y tn thei r potentia l or reactivit y 

to form ozone . The flr9 t effor t to apply a 

practica l hydrocarbon reactivit y scal e occurred 

In 196 6 wit h th e implementatio n o f a 

regulation , known a s Rul e 66 , by the Los 

Angeles Air Pollution Control Distric t to limit 

organi c emission s on th e basi s of thei r 

capacity to form photochemical smog Since 

then , othe r reactivit y scale s hav e bee n 

proposed(l''~21 ) an< i a non-methane exhaust 

hydrocarbon vehicl e standard forms a part of 

the Californi a ai r qualit y program, on the 

basl3 that methane ts essentiall y inert. 

Reactivit y scale s are developed with the 

goal of determining which hydrocarbons add to 

the ambient ozone/oxldant problem and which are 

of littl e or no consequence. Candidates tn the 

latte r category might Include methane, ethane, 

propane , acetylene , benzene , and methanol. 

However, a lis t of compounds that may be of 

littl e consequenc e fo r singl e day ozon e 

episodes (singl e days In which ambient ozone 

concentrations exceed the standard) might be of 

some consequence In multi-day episodes. In 

multi-day episodes, stagnant air , lastin g for 

several days, traps emissions and allows more 

time for reaction s to occur and for ozone 

concentrations to build up before significan t 

dilutio n can take place . In any event, lower 

reactivit y tends to reduce the rate of ozone 

formation , such tha t highe r ambient ozone ' 

concentrations would be less likely . 

T he mode l develope d In thi s stud y 

calculate s photochemical reactlvtt y of gasoline 

vapor on the basis of hydrocarbon OH reaction 

rate data. The use of OH reaction rate data 

for hydrocarbon reactivit y classificatio n has 

been proposed previously.(-0.21 ) i n contrast 

to other scale s based on simulated laboratory 

yield s of ozone, i t focuse s directl y on the 

actual Initia l rate s of photooxldatlon, a major 

rate controlling step for ozone formation. Such 

rat e data for hydrocarbons of Interest are 

liste d in Table 1.(22,23 ) 

TABLE I 

OH REACTIVITY RATE DATA 

Compound -

M<*r.h*n* 

Ethan« 

Propane 

Hut t\n** 

P'*nt.»«** 

2-<nrthyl- 2 butan e 

reltinnl 

McttiAnn I 

B»nr»ne 

Tolu^n* 

o-xvlen f 

[sopren f 

OH Reactio n 

Rflf* 

(cm*-"in 

1 

2 . 7 

1 . 5 

7. . 4 

1.7 

l.fl 

fl 

"... \ .0 

I . ? 

fs.m 

1 .2 

1. 1 

9 . 9 

ecule"W"1) 

X 

It 

* 

X 

X 

X 

< 

X 

X 

X 

X 

X 

X 

X 

,0-1 5 

m-ii 

io-<2 

m-<2 

IO-' 2 

n-' i 

11-1 2 

10-1 2 

,0-1 2 

10-1 2 

10" " 

10-11 

io-'< 

OH 

<«€ 

t o 

Reactio n 

A. Relativ e 

Methane 

I 

14 

2 4 0 

JOO 

n« 

4*5 

10,OOO 

41*. 

125 

HO 

'2 5 

1,50 0 

1.1B0 

1 2 . IRO 
860532 

shown i n Tabl e 4 . Thus, equation s wer e 

developed , usin g secon d orde r La Grange 

polynomials, as discussed earlier, , to account 

for volume and densit y changes with temperature 

for each major fuel constituent. Temperature 

corrected densit y values are used during the 

model iteratio n process i n estimatin g Fuel 

vapor pressure . When measured relativ e volume 

data were not availabl e for a component, the 

relativ e volume equatio n for a chemicall y 

similar component was used. 

TABLE 4 

RELATIVE V0LU1ES OF VARIOUS COMPOUNDS AS 

FUNCTIONS OF TEMPERATURE AT 

ATMOSPHERIC PRESSURE* 

distinctl y differen t bas e fuels for which 

measured fuel parameters are available . 

MODEL OUTPUT FORMAT - Tables A-l through 

A-6 in Appendix A Illustrat e the format For the 

display of the results obtained for the three 

base fuels using the fue l composition data 

obtained from the CRC program (25 ) § a fue ^ 

temperature of 37.8°C (100"F) and a vapor/fuel 

rati o of 4 (used in RVP calculations) . In 

Appendix A there are two table s for each fuel, 

thos e endin g l n odd number s lis t fue l 

characteristic s whil e thos e ending In even 

numbers lis t fue l vapor characteristics. A fuel 

summary als o Is Included at the bottom of even 

numbered tables. With this background, model 

output results wil l now be discussed. 

DISCUSSION OF RESULTS 

Relativ e Volume a t Temperature , OeRrecs C 

Compound 0"C "MT'C 50 "C 60 "C 95'C 

Octane I.000 0 

Benspne I.000 0 

Methanol I.000 0 

* Reference 14 

1.0595 

1:0610 

t.121 0 

1.0483 I.on/ 

HEAT OF COMBUSTION - Gasolin e heatin g 

value , expressed as BTUs per gallon , Is a fuel 

parameter of general Interest. Published molar 

heating values(^ ) were Incorporated into the 

model to calculat e fue l energy content assuming 

the final products of combustion are gaseous 

carbon dioxide and gaseous water at 25"C and 

atmospheri c pressure , startin g with liqui d 

constituent s a t 25°C and a t saturatio n 

pressure . 

MODEL APPLICATION 

FUEL DATA - To validat e and to Illustrat e 

the application of the""model developed ln this 

study-f gasolin e fue l compositio n dat a and 

specification s wer e obtaine d fro m a 

Coordinating Research Council (CRC) sponsored 

study conducted at the National Institut e for 

Petroleu m and Energ y Researc h (NIPER) 

specificall y for the purpose of determining the 

fue l benzene effec t on automotiv e benzene 

emissions.^5 ) Tn tna C study, detailed data 

were availabl e for 9 fuels. The study used 2 

low benzene bas e fuels, each of which were 

divided int o 4 separat e portions and spiked 

with different levels of benzene, resulting In 

a matrix of 8 different fuels In addition to a 

certificatio n referenc e fue l referred to as 

Indolene 8523. Tables A-l, A-3, and A-5 in 

Appendix A contain the fue l composition data 

(In Column 2) for the Indolene and the two 

specia l low benzene base fuels (25% aromatics 

and 40% aromatics) used ln that 3tudy(25). 

These data are particularl y useful for model 

validatio n becaus e the y relat e t o thre e 

FUEL RVP - Details of the RVP measurement 

a r e describe d In ASTM Method D323 . The 

technique reports fue l vapor pressure measured 

at 37.8°C (100°F) in a container having a 4 to 

1 ratio of ai r to liquid . Air pressure is not 

Included eithe r In the model calculatio n or in 

the RVP measurement. Figure 2 compares the 

calculated fuel RVP with measured RVPC25) ua t n g 

the equivalent micro vapor pressure technique 

for the fuels of the CRC study . Measured 

microvapor pressures have been corrected to the 

equivalent RVP using the correlatio n method 

described In ASTM D2551. Fuels 8531 to 8534 

represent the 25% aromatic base fuel with four 

differen t levels of benzene. Fuels 8541 to 

8544 represent the 40% aromatic base fuel with 

simila r addition s of benzene . As found in 

Table 5 the measured and calculated RVP values 

a r e withi n 5%. Bot h th e measure d and 

calculated values Indicat e a decrease In RVP' 

FIGURE 2 

Calculated And Htasursd Raid Vapor Praaaura 

Cilculeted RVP 

Measured RVP 

Fuel Vapor Preseure. pit 

Fuele >S3> te M M eere tlenee* free tfie lee 

ereeatlc eeee OSfl . Fuela S941 te SS44 eere 

oleneee frea tte Men ereeatlc eeee (40U. 
860532 

BENZENE LEVELS - Figure 5 compares measured 

fuel tank benzene vapor levels above the liquid 

fue l a t a temperatur e of 85"F(25 ) wit h 

calculated values. There is good agreement 

between measured and calculated values except 

for the low benzene fuels, possibl y due to 

measurement Inaccuracie s at the ver y low 

benzene levels. 

REACTIVITY - Ao a frame of reference , Table 

6 list s 21 hydrocarbons that have been shown to 

account on average for about 92 percent of 

ambient gasoline vapors around marketing plants 

where gasoline Is loaded Into tank trucks for 

deliver y t o servic e statlons(2 - ) . The 

TABU! A 

MEASURED OASOLINE VAPOR COMPONENrS, MOLE 7.* 

FIGURE 9 

Calculated and Maaaured Fual Tank Banzana Vapora 

For a Fual Teaperature of 85 Degrees F 

Compound 

Propane 

formal butane 

tsobutane 

Isopentane 

Normal pentane 

Cyclopentane 

2,3-dlmethylbutane 

2-methylpentane 

3-nethylpentane 

formal henane 

Methyl cyclopentane 

2,4-dlmethylpentane 

2,3-dtmethylpentane 

2,2,4-trlmethylpeat a 

Iaobuty'ene 

1-meehyl-l-buccne 

cl»-2-pentene 

2-methyl-2-butene 

Benzene 

Toluene 

Xylene (p,m,o) 

Total* 

Correction 

Point, C 

- 42.1 

- 0.5 

- U. 8 

27.9 

16.1 

49. 1 

58. 0 

60.3 

63. 1 

68.7 

71.8 

80. 1 

89.8 

vs 99.2 

- 6.9 

31.2 

17.0 

18.6 

80.1 

no.6 

142.0 

for ralsstni 

Reactivit y 

240" 

118 

300 

480 

475 

713 

760 

425 

425 

613 

663 

763 

763 

525 

5880 

6250 

6000 

10000 

150 

725 

2700 

I HC — 

* 

* 

* 

* 

* 

« 

* 

* 

* 

* 

* 

* 

* 

* 

Mole I 

0.8 

38.1 

5.2 

2 2 . 

7.0 

0.7 

0.7 

2.1 

1.6 

1.5 

1.3 

0.4 

0.7 

0.5 

1.1 

1.6 

1.2 

1.7 

0.7 

1.8 

0.5 

92.1 

-> 

Relative 

Reactivit y 

1.9 

129.8 

15.6 

109.9 

33.3 

5.0 

5.3 

8.9 

6.8 

9.2 

8.6 

3.1 

5.3 

2.6 

64.7 

100.0 

72.0 

170.0 

1.1 

13.1 

13.5 

779.7 

346.6 

Reference' 26. 

Estimated value. 

MllloraM Benzene Per Gallon 

no 

• <««« + 

Fuel Identification Muster and Benzene Voluae X 

Fuele * an i te * 0B34 reereeent the lea 

ereeatlc beoe BSD . Fuel, f 0941 to I 0S44 

reereeent the Men ereeatlc beee I40H . 

reactivity of these hydrocarbons is estimated 

to be about 350. Table "7 summarizes the vapor 

reactivity calculations made for the 9 fuels of 

the CRC study. Reactivity estimates for the 

low aromatic special fuels are lower than 

comparable reactivity found near marketing 

plants whereas the reactivity of the high 

aromatic fuels are about the same. The 

Indolene fuel used In the CRC program had a 

significantly higher reactivity due largely to 

the quantity of C4 olefin in the vapor phase 

(Table A-2) . Figure 6 displays the OH 

reactivity of completely vaporized fuel as an 

FI6URE 6 

Fuel Tank Vapor And Coapletely Vaporized Fual 

OH Reactivity Relative To Methane 

SUMMARY Of FUEL CALCULATIONS 

VAPOR 

"»• PR'JSSUR*. 

FUEL (psi ) 

8511 

USII 

6511 

8514 

8541 

8542 

8543. 

8544 

INDOLF.NF. 

852) 

11.10 

6.41 

I0.«>1 

8.49 

10.79 

8.17 

10.65 

8.2? 

9.5 6 

7.44 

9.4 ) 

7.31 

9.32 

7.24 

9.22 

7.16 

8.90 

6.89 

Fl*L 

rr.Mpr.nATime 

CF) 

too 

85 

100 

83 

100 

83 

100 

93 

100 

85 

100 

85 

IOO 

85 

100 

85 

100 

85 

VAPOR/ 

I.10.UI0 

RATIO 

VAPOR 

REACTIVITY 

(CHf I) 

4 70 

467 

468 

465 

466 

464 

465 

462 

796 

802 

792 

798 

788 

795 

784 

791 

2)8 0 

2410 

REFUELING LOSS 

(C/CAL) 

Calculate d Measured 

7.4 

5 . 9 

7 . ) 

3.8 

7 . 2 

5 . 7 

7 . 1 

5 . 6 

6 . 3 

5 . 0 

6.3 

4 . 9 

6.2 

4 . 9 

6. 1 

4 . 8 

6 . 0 

S.I 

6 . 1 

6 . 1 

6 . 0 

5.9 

5.5 

5.2 

5.3 

5 . 5 

Coapletely Vepertzed 

Fuel Reactivity 

Fuel Tenk Viper 

Reactivity 

Fuel Identification Nuaber 

Fuele # ani te * SBM ray wont the lee 

tic Mee OSO . Fuele # 054J te * 0044 

the blah areeetlc eeee I40SJ . 
860532 

P.26 

1 1 . van Nes, K. , and van Westen , H. A. , 

"Aspects of the Constitutio n of Mineral 

Oils, " Elsevie r Publishing Co., Inc. , 1951, 

104. 

12. Reid , R. C. , Prausnlty , J . M. , and 

Sherwood, T. K., "The Properties of Gases 

and Liquids, Third Edition, " McGraw-Hill 

Book Company. 

1 3 . CRC Handbook of Chemistry and Physics, 64th 

Edition , CRC Press , Inc . Boca Raton , 

Florida , 1983-1984. 

14. American Institut e of Physic s Handbook, 

Second Edition, McGraw-Hill. 

1 5. Brunelle , M. F. , Dickinson, J . E. , and 

Hamming, W. J. , "Effectiveness of Organic 

Solvents ln Photochemical Smog Formation," 

Solvent Project, Final Report, Los Angeles 

County Air Pollution Control District , July 

1966. 

2 3 . Mill , T. , Winterle , J. S. , Davenport, J. 

E . , "Validation of Estimation Techniques 

for Predicting Environmental Transformation 

of Chemicals," Final Report, EPA Contract 

No. 68-01-6269, SRI International, Menlo 

Park, California . 

2 4 . Cox , F. W., "Physica l Propertie s of 

Gasoline/Alcoho l Blends," Department of 

Energy, BETC/RI-79/4, September 1979. 

2 5. Automotive Benzene Emissions, CTC-APRAC 

Project No. CAPE-35-83, Preliminary Report, 

presented to the California Air Resources 

Board and the U.S. Environmental Protection 

Agency, October 8, 1985. 

2 6 . McDermott, H. J. , Kllllany , Jr. , S. E. , 

"Quest For a Gasolin e TLV," American 

Industria l Hygien e Associatio n (39) , 

February, 1978. 

16. Altshuller , A. P. , "An Evaluatio n of 

Techniques for the Determination of the 

Photochemica l Reactivit y o f Organi c 

Emissions," JAPCA, 16, 257, 1966. 

17. Trijonis, J. C , Ar ledge , K. W., "Utilit y 

of Reactivit y Criteria In Organic Emission 

Control Strategie s for Los Angeles," TRW 

Environmental Services, Redondo Beach, 

California , December, 1975. 

18. "Proceeding s of the Solven t Reactivit y 

Conference," EPA-650/3-74-010, November, 

1974. 

19. Jackson, M. W., and Everett, R. L. , "Effect 

o f Fue l Compositio n on Amount an d 

Reactivit y of Evaporative Emissions," SAE 

PT-14, 802, 1967-70. 

2 0. Niki, H., Daby, E. E. , and Weinstock, B. , 

"Mechanism s of~Smo g Reactions, " ACS 

Advance s in Chemistr y Serie s 113 , 16 , 

Photochemical Smog and Ozone Reactions, 

1972. 

2 1 . Pitts, J. N., Jr. , Lloyd, A. E. , Winer, A. 

M., Darnall , K. R. , and Doyle , G. J. , 

"Developmen t and Applicatio n o f a 

Hydrocarbon Reactivit y Scal e Based on 

Reaction With the Hydroxyl Radical, APCA 

Pape r #76-31.1 , 69t h Annual Meeting , 

Portland, Oregon, June 27-July 1, 1976. 

2 2 . Atkinson, Ri and Lloyd, A.C , "Evaluation 

o f Kineti c and Mechanisti c Dat a fo r 

Modeling of Photochemical Smog," Journal of 

Physical and Chemical Reference Data, Vol. 

13, No. 2 , 315-444, 1984. 
P.27 

860532 11 

Tabli A-3 

Fue l Characteristic s For Fue l » 353 1 (25 1 aromatlcs l 

Calculate d Fue l Vapor Pressur e of ll. l psi . or S73.9 8 • • H9 

At fue l temperatur e 100.0 0 degree s F , o r 37.7 8 degree s C 

Component 

C3H6 

C3H8 

C4H10 

C4H8 

C4H10 

CSH12 

C5H10 

CSH12 

C5H10 

C6H14 

C6H12 

C6H14 

C6H12 

C6H6 

C7H16 

C7M14 

C7H16 

C7H14 

C7H8 

C8HI8 

C3H16 

C8H18 

C3H16 

C8H10 

C9H20 

C9H18 

C9H20 

C9H12 

10H22 

10H22 

C10H8 

10H14 

UH24 

11H24 

UH16 

Original 

Fuel 

Volume 

Percent 

0.030 

0 310 

3.970 

0.080 

1.940 

3.010 

0.060 

25.340 

0.060 

1.670 

0.130 

0.030 

O-.020 

0.020 

4.630 

0.000 

0.010 

0.020 

6.500 

30.550 

0.000 

0.000 

0.030 

6.960 

1.330 

0.000 

0.000 

9.120 

0.390 

0.000 

0.000 

2.9S0 

0.030 

0.300 

0.180 

Original 

Fuel 

Mole 

Percent 

0.048 

0.477 

4.899 

0.111 

2.524 

3.402 

0.073 

28.729 

0.083 

1.653 

0.136 

0.030 

0.023 

0.029 

4.067 

o.noo 

0.009 

0.020 

7 .988 

25.017 

0.000 

0.000 

0.027 

7.S38 

1 .342 

0.000 

0.000 

8.865 

0.262 

0.000 

0.000 

2.490 

0.019 

0.000 

0.139 

Final 

Fuel 

Hole 

Percent 

0.037 

o.iai 

4.511 

0.103 

2.387 

3.357 

0.072 

28.547 

0.083 

1.664 

0.137 

0 030 

0.023 

0.030 

4.113 

0.000 

0.009 

0.021 

S. 105 

25.381 

0.000 

0.000 

0.028 

7.656 

1.363 

0.000 

0.000 

9.007 

0.266 

0.000 

0.000 

2.530 

0.019 

0.000 

0.141 

Fuel 

Vapor 

Pressure 

- PSI 

0.081 

0.715 

3.211 

0.065 

1.223 

0.687 

0.010 

4.440 

0.008 

0.111 

0.008 

0.002 

0.001 

0.001 

0.139 

0.000 

0.000 

0.000 

0.084 

0.278 

0 OOO 

0.000 

0.000 

0.020 

O.OOS 

0.000 

0.000 

0.006 

0.000 

0.000 

0.000 

0.001 

0.000 

0.000 

0.000 

Percent 

Fuel 

Vapor 

Pressure 

0.734 

6.447 

28.933 

0.589 

11.018 

6.191 

0.093 

40.004 

0.074 

0.999 

0.074 

0 014 

0.009 

0.009 

1.253 

0.000 

0.001 

0.003 

0.752 

2.508 

0.000 

0.000 

0.002 

0.180 

0.045 

0.000 

0.000 

0.053 

0.003 

0.000 

0.000 

0.011 

0.000 

0.000 

0.000 

Percen t 

Component 

8TU 

C3 olefi n 

C3 n-p«raffi n - -

isoparaffi n -

olefi n 

n-para ff i n 

isoparaffin -

olefi n 

n-paraff1 n - -

napthen e 

isoparaffi n -

olefi n 

n-paraffi n - -

napthen e 

aromati c 

[7 isoparaffi n • 

[7 olefi n 

C7 n-paraffin --

C7 napthene 

C7 aromatic 

C8 isoparaffin -

C8 olefin -

C8 n-paraffi n - -

C8 napthen e 

C8 aromati c 

C9'isoparaffi n -

C9 olefi n 

C9 n-paraffi n - -

C9 aromati c 

CIO isoparaffi n 

CIO n-paraffi n -

10 napthen e 

10 aromati c 

11 

11 

Isoparaffi n C 

n-paraffi n C 

I t aromati c - C 

0.0 2 

0.2 4 

3.1 6 

0.0 7 

1.6 3 

2.7 0 

0.0 6 

22.8 7 

0.0 6 

1.56 

0.1 ] 

0.0 3 

0.0 2 

0.0 2 

4.4 4 

0.0 0 

0.0 1 

0.0 2 

7.1 2 

31.1 2 

0.0 0 

0.0 0 

0.0 ] 

7.8 1 

1 86 

0.0 0 

0 -.00 

10.7 7 

0.4 0 

0.0 0 

0.0 0 

3 40 

0.0 3 

0.0 0 

0.2 1 

99.89 0 100.00 0 100.00 0 11.10 0 100.00 0 100.00 

Table A-4 

Fue l Vapor Characteristic s For Fue l I 653 1 (25 1 aromatics ) 

Vapor Grams Vapor Fuel 

Mole-Vo l Vapor / X Vapor 

Component Percen t ga l vap . Mass Reactivit y 

C3 olefi n C3H6 0.73 4 0.0346 3 0.46 6 22. 9 

C3 n-paraffi n C3H8 6.44 7 0.3186 8 4.29 0 15. 5 

C4 isoparaffi n C4H10 28.93 3 1.8S54 2 25.38 2 86. 8 

C4 olefi n C4H8 0.58 9 0.0370 5 0.49 9 34. 6 

C4 n-paraffi n C4H10 11.01 8 0.7179 8 9.66 6 17. 2 

CS isoparaffi n CSrl 12 6.19 1 0.5008 5 6.74 3 29. 7 

CS olefi n CSMIO 0.09 3 0.0073 4 0.09 9 9. 3 

CS n-paraffi n .--- - C5H12 40.00 4 3.2361 9 43.56 7 190. 0 

C5 napthen e CStUO 0.07 4 0.0058 6 0.07 9 O.S 

C6 isooaraffi n C6H14 0.99 9 0.3965 2 1.29 9 4 2 

C6 olefi n C6M12 0.07 4 0.0070 1 0.09 4 6. 5 

C6 n-paraffi n C6H14 0.01 4 0.0013 1 0.01 8 0. 1 

C6 napthen e C6H12 0.00 9 0.0008 8 0.01 2 0. 1 

C6 aromati c C6H6 0.00 9 O.OQ075 0.01 0 0. 0 

C7 ftoparaffi n C7H16 1.25 3 0.1408 4 1.896 9. 6 

C7 olefi n C7H14 0.00 0 0.0000 0 0.00 0 0. 0 

C7 n.paraffi n C7N16 0.00 1 ' 0.0001 5 0.00 2 0. 0 

C7 naptnen e C7H14 0.00 3 0.0003 1 0.00 4 0. 0 

C7 aromati c C7H8 0.7S 2 0.0777 3 1.046 5. 5 

CS isoparaffi n C8H18 2.50 8 0.3212 0 4.32 4 13. 2 

CB olefi n C8H16 0.00 0 0.0000 0 0.00 0 0. 0 

Ca n-paraffi n C3H18 0.00 0 0.0000 0 0.00 0 0 0 

C8 napthen e --- - C3H16 0.00 2 0.0002 0 0.00 3 0.0 . 

C8 aromati c C8H10 0.18 0 0.0213 7 0.28 8 3. 3 

C9 Isoparaffi n C9M20 0.04 5 0.0064 4 0.08 7 0. 5 

C9 Olefi n C9H18 0.00 0 0.OOOOO 0.00 0 0. 0 

C9 n-paraffi n C9H20 0.00 0 0.0000 0 0.00 0 0. 0 

C9 aromati c -.-- - C9H12 0.0S 3 0.0072 0 0.09 7 0. 7 

CIO Isoparaffi n C10H22 0.00 3 0.0004 8 0.00 6 0. 0 

CIO n-paraffi n C10H22 0.00 0 0.0000 0 0.00 0 0. 0 

CIO napthen e C10H8 0.00 0 0.0000 0 0 000 0 0 

CIO aromati c C10H14 0.01 1 0.0016 7 0.02 2 0. 2 • 

C l l isoparaffi n C11H24 0.00 0 0.0000 1 0.00 0 0. 0 

C-11 n-paraffi n C11M24 0.00 0 0.0000 0 0.00 0 0. 0 

C l l aromati c CUH1 6 0.00 0 0.0000 4 0.00 0 0. 0 

TOTALS 100.00 0 7.4281 0 100.00 0 470. S 

The averag e molecula r aeigh t of liqui d fue l I s 93.9 1 Fina l 94.3 5 

The averag e molecula r aeigh t of th e vapo r i s 66.2 5 

The origina l vapor/liqui d rati o I s 4 The fina l I s 4.0134 8 

The origina l fue l mass pe r ml i s .7196 9 The fina l .7212 8 

The aoove fue l densit y I s calculate d fo r 20 degree s C 

The origina l fue l reactivit y relativ e to methane * 1 i s 753. 1 

The refuelin g los s aoul d oe 7.42 6 grams per U.S . gallo n 

Thi s refuelin g los s I s 112.1 2 millimole s per U.S . gallo n 

The liqui d fue l hydrogen/carbo n rati o is - 1.97 

The vaoo r hydrogen/carbo n rati o i s 2.4 1 

The fue l STU per gallo n i s U217 7 
860532 13 

APPENDIX B 

1 rem *** Vapor pressure program, nZ Is 9 of 

components. 

2 rem *** Line numbers optional for Fortune 

32-16 except goto. 

5 nZ-35 

dim 

narae$(nZ), lb(n*'> ,mf(nZ) ,raw(nZ) ,vmf(nZ) ,ovmg( 

nX),si(nZ),den(nZ) 

dim 

bl(nZ),lp(nZ,3),vap(nZ,3),pmass(nZ),ml(nZ),p 

vk(nZt 

dim tk(nZ,3),btc(nZ'»,mfe(nZ),rea(nZ'>,fuelr(nZ) 

dim 

pres(nZ>,rvp(nZ>,percent(nZ),react(nZ>,astra t 

Z(l9),astmp(19 ) 

dim 

ract(l9),vract(l9),vmfe(nZ),gra(nZ),volp(nZ ) 

,racora(nZ),hn(nZ),cn(nX) 

dim tcden(nZ),mhv(nZ) 

rem *** Read specific component data *** 

300 for 1 - I to nZ 

read 

name$(l),mw(l),den(i),hn(l),cn(i),btc(l),react( 

l).mhv(l) 

next l 

prin t "Ente r Fuel Identificatio n name. "; 

Input fuelname$:print 

700 Input "Ente r fuel temperatur e In degree s F 

**;tempf 

710 tempe - 5*(tempf - 32)/ 9 

720 terapk - tempe + 273.16 

722 prlnt:print:prln t 

r e m ******* DENSITY CALCULATIONS *********** 

vrvo = 1.0311837 

vtvo -

1.1182172-0.002213587*tempk+(6.5192982e-6)*t 

empk©2 

for l- l to 12 

tcden(l)-den(i)*vrvo/vtv o 

next 1 

for 1=15 to 17 

tcden(1)-den(1)*vrvo/vtv o 

next 1 

vrvo - 1.0250639 -» 

vtvo—-

0.8581784-0.000107003*tempk+(2.2923977e-6)*t 

erapk©2 

for 1-13 to 14 

tcden(l)-den(l)*vrvo/vtv o 

next I 

for 1-18 to 19 

tcden(l)-den(l)*vrvo/vtv o 

next 1 

for 1-23 to 24 

tcden(l)-den(l)*vrvo/vtv o 

next 1 

tcden(28)-den(28)*vrvo/vtv o 

for 1-31 to 32 

tcden(l)-den(l)*vrvo/vtv o 

next 1 

tcden(35)=den(35)*vrvo/vtv o 

vrvo-l.0235725 

vtvo-0.8803969-0.000197926*tempk+(2.327485e-6)* 

tempk©2 

for 1-20 to 22 

tcden(1)-den(1)*vrvo/vtv o 

next I 

for 1-25 to 27 

tcden(l)-den(l)*vrvo/vtv o 

next i 

for 1-29 to 30 

tcden(l)-den(i)*vrvo/vtv o 

next 1 

for 1-33 to 34 

tcden(l)-den(l)*vrvo/vtvo 

next i 

vrvo-l.02487122 

vtvo-0.77441457+0.0004929225*tempk+(I.2l875e-6) 

*tempk«2 

input "Enter air volume fraction above liquid. 

Use 4 for actual RVP. ";vo 

print 

print "Are your data mole percent or volume Z 

?" 

723 Input " Enter mp or vp > ";qz$:print 

727 print rprlnt 

if qz$-"vp" then goto 761 

if qz$-"mp" then goto 730 

print:goto 723 

730 for I - I to nZ 

740 print "Enter mole percent ";name$(i);" " 

750 input " 

> ";pet 

mf(l)-pet/l00 

760 next I 

for I - I to nZ 

mcom(l) • mf(l) 

gra(l) - mcora(i)*mw(i) 

rem ** In the next line volp(l) - ml of 

component I for 1 mole of all components 

volp(l) - gra(l)/den(i) 

next 1 

tomol • 0 

for I - 1 to nZ 

tomol - tomol + volp(i): rem ** Total ml of 

components 

next 1 

for 1 - 1 to nZ 

volp(l) - 100 * volp(l)/tomol: rem ** Volume 

percent calculation 

next I 

tomol - 0 

for 1 - I to nZ 

tomol - tomol + volp(i) 

next 1 

goto 810 

761 for I - I to nZ • 

print "Enter volume percent ";name$(l);" " 

Input 

> ";volp(l) 

next t 

for 1 - 1 to nZ 

gra(l) - voip(l)*den(i): rem ** Density at 20 

degrees C 

mcom(l) - gra(l)/mw(i) 

next i 

tomol - 0 

for I - I to nZ 

tomol - tomol + mcora(l) 

next 1 

for i - 1 to nZ 

mf(l) - mcoia(i)/tomol 
P.29 

860532 15 

826 trvp - trvp + rvp(i):nex t i:fo r 1 - I co 

nZ.-percent(l) - I00*r'vp(l)/trvp 

827 next I 

828 tpres =• lnt(le2*tpres +.5)/le2 

829 trvp - lnt(le2*trvp + .5)/le2:lprlnter 

width 120: rem ** Fortune file 

rem ** print command- at specified width 

print:print:prInt:print:prlnt:print:print 

print" Fuel 

Characteristics For Fuel ";fuelname$:prlnt 

print" Calculated Fuel Vapor Pressure 

of ";trvp;" psi, or ";tpres;" mm Hg" 

mat2$-" At fuel temperature 

9999.99 degrees F, or 999.99 degrees C" 

856 print using mat2$;tempf,tempe 

890 print 

tmfe - 0:tmfae - 0 

for I- 1 to nZ 

mfe(l) - mt(l) - sl(l): rem ** Final fual 

mllllmoles per component I 

tmfe - tmfe + mfe(l): rem ** Total final fuel 

mllllmoles 

next I 

for I - 1 to nZ 

mfe(l) - mfe(l)/tmfe: rem ** Mole fraction -

final fuel 

tmfae - tmfae + mfe(l) 

next I 

sumbl • 0:btugal - 0 

for 1 - 1 to nZ 

mhv(l) - 3.9685*mf(l)*mhv(i) 

gra(l) - mf(i)*mw(l) 

bl(l) - gra(l)/den(i) 

sumbl - sumbl + bl(l) 

next 1 

for I - 1 to nZ 

mhv(t) - 3785.4*mhv(i)/sumbl 

btugal - btugal + mhv(l) 

next 1 

print " 

Original Final Fuel 

Percent" 

print " 

Fuel Fuel Vapor 

Component" 

print " 

Mole Mole Pressure 

prlnt~-" Component 

Percent Percent psi 

Contribution" 

print "——»—— — ssAjaasaasss 

matl$-"/ 

999.999 999.999 999.999 

al$ -"/ 

9.9999 999.999 9999999.99" 

mat5$-'7 

9.99999 999.999 9999999.9" 

a2S - "I 

9.999999 999.999 9999999.9" 

btugalp » 0 

940 for 1 - I to nZ 

btup = 100*mhv(l)/btugal 

btugalp = btugalp + btup 

950 print using matl$; 

/ 9r<.999 

r 

/ 9.9999 

1999.999 

I 9.9999 

natne$(l).volp(l),l00*mf(l),l00*mfe(l),rvp(l) 

,percent(l),btup 

960 next I 

970 lmf - 0:vpp - 0:vpmm » 0:perp • 0:tvmf -

0:tovmg=0:t pmass-0 

980 for 1=1 to nZ 

990 lmf » lmf + mf (1) :v'pp=vpp + 

rvp(l):vpmm-vpmm + pres(l) 

1000 perp • perp + percent(l):nex t 1 

prin t "aaaa-aaa a a a isiasaaaaaMaaa q « a mm aaisi.aai aaai 

a a aaaaia-aa a aaaaaaa a 

• asasa a a a aaaaaai a 

• a a aaaaaaa a aa a 

a .** a a a '* 

hhh$ -" TOTALS 999.999 

99.99999 999.999 9999999.9" 

ahh$ -" TOTALS 99*.W 

999.999 999.999 999.999 999.999 

1020 print using 

ahh$;tomol,100*lmf,100*tmfae,trvp,perp,htuga 

lp 

1035 tvmf » 0:tovmg - 0:tpmass - 0 

1030 for 1 - 1 to nZ 

1040 vmf(l) - si(l)/s 

1050 ovmg(l)- sl(l)*mw(l)/1000 

1060 tvmf - tvmf + vraf(l) 

1070 tovmg - tovmg + ovmg(l) 

1080 next l:vr - 0:fract - 0 

1090 for I - 1 to nZ 

1100 pmass(l) - 100*ovmg(l)/tovmg 

1105 tpmass - tpmass + pmass(I) 

fuelr(i) - mf(l)*react(l) 

fract - fract + fuelr(i) 

fract - lnt(lel*fract + .5)/lel 

rea(l) - vmf(i)*react(l) 

vr - vr + rea(l) 

1110 next I 

1115 for l-l to I5:prlnt:next I 

print" Fuel Vapor Characteristics 

For Fuel ";fuelname$ 

Original 

Percent 

Fuel 

Fuel 

Volume 

Vapor BTU" 

Percent 

Pressure 

print 

print" 

Grams 

print" 

Vapor / 

print" 

gal vap. 

print"-—-* 

aaaaaaa a 

1118 for l1120 print 

Vapor Fuel" 

Z Vapor" 

Component 

Mass Reactivity" 

-aaaa=ia3aaaasa.a=ia.a.aaaaaaaa 

asaaaaasa a^maaa-m-aaaa-m' 

1 to nZ 

using 

Vapor 

Mole-Vol 

Percent 

aaaaaaata 

mat5$;name$(l),lOO*vmf(l),3785.4*ovmg(l)/vat,pm 

asc(l),rea(l) 

1130 next I 

print " —— — — — ——aaaaaaasiaaaa s aaaaaa a 

1140 print using 

hhh$;I00*tvraf,3785.4*tovmg/vat, tpmass,vr 

1150 avmwl - 0:avmwv = 0:avmwlf - 0 

1160 for I - 1 to nZ 

1170 wer - mf(l)*mw(i) 

1172 www - mfe(l)*mw(l) 

1180 war - vmf(l)*mw(l) 

1190 avmwl - avmwl + wer 

1200 avmwv • avmwv + war 

1205 avmwlf - avmwlf + www 
P.30 

GMR-5753 

Rapid Post-Exposure Decay of a- -Globulin and Hyaline Droplets 

in the Kidneys of Gasoline-Treated Male Rata 

Bhagwan D. Garg and Michael J. Olson 

Biomedical Science Department 

General Motors Research Laboratories 

Warren, Michigan 4.3090-9058 

Williaa F. Demyaa and alrun K. Roy 

Department of Biological Sciences 

Oakland Unirersity 

Rochester, MI 48063 

Running Title: Recorery froa Gasoline Nephropathy 

Address iaj Correspondence: Bhagwan D. Garg 

Biomedical Science Department 

General Motors Research Laboratories 

30500 Mound Road 

Warren, MI 48090 

(313) 986-1652 

Submitted for Publication in 

Journal of Toxicology and 

Environmental Health 
P.31 

ABSTRACT 

Rapid Post-Exposure Decay of o^-Globulin and Hyaline Droplets in the Kidneys 

of Caso line-Treated Male Rats. Garg, B.D., Olson, M.J., Demyan, W. and Roy, 

A.K. (1987). J. Toxicol. Environ. Health 

Unleaded gasoline induces accumulation of hyaline droplets in epithelial 

cells of the renal proximal convoluted tubules only in malm rats. Synthesis of 

the male rat-specific protein, a- -globulin, a constituent of renal hyaline 

dropleta, occurs in the liver and is unaltered by gasoline. Renal a* -globulin 

content incressed to 210X of control vslues after a single oral doae of 

gasoline (2.0 mL/kg); maximal levels (320% of control) were attained following 

gasoline administration for three days. The conjoint effect of post-exposure 

recovery and estradiol (inhibitor of hepatic a. -globulin synthesis) was 

determined in male rats. Gasoline alone (2.0 mL/kg, p.o., 9 days) increased 

renal a- -globulin 530 X with concurrent proliferation of hyaline droplets. 

Within 3 days of terminating gasoline administration, renal a~ -globulin 

content decreased to the same level as that in unexposed rsts. However, the 

number of renal hyaline droplets did not return to the pre-treatment level 

until days 6 to 9 of recovery. On post-exposure days 3, 6 snd 9, estradiol 

treatment (1 mg/kg, s.e, 4 days, starting on the 9th day of gasoline 

treatment) decreased rensl a. -globulin content to 75X, S9X and 48%, 

respectively, of thst in rsts not receiving hormone treatment. Hepatic tt2u~ 

globulin in estradiol-treated rats decreased by 74X, 97X and 96% at the same 

intervals. Estradiol treatment also appeared to increase the rate at which 

normal renal morphology was restored. Thus, accumulation of o^-gIobuI in and 

hyaline droplets after subacute exposure to gasoline is rspidly reversible and 

apparently dependent on continuous exposure to gasoline snd maintenance of the 

normal rate of hepatic tt_ -globulin synthesis. 

• 2 -
P.32 

INTRODUCTION 

Isopsrsffinic hydrocarbons and mixtures of these compounds have been shown 

to induce a unique nephropathy in male rats (Phillips and Egan, 1984; Garg et 

al., 1986; Kanerva et al., 1986; Viau et al., 1986). An inhalation bioassay 

protocol conducted as psrt of the toxicological assessment of unleaded gssoline 

indicated thst chronic exposure csused nephropsthy snd kidney tumors. The 

results of this study, in which B6C3F. mice and F-344 rats were exposed to 

wholly volatilized gasoline for 24 months, showed that gasoline vapor increased 

the incidence of non-neoplastic renal pathology and renal cortical carcinoma in 

male rats only (Busey and Cockrell, 1984; MacFarland et a I., 1984). Because of 

the wide-spresd use of unleaded gasoline and a trend toward greater use of 

self-service refueling (Environmental Health Letter, 1985), significant 

attention has been focused on the possibility that recurrent gasoline exposure 

of humans may result in adverse health effects. 

Although the mechanism of the sex and species-specifie toxicity and 

carcinogenicity of gasoline vapors remains obscure, resesrch has shown that it 

does not seem to act as a renal carcinogen by producing somatic mutation 

(Conaway et al., 1983; Richardson et al., 1986; Loury et al., 1986a; Loury et 

a I., 1986b). An alternative hypothesis concerning the tumorigenie potential of 

gasoline is bssed on the observation that following short-term exposure to 

gssoline or relsted hydrocsrbon mixtures, the epithelium of the renal proximal 

convoluted tubules (PCT) of male rats shows evidence of cell death, exfoliation 

and restorative cell proliferation (Busey and Cockrell, 1984; Haider et al., 

1984). Immediately preceding these events, PCT epithelial cells, especially 

those of the S2 segment, show a marked increase in the number and size of 

cytoplasmic hyaline droplets (Haider et al., 1984; Garg et al., 1986; Olson et 

a I., 1986; Short et a I., 1986). Further, it has been observed that the same 

population of cells which accumulates hyaline droplets subsequently shows an 
P.33 

increased rate of DNA synthesis (Short et a I., 1986). The concept of chronic 

chemical Iy-induced cytotoxicity and cell proliferation as a precursor event in 

carcinogenesis has been invoked to explain the carcinogenic effect of gasoline 

(Loury et si., 1986b) as well ss other chemicsIs (Zeiss et si., 1986). 

Interest in the potent-islly adverse health effects of gssoline led us to 

develop s short-term model for examining the toxicity of gasoline rn rats (Garg 

et al., 1986). Our initial research showed that a major component of gasolineinduced hyaline droplets is the androgen-dependent rat urinary protein a~ -

globulin (Olson et si., 1986). Electron microscopic studies hsve revested that 

gasoline-induced hyaline droplets are the ultrastructural equivalent of 

phagolysosomes (Garg et al., 1986). Accumulation of this protein due to 

defective renal lysosomal degradation has been proposed ss sn integral event in 

gasoline-induced cell death in the kidney (Kloss snd Bus, 1985). We hsve 

attempted to understand if and how gasoline sIters the rensl metabolism of a. -

globulin and whether such alterations contribute to the nephropsthy associated 

with fuel hydrocarbon exposure. This report describes the relationship between 

durstion of gasoline administration and the renal accumulation of a« -globulin. 

Further, the rate of decay of hyaline droplets snd accumulated a« -globulin 

after termination of gasoline exposure,was determined. 

-4-
P.34 

MATERIALS ANO METHODS 

Experimental animals and chemicals 

MaLe Fischer 344 rats (CDFR(F-344)/Crl BR; Charles River Laboratories, 

Wilmington, MA) weighing approximately 250 g were used throughout these 

studies. Rats were maintained in conditions consistent with AAALAC 

recommendations with Purina 5012 chow and tap water freely available during the 

entire period. For time-course experiments rats were administered physiological 

saline (2.0 mL/kg) or a commercially available unleaded gasoline (2.0 mL/kg) by 

intragastric intubation once daily and killed 18 hours after the final 

treatment. In hyaline droplet decay studies rats were treated with gasoline 

(2.0 mL/kg, p.o., 9 days) or gasoline plus estradiol (1 mg/kg in a propylene 

glycol vehicle (Roy et al., 1975), s.e, 4 days, starting on the final day of 

gasoline treatment). Following conjoint gasoline and hormone treatment, animals 

were serially sacrificed at 3, 6 and 9 days after the last dose of gssoline. 

Controls receiving saline and rats treated with gasoline slone were also 

allowed to recover for 3 to 9 days after termination of treatment. All animals 

were sacrificed between 8:00 and 10:00 am. 

Radioimmunoassay of 2„ -globulin in kidney and liver 

The cytosolic fractions of kidney and liver homogenates were prepared (Roy, 

1977) from samples of the perfused organs (see below) and asssyed for <-*-

globulin by double-antibody radioimmunoassay as described by Roy (1977). The 

quantity of <U -globulin in the cytosolic fraction was expressed relative to 

total protein which was determined by the method of Lowry et al. (1951). 

Histological procedures 

Eighteen hours after the final dose of gasoline or after the period of 

recovery, rats were anesthetized with an intraperitoneal injection of 

pentobarbital, perfused via the heart with cold 0.9% sodium chloride solution 

and the kidneys and livers were removed, fixed by immersion in 10% neutral 

-5-
P.35 

buffered formalin, processed routinely and embedded in paraffin. 4 /un sections 

of paraffin-embedded kidney were stained with Mallory's Heidenhain method for 

visualization of hyaline droplets. 

-6-
P.36 

RESULTS 

Dependence of renal flp -globulin accumulation on duration of gasoline 

adminJstration 

Experimenta were conducted to determine whether renal accumulation of a -

2u 

globulin resulted immediately upon initiation of gasoline administration or 

required multiple treatments. Male rats were treated orally with gasoline (2.0 

mL/kg) once daily for 1, 3, 5, 7 or 9 days; 18 hours after dosing, liver and 

kidney tissues were collected for determination of a« -globulin content and 

histologic evaluation. Following a single dose of gasoline, renal a- -globulin 

levels were increased to 210% of control values after 18 houra (Table 1) . 

Maximal elevation of a. -globulin content (320% of control) in the kidney 

occurred after 3 days of gasoline administration. Continued treatment 

maintained, but did not significantly alter, the elevated level of renal a* -

globulin (Table 1) . The hepatic content of a. -globulin waa not different from 

control at any sampling time during 9 days of gasoline treatment of male rata. 

Post-exposure decay of renal a? -globulin and hyaline droplets accumulated 

during gasoline exposure 

Since most exposures to fuel hydrocarbons are intermittent, studies to 

assess the effect of a variable period of post-exposure recovery on indices of 

gssoIine-induced nephropsthy were conducted. Similar to the studies already 

presented, orsl administration of gasoline for 9 days caused a marked increase 

in the renal, content of a. -globulin while the level of hepatic globulin 

protein was not different from controls (Fig.1). Correlated morphological 

studies showed thst increased renal a« -globulin was accompanied by 

accumulation of hyaline droplets in PCT epithelial cells, exfoliation of cells 

into tubule lumina and infrequent development of foci of epithelial cell 

proliferation (Fig.2). Analysis of renal tissue from rats s11 owed 3 dsys of 

recovery following 9 days of gasoline exposure showed that Bjy-globulin levels 

- 7-
P.37 

were decreased sbout 75% from the high levels observed during exposure and did 

not differ froa levels in rats receiving saline only (Fig.l). After recovery 

for 6 or 9 days, renal a« -globulin was similar to that observed at the 3 day 

recovery interval. Examination of renal tissue sections from rsts allowed 3 

daya post-exposure recovery showed thst the number of hysline droplets was 

reduced from peak levels observed immediately after gasoline treatment (Fig.3). 

However, the complete resolution of hyaline droplet accumulation appeared to 

require 6 to 9 days (Fig.4). 

Effect of estradiol on hepatic and renal a^, -globulin and hyaline droplets 

during recovery from gasoline treatment 

Treatment of male rats with estradiol decreased the hepatic content of a„-

globulin by 74% by post-gasoline exposure day 3 compared to rats receiving 

gasoline without hormone treatment (Fig.l). The suppressive effect of estradiol 

on hepatic synthesis of a. -globulin increased by post-exposure dsys 6 and 9, 

although estradiol treatment was discontinued on post-exposure day 3. Thus, 

levels of a. -globulin in the livers of estradiol-treated rats were only about 

3.5%.of values observed in rats allowed to recover from gasoline administration 

without hormone for 6 and 9 days (Fig.l). Reflecting the effects of estradiol 

on hepatic synthesis of a« -globaliny the renal content of this protein was 

decreased by 25%, 41% and 52% on days 3, 6 snd 9 post-gasoline exposure when 

compared to tha kidneys of rats not receiving the hormone (Fig.l). Histologic 

analysis of kidney tissues from estradiol-treated rats revealed that hormone 

treatment also appeared to accelerate the rate of removal of gasoline-induced 

hyaline droplets such that, compared to rats not receiving the hormone, hyaline 

droplet number and size were near control levels at 3 days post-exposure 

(Fig.5) and by 6 dsys of recovery, renal PCT were not different in appearance 

from ssline-trested controls (not shown). 

-8-
P.38 

TITIOO o&cStf |°)Ppoo(n)feo to® ffundoEoato1 noo ©Iboopvatiens in tho study of 

gas©lino°5ifc°fc#gcd hyolino dp@®lot nophpepathy of rats . Thoso aro; 1) tho 

dio<s@wopy tlniot o oonglo odaonootratiiQin) ©ff «goo©Iino io suffffoeiopt t© indue© 

o3cgira5•{?otsoffafe poaol potoatoQa ©ff s^ °gfl@fo<al on, on^) 2) e9 °gl!@@ial in ond hyolosa© 

dp®(°>loto o<§€isaiyi I ofec^l <°!iaP0(n)@ goo© II Sao ii(n)fe©no<sofeii®in) dJoo^Qor. po(°>odly ohon 

goo©I ono o<fciofa3oferofej©fa os topaoaotcd ond tho op®ntono®us poto @f poeevopy froa 

fjyoB 5ao dp®@l)ot (nio(°>!n)P©(p>athy oo oeeollopotod fey tpoofesont @f nolo poto aith 

•ootpodi®!. 

Synthoooo ond oaepoti®n ©ff e« =§1©km ion ©eeupo at high potos in oolo pots 

(ffciuhaus ot a!. , 19§1). Thooo popoeotops apo not altopod fey hydp@eapfe©n 

ospesuro (Gopg ©t ol. , 1986; 01a@n ot a I. , X8@<S; Voau ot a I. , 5L9H6). 

Fupthopraoro, tho pos@rpti©n ©ff thi o globulin (p)p®to5n io gpoator than 8053 

offffoeiont amid net i capo i pod duping hydp©eapb©n°indu<e©d hyolino dp©g>8ofe 

nophp@pothy (Voau ot al. , 198®). Thoao ©fesopvatieno in conjunction'aith tho 

ti£3a-=dopondoneo of e« -globulin accumulation (Toblo 1) suggest that gaselin© 

caus©s a papid dirainutien ©f tho p©st°Pos©ppti©n ppocossing of e« -globulin in 

mmWm. 

ronol PCT op i tho I i a I colls . Tho ppoeooo raoehonossa of such an offset is untxnoon 

but oay feo duo t@ eauoos ine Dud ing otpuetupal isodif ieafeion ©f e„ -glebulin 

(KI©so and Bus, 19®S)„ onhifeoto@n ©ff ®P®to@lytoc onxyroo activity , altopatoen of 

m-r 

felruo ontpalyo®o®cal) $!. ©p altopoto@n ©ff ponol onopgy csotab©! osffii. Xt has feo©n 

sh®an PC@csCillj7 tlhiofe soa°spo<§ o ff o e phopcoeekinotie difforoncos result in 

®p@§®[nK°)C^] rofecsafeiiefii ©ff 2„2„4°tp5cothyl^ontono (TOP), a nephrotoxic gao@lin© 

eenotituont,, in tho bidnoys @f eolo pots (Chopbonn©au ot a I. , 1986). TOP 

spcciffieally oncroooos hyolino dp@®tot ff@PQoto@n and aeeuraulatien off e« -

glefeulon in tho S2 sogaont ©ff tho ponol pp@aooal tubulos of eaalo rats (Sfeonard 

ot ol. , 1988). Ronol lowols ©ff TOP OOPO GOHSKOI 12 hp afftop a singlo ©rai dose 

©ff TOP and at 24 ond 4® hp OOPO 948 and 45S3 ©ff souiraal (Charbonnoau ot al. , 

- 9 -
1986). In contrast, peak TMP concentrations in the kidneys of female rats were 

only 46% of those found in males and were attained 8 hr after administration 

by 24 and 48 hr post-TMP exposure, levels were 53% and 15%, respectively, of 

maximal. Thus, peak levels of hydrocarbon retained in the kidneys of male rats 

correspond tempore M y to-the point st which accumulation of a« -globulin snd 

hyaline droplets were noted in our study. It may be concluded from this that 

renal a. -globulin accumulation is dependent on s defect in a« -globulin 

catabolism which is associated with persistent high levels of hydrocarbon. 

Rapid dissppearanee of a. -globulin from the kidney was observed following 

termination of gasoline administration (Fig.l). The data show clearly that 

after only 3 days of recovery from gasoline intoxication, a„ -globulin 

accumulated in the kidney had dissipated. Moreover, the renal content of a. -

globulin did not differ between rats allowed 3, 6 or 9 days recovery from 

gssoline. Thus, it is likely that reduction in abnormal levels of a, -globulin 

occurred more quickly than 3 days. In spite of the lack of specific information 

on renal a. -globulin concentration on days 1 and 2 post-exposure, it is 

obvious that the accumulation of a« -globulin caused by gasoline exposure is a 

readily reversible phenomenon. Qualitative observation of the histological 

appearance of the kidney during recovery after gasoline administration (Figs.3 

mlmV 

and 4) supports this conclusion since hyaline droplets, for which a- -globulin 

is s biochemical marker, are greatly reduced in number and size by 3 dsys of 

recovery. Hyalfee droplet number continued to decrease on recovery days 6 snd 

9; by 9 dsys of recovery there was no apparent visusi difference between kidney 

tissue from ssIine-trested rats and those allowed 9 days of recovery from 

gssoline administration. Stonard et al. (1986) observed that the accumulation 

of a2 -globulin caused by a single dose of TMP was msximal 48 hours after 

administration. However, in that study the renal content of ajy-g'obulin 

returned to pre-exposure levels only after seven days. The differences in the 

-10-
P.40 

rate of disappearance of a. -globulin in our study and that reported by Stonard 

et al. (1986) with TUP may be attributed to the fact that TUP is a potent 

nephrotoxin and our study used whole gasoline with a corresponding decrease in 

absolute TUP dose. 

Evidence thst hysline droplet nephropsthy in male rata ia a sex-specific 

event dependent on continuous availability of high levels of a« -globulin vss 

derived from an experiment on the effect of estradiol on recovery from 

hydrocarbon-induced nephropathy (Fig.l). As shown previously, a~ -globulin 

synthes is in the Ii ver is h i ghIy dependent on testosterone (Roy et a I., 1975). 

When androgenic activity is decreased by castrstion or administration of 

estrogenic hormones, a. -globulin synthesis declines dramatically (Roy et al., 

1975). In our experiments, subcutaneous injection of estradiol caused a rapid, 

almost complete disappearance of hepatic a. -globulin (Fig.l). Although renal 

a- -globulin levels were not as dramatically altered by estrsdiol ss those of 

liver, a significant decrease in the renal concentration of thia protein was 

observed compared both to saline-treated controls and to rata allowed to 

recover from gasoline-induced nephrotoxicity without hormone treatment (Fig.l). 

Because a. -globulin resorption from the urinary ultrafiItrate is very 

efficient (Viau et a I., 1986), it was not expected thst the estrsdiol-medisted 

alteration in renal concentration of a- -globulin would be of the same 

magnitude as" that observed in liver. The important point, however, is thst 

levels of tu-globulin in the kidney are directly related to those in liver and 

that the norma Ily high circulating levels of a- -globulin in male rats must be 

msintained in order for renal a. -globulin accumulation to occur in response to 

hydrocarbon intoxication. This point is further supported by work of Ksnervs et 

al. (1986) and Hobson et al. (1986) showing that immature or castrated male 

rats which hsve no or greatly diminished a« -globulin synthesis do not develop, 

hyaline droplet nephropathy when exposed to hydrocarbons similar to those found 

-11-
P.41 

in unleaded gasoline. 

It is possible that the normal rate of protein catabolism in renal 

lysosomes-, which may be impaired by gasoline, is restored after gasoline 

withdrawsl snd accumulated proteins, including a~ -globulrn, are 

proteolytics!ly digested. An alternative possibility is that PCT epitheIia I 

eel la containing accumulated a. -globulin may be irreversibly damaged and 

during the firat 3 daya following gasoline exposure, these cells sre then 

exfolisted. Although either of these mechanisms would sccount for results shown 

in Figure 1, they hsve different mechanistic implicstions. While the first 

possibility would support the reversible nsture of fuel hydrocarbon-induced 

nephropsthy, the second suggests that the rapid onset of an irreversible 

toxicity contributes to cytotoxicity and increased epithelisi cell replication 

(Short et al., 1986). Further experiments are needed to differentiate between 

these possibilities. 

Thus, the data presented in this report strongly suggest thst gasolineinduced a. -globulin and hyaline droplet accumulation in male rats is rapidly 

inducible, is readily reversible and dependent on maintenance of normal 

constitutive levels of circulating a. -globulin. This suggests further that 

significsnt nephrotoxicity due to gasoline exposure would be likely to occur 

only in snimal species with high-level proteinuria. Since male rats are known 

to exhibit physiologic proteinuria, it is possible thst these snimals are 

unususlly susceptible to gssoIine-induced nephropsthy. 

•12-
P.42 

ACKNOWLEDGEMENT 

The authors are grateful for the assistance of Scott Webb, Dr. Kathleen 

Smiler, Alexandra Brady and Linda Li of the Biomedical Science Department, GM 

Resesrch Lsboratories snd Michael Mancini, Or. C.V.R. Murty and Or. Fazlul 

Sarkar of the Department of Biological Sciences, Oakland University. Partial 

funding of the resesrch equipment used for some of the experiments wss provided 

by NIH grant AM-14744. 

-13-
P.43 

REFERENCES 

Busey, N.M., snd Cockrell, B.Y. (1984). Non-neoplastic exposure-related renal 

lesions in rsts following inhalation of unleaded gasoline. In Renal Effects 

of Petroleum Hydrocsrbons. (M.A. Mehlmsn, CP . Hemstreet, III, J.J. Thorpe 

snd N.K. Wesver, eds.), pp. 57-64. Princeton Scientific Publishers, Inc., 

Princeton, N.J. 

Chsrbonneau, M., Lock, E.A., Strasser, J., and Bus, J.S. (1986). Disposition of 

[

14C]-trimethylpentane (TMP) 

Pharmacologist 28, 178 (abstr) 

[ C]-trimethylpentane (TMP) in male and female Fischer 344 rats. The 

Conawsy, CC , Schreiner, C.A., and Cragg, S.T. (1983). Mutagenic evaluation of 

petroleum hydrocarbons, in Proceedings of the Symposium: The Toxicology of 

Petroleum Hydrocarbons. (H.N. MacFarland, C.E. Holdsworth, J.A. McGregor, 

R.W. Call and M.L. Kane, eds.), pp. 128-138. American Petroleum Institute, 

Washington, DC. 

Environmental Health Letter (1985). Health Effects Institute report on gasoline 

vapors. 24 (Oct. 15), 8. 

Gsrg, B.D., Olson, M.J., Sarkar, F.H. and Roy, A.K. (1986). Renal hyalin 

droplet snd a. -globulin accumulation after gasoline administration to male 

rats. The Pharmacologist 28, 210 (abstr). 

Hslder, CA., Warne, T.M., and Hatoum, N.S. (1984). Renal toxicity of gasoline 

and related petroleum naphthas in msle rata. In Renal Effects of Petroleum 

Hydrocarbon*, (M.A. Mehlmsn, CP . Hemstreet, III, J.J. Thorpe and N.K. 

Wesver, eds.), pp. 73-88. Princeton Scientific Publishers, Inc., Princeton, 

N.J. 

Hobson, D.W., Msttie, D.R., Bruner, R.H., Uddin, D.E., Eurell, T., and Olson, 

C T . (1986). Effect of castration on the development of 2,3,4-

trimethylpentane-induced hydrocarbon nephropathy in the male F-344 rat. The 

ToxicoIogist 6, 173 (abstr). 

•14-
P.44 

Kanerva, R.L., Ridder, CM., Stone, L.C, and Alden, C.L. (1986). 

Characterizstion of spontaneous and decalin-induced hyaline droplets in 

kidneys of adult male rats. Fd. and Chem. Toxicol, in press. 

Kloss, M.W., and Bus, J.S. (1985). Hydrocarbon-mediated nephrotoxicity. 

C.I.I.T. Activities S, 1-8. 

Loury, D.J., Smith-Oliver, T., and B.E. Butterworth (1986a). Induction of DNA 

repair and replicative synthesis (RDS) following exposure to unlesded 

gasoline. The Toxicologist, 6, 227 (abstr). 

Loury, D.J., Smith-Oliver, T., Strom, . S., Jirtle, R., Michslopoulos, G., snd 

Butterworth, B.E. (1986b). Assessment of unscheduled and replicative DNA 

synthesis in hepatocytes treated in vivo and in vitro with unleaded gasoline 

or 2,2,4-trimethylpentane. Toxicol. Appl. Pharmacol. 85, 11-23. 

Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951). Protein 

measurement with the Folin phenol reagent. J.Biol. Chem. 193, 265-275. 

MacFarland, H.N., Ulrich, C.E., Holdsworth, C.E., Kitchen, D.N., Hslliwell, 

N.H., and Blum, S.C. (1984). A chronic inhalation study with unlesded 

gasoline. J. Am. Col I. Toxicol. 3, 231-248. 

Neuhaus, O.W., Flory, W., Biswas, N., and Hollerman, C.E. (1981). Urinary 

excretion of a. -globulin and albumin by adult male rata following treatment 

with nephrotoxic agents. Nephron 28, 133-140. 

Olson, M.J., Garg, B.D., Murty, C.V.R., and Roy, A.K. (1986). Accumulation of 

a. -globulin in the renal proximal tubules of male rats exposed to unlesded 

gasoline. Toxicol. Appl. Pharmacol., submitted. 

Phillips, R.D. and G.F. Egsn (1984) Effect of C1 Q - C u isopsraffinic solvent 

on kidney function in Fischer 344 rats during eight weeks of inhslation, 

Toxicol. Appl. Pharmacol. 73: 500-510. 

-15-
Richsrdson, K.A., Wilmer, J.L., Smith-Simpson, D., and Skopek, T.R. (1986). 

Assessment of the genotoxic potential of unleaded gssoline and 2,2,4-

trimethyIpentane in human lymphoblasts in vitro. Toxicol. Appl. Pharmacol. 

82, 316-322. 

Roy, A.K., McMinn, D.M., and Biswas, N.M. (1975). Estrogenic inhibition of the 

hepatic synthesis of a- -globulin in the rst. Endocrinology 97, 1501-1508. 

Roy, A.K. (1977). Esrly events in the steroidsl regulstion of a- -globulin in 

the rst liver. Eur. J. Biochem. 73, 537-543. 

Short, B.G., Burnett, V.L., and Swenberg, J. (1986). Histopsthology and cell 

proliferation induced by 2,2,4-trimethyIpentane in the male rat kidney. 

Toxicol. Pathol. 14, 194-203. 

Stonard, M.D., Phillips, P.G.N., Foster, J.R., Simpson, M.G., snd Lock, E.A. 

(1986). a- -Globulin: Measurement in rat kidney following administration of 

2,2,4-trimethyIpentane. Toxicology 41, 161-168. 

Viau, C , Bernard, A., Gueret, F., Maldaugue, P., Gengoux, P., and Lauwerys, R. 

(1986). Iaoparaffinic sol vent-induced nephrotoxicity in the rst. ToxicoIogy 

38, 227-240. 

Zeiss, L., Crouch, E.A.C, and Wilson, R. (1986). A possible relationship 

between toxicity and carcinogenicity,. J. Am. Coll. Toxicol. 5, 137-151. 

•16-
P.46 

Table 1 

Effect of Duration of Oral Dosing With Gssoline on RensI snd Hepstic 

Om -Globulin Content 

Treatment Renal a- -Globulin Hepatic a« -Globulin 

0*g/mg total protein) 

Saline, 9 days 20.65 * 0.91

 1 0.80 * 0.19 

Gasoline x ld 44.17 * 4.01 "'** 0.82 * 0.13 

" x 3d 66.56 * 11.13 "»* * 0.79 • 0.16 

• x 5d 69.04 * 14.12 *' 1.08 * 0.06 

• x 7d 82.74 * 34.40 ** 0.97 * 0.16 

• x 9d 63.77 * 13.86 ** 0.84 * 0.02 

Gssoline (2.0 mL/kg) or sn equivalent volume of saline were administered by 

oavage once daily and tissues for assay were obtains^ 18 hours sfter dosing, 

mean * S.E.M., n » 3 rats per treatment group. different from salinetreated group, p < 0.01 (Dunnett's test). ** different from preceding value, p 

< 0.01 (Duncan's test). 

•17-
P.47 

FICURE LEGENOS 

Figure 1. Effect of recovery time and estradiol treatment on renal a~ -globuNn 

content after termination of gasoline exposure. Rata were treated 

with vehicle alone (X) , 2.0 mL/kg gasoline, p.o. for nine days (s)) 

-or gaaoline plus estradiol (1 mg/kg, s.e.) for four days beginning on 

day nine of gasol ine administration. ( O ) • Data are expressed as mean 

* SEM a. -globulin content in liver and kidney. Arrow indicates 

terminstTon of gasoline treatment of rata. Significantly different 

from all other group means, P < 0.01 (Duncan's test). Different 

from corresponding value for group not receiving estradiol, P < 0.05, 

P < 0.01 (Duncan's test). 

Figure 2. Photomicrogrsphs of rensi tissue sections from ssline-treated (2.0 

mL/kg, p.o., 9 days; Panel A) and gasoline-trested (2.0 mL/kg, p.o., 

9 days; Panel B) rats. Enhanced hyaline droplet (<4^») accumulation, 

correlated to an increase in a. -globulin content, is obvious in the 

kidney of the gasoline-treated "animal. Mallory's Heidenhain stain. 

50X. 

Figure 3. Photomicrograph of kidney tissue from a gasoIine-trested (2.0 mL/kg, 

p.o., 9 days) rat allowed 3 days post-exposure recovery. Compare to 

Fig.2 for effect of recovery time on resolution of hysiine droplet 

nephropathy. Mallory's stain. 50X. 

Figure 4. Effect of 9 days post-gasoline exposure recovery on hysiine droplet 

(^•) number in renal PCT epithelial cells. Ms I lory's stain. 50X. 

Figure 5. Acceleration of recovery from gasoline-induced (2.0 mL/kg, p.o., 9 

days) hyaline droplet nephropathy by estradiol treatment. Estradiol 

(1 mg/kg, s.e.) was administered daily for 4 days beginning on .the 

ninth dsy of gasoline treatment. Renal tissue wss collected on the 

final day of estradiol treatment (thus si lowing 3 days recovery from 

gasoline). Compare to Figs. 2 and 3 to judge estrsdiol effect. 

Ms I lory's stsin. 50X. 

•18-
P.48_ 
P.49 

P.51 

0 9 

TIME (DAYS) 

* S ' 
P.52 

GMR-6785 

IMMUNOELECTRON MICROSCOPIC LOCALIZATION OF ALPHA-2U-GL0BULIN 

IN THB MALE RAT KIDNEY AFTER OASOLINE TREATMENT 

B. D. Garg* 

M. J. Olson* 

L. C. Li* 

M. A. Mancini** 

A. K. Roy** 

•Biomedical Science Department 

General Motors Research Laboratories 

Warren, MI 48090-9058 

••Department of Biological Sciences 

Oakland University 

Rochester, MI 48063 

For Presentation at the 

Electron Microscopy Socisty of America Meeting 

Baltimore, Maryland 

August 2-7, 1987 
P.53 

IMMUNOELBCTRON MICROSCOPIC LOCALIZATION OF o^-GLOBULIN IN THB MALE RAT KIDNEY 

AFTER GASOLINE TREATMENT 

B.D. Garg*, M.J. Olson*, L. Li*, M.A. Mancini** aad A.K. Roy** 

Biomedical Science Department, GM Research Laboratories, Warren, MI 48090 * 

Department of Biological Sciences, Oakland University, Rochester, MI 48063 •• 

Exposure of male rata to gasoline and other volatile hydrocarbons causes acute 

nephrotoxicity accompanied by accumulation of hyalin droplets (HD) in 

epithelial cells of the proximal convoluted tubules (PCT) . fe hare shown 

that HD accumulated after gasoline intoxication are partially composed of a« -

globulin ' , a protein found exclusively in adult male rate . Ultrastructural 

examination of PCT during gasoline intoxication revealed HD to be enlarged 

phagolysosomes . It is hypothesised that metabolites of gasoline hydrocarbons 

bind rerersibly to a„ -globulin, perhaps limiting lysosomal biodegradation of 

this protein . a* -Globulin accumulation may also occur due to mechanisms such 

as hydrocarbon-mediated inhibition of lysosomal proteolytic ensyms activity. 

Ultrastructural examination of PCT during gasoline intoxication revealed HD to 

be enlarged phagolysosomes These studiss were conducted to determine the 

distribution of a« -globulin within epithelial cells of PCT during gasoline 

intoxication. 

Male F-344 rats were administered unleaded gasoline or 0.9% NaCl solution (2.0 

•L/Kg, p.o., once daily, nine days) and 2 am slicss of saline-perfused kidney 

were fixed by immersion in 2.5% glutaraldshyde, 0.1 M sodium cacodylate (pH 

7.4). For iamunolocalisation of a« -globulin, thin sections on uncoated nickle 

grids were incubated with saturated sodium metaperiodate, washed sequentially 

in deionised water and Dulbecco's phosphate buffered saline (DPBS), then placed 

in 3% noraal goat serum/0.IX bovine serum albumin/DPBS. Then, polyclonal rabbit 

IgG against rat a„ -globulin (1-60 Ig/aL) was appliad to ths sections. After 

washing in DPBS, sections were treated with colloidal gold-conjugated goat 

anti-rabbit IgS for 30 min, washed in DPBS and deionised water prior to uranyl 

acetate/lead citrate ataining and electron microscopy. 

In salins-trsated rats, a faw phagolysosomes (2 /tm diam.) of uniform siss with 

round or oral contours were pressnt in ths cytoplasm of PCT spithslial cells 

(Fig. 1). There was a narked increase in the sise (9 ftm) and number of 

phagolysosomes in epithelial cells of the PCT following treatment of rats with 

unlsaded gasoline. Many of ths enlarged phagolysosomes were angular in 

sectional profile and frequently contained electron-dense crystalloid bodies 

(Fig. 2). There were no other alterations visibls in subcellular organelles 

with ths exception of a few cells showing araas of cytoplasmic clearing. The 

imaunogold technique specific for a« -globulin showed exclusive localisation of 
P.54 

this protein in the matrix of phagolysosomes; no a« -globulin was observed 

outside the phagolyaosoaes. Smaller phagolysosomes possessed uniform 

distribution of au -globulin within thsir matrices. Large irregular 

phagolysosoass, however, had deposits of a* -globulin localised almost 

exclusively orer the electron-dense crystalline inclusions, suggesting an 

aggregation of a2 u "8l°^u ^

n *a these structures (Figs. 3,4). 

Phagolysosomes (secondary lysosoass) ars formed by association of sndocytotic 

vacuoles containing engulfed proteinaceous material froa the urinary 

ultrafiltrate with primary lysosoass. The iaaunogold technique showed 

prsferential localisation of a. -globulin within phagolysosomes. Furthermore, 

as electron-dense crystalloid phagolysosome! inclusions formed, a„ -globulin 

became localised only within these condensed areas of the matrix. Thus, it is 

concluded that a defect, induced by gasoline, of renal lysosomal proteolysis 

results in sequestration of large amounts of ou -globulin in cells of the PCT. 

Furthermore, a« -globulin forms a major component of the intralysosoaal 

crystalline inclusions characteristic of hyaline droplet nephropathy caused by 

hydrocarbons. 

References 

1. CA. Haider et al., Renal Effecte of Petroleua Hydrocarbons, Princeton, 

NJ: Princeton Scientific Publishers, Inc. (1984) 73. 

2. I.M. Busey and B.Y. Cockrell, op. cit., (1984) 57. 

3. M.J. Olson et al., Toxicol. Appl. Pharmacol. (1987) in press. 

4. B.D. Garg et al., The Pharaacologist (1986) 28, 210. 

5. A.K. Roy, Burop. J. Biochea. (1977) 73, 537. 

6. M. Charbonneau et al., The Toxicologiat (1987) 7, 89. 

Figure Legends 

FIG.l. —Bpithslial call of PCT froa a control rat showing round or oral 

cross-sectioned phagolyaosoaes (-*>•). x2,400 

FIG.2. —Ths Euaber and sis* of phagolysosomes was markedly increased by 

treatment with gasoline. Many of the organelles have assumed 

rectangular and irregular shapes aad contain electron-dense 

crystalline cores (-• ). 

FIG.3. —Two large phagolysosomes are labeled with gold-tagged antibody to 

tt. -globulin (punctate electron-dense material) demonstrating ths 

presence of this protein in tho phagolysosomes. Note absence jj|: 

extralyaosomal labeling. x2,400 . ' • -" 

FIG.4. —Higher magnification of phagolysosomes showing electron-dense 

antigen-antibody complexes labeling a^-globulin. x2,400 
P.55 
P.56 

^1» " W * a-iS* " *3<L . mm .' 
P.57 

GMR-5597 

.*?•••• • 

HYAUNE DROPLET AND 

0£2u. -GLOBULINACCUMULATION (N W E KIDNEYS OF 

MALE RATS: AFTER GASOUNE 

ADMINISTRATION 

AV ' M. J. Oteorv 

B. D.Gafg 

Department 

^•i^r^'^'-'r f 

:..-.*..' 

October 27,1986 

• lesearch L ' 

Warren. Michigan 48090 

MAY BE OlSTRlBUTEO OUTSIDE GENERAL MOTORS . Not to be Reproduced Without Permission 
P.58 

GMR-5597 

HYALINE DROPLET AND a2u-GL0BULIN ACCUMULATION IN THE KIDNEYS 

OF MALE RATS AFTER GASOLINE ADMINISTRATION 

M.J. Olson and B.D. Garg 

Biomedical Science Department 

General Motors Research Laboratories 

Warren, MI 48090 

F.H. Sarkar and A.K. Roy 

Biological Sciences Department 

Oakland University 

Rochester, MI 48063 

Presented October 28, 1986 at the 16th Conference on Toxicology, 

Dayton, Ohio 
ABSTRACT 

Saturated branched-chain aliphatic hydrocarbons, found in motor fuels, induce a 

unique nephrotoxicity in male rats. Unleaded gasoline administered to male rats 

(0.04 - 2.0 mL/kg body weight, p.o.) for nine days caused a marked increase in 

th© number and size of hyaline (protein resorption) droplets in epithelial 

cells of the proximal convoluted tubules (PCT) and increased cellular 

exfoliation at high dose levels. No other treatments© I ated pathological 

effects were observed in the glomeruli, distal tubules or medulla. The renal 

content of ts« -globulin, a protein synthesized in the liver and excreted in the 

urine of male rats, was determined by radioimmunoassay to be increased in a 

dose-related manner; maximal accumulation was 440% of control. Subsequently, we 

observed that both hyaline droplets and e„ -globulin were increased 

significantly after a single dose of gasoline (2.0 mL/kg,p.o.). Immunoperoxidase staining of kidney tissue for a« -globulin revealed increased protein 

content in gasoline-treated rats and Targe accumulations of antigen were 

localized in some PCT epithelial cells which contained hyaline droplets. The 

hepatic concentration of a„ -globulin and the quantity of a„ -globulin mRNA in 

liver were not altered by gasoline. Accumulation of a., -globulin was reversed 

within three days of terminating gasoline administration. A hydrocarbon-induced 

defect in the renal lysosomal degradation of low molecular weight proteins, 

rather than increased synthesis or defective metabolism of a« -globulin, 

appears to cause hyaline droplet accumulation. 

INTRODUCTION 

Some petroleum hydrocarbon mixtures used as motor fuels, solvents, paint and 

varnish additives, degreasing agents and industrial intermediates cause dosedependent alterations in the form and function of the renal cortex following 

subacute or chronic exposure (1). The importance of understanding hydrocarbonmediated nephropathy increased greatly following the observation that rats 

exposed for 18 or more months to wholly volatilized unleaded gasoline exhibited 

an increased incidence of renal carcinoma (2). The occurrence of tumors was 

sex- and species-specific; male rats only exhibited treatment-related renal 

neoplasia. Analysis of gasoline and some of its nephrotoxic constituent 

hydrocarbons in short-term assays for genotoxicity has produced equivocal or 

negative results (3). Thus, research has been directed toward elucidating the 

mechanisms by which hydrocarbons induce overt nephrotoxicity in the hope that 

an epigenetic event related to toxicity, such as an increased rate of 

restorative cell replication, may explain the occurrence of renal tumors. 

The primary site of toxicity of hydrocarbon mixtures similar to gasoline, 

such as jet fuels, C1Q-C„_ isoparaffinic solvent or Stoddard solvent appears to 

be epithelial cells oV tiie renal PCT and, by light microscopic examination, 

presents as an accumulation of protein resorption droplets (1). Although a 

constitutive population of droplets exists, overabundance of these droplets, 

also known as hyaline droplets, is interpreted as a pathological alteration. 

Recently, it was shown that the renal content of a„ -globulin, a major urinary 

protein found exclusively in mature male rats \*)» wa s increased many-fold 

during exposure to either saturated isoparaffinic solvent or decahydronaphthalene (decalin). a„ -Globulin has been linked inferentially to the 

development of hyaline droplet nephropathy since volatile hydrocarbon-induced 

nephropathy is limited to male rats. Species, which lack a_ -globulin (mice, 

-2-
P.60 

dogs, monkeys), show no evidence of hyaline droplet accumulation in either sex'. 

The studies described here were performed to characterize histological and 

biochemical changes in the kidneys of male rats after short-term treatment with 

unleaded gasoline. Oral administration was used in an attempt to develop a cost 

and time-efficient method for inducing the early changes characteristic of 

hydrocarbon-induced nephropathy. Specific analysis of th© kidneys was performed 

to.examine whether a. -globulin was accumulated during gasoline intoxication 

and to determine the subcellular disposition of this protein within the kidney. 

MATERIALS AND METHODS 

Experimental animals and chemicals 

Male Fischer 344 rats (CDF (F-344)/CrI BR; Charles River Laboratories, 

Wilmington, MA) weighing approximately 250 g were used throughout these 

studies. Rats were administered physiological saline or a commercially 

available unleaded gasoline by intragastric intubation. In some experiments, 

animals were allowed to recover for a variable period after termination of 

gasoline administration. 

Histological procedures 

Rats were anesthetized with pentobarbital, perfused via the heart with cold 

0.9% sodium chloride and a complete necropsy was performed. Kidneys and livers 

were removed, fixed by immersion in 10% neutral buffered formalin, processed 

routinely and embedded in paraffin. Tissues for immuno-localization of a„ -

globulin were fixed by immersion in 3.6% formalin, 0.2 M mercuric chloride fpH 

7.4) then processed and embedded in paraffin. 

Serial sections of kidney were stained with Mallory's Heidenhain method or 

immunostained for a„ -globulin with the avidin-biotin procedure (5). Briefly, 

the tissue was deparaffinized, rehydrated and incubated in a solution of 3% 

normal goat serum, 3% bovine serum and 2% gelatin. Sections were then incubated 

with polyclonal anti=e„ -globulin (rabbit IgG) for 30 min and washed with 

phosphate buffered saline (PBS; 0.14 M NaCl, 0.01 M Na-phosphate, pH 7.4). The 

sections were further incubated for 30 min with biotinylated anti-rabbit IgG 

(goat IgG) and washed. A final 30 min incubation with freshly prepared avidinbiotin reagent was followed by extensive washing in PBS. The tissues were then 

placed in 0.05 M Tris-HCI, pH 7.0, containing 0.0014 U 3,3'-diaminobenzidine 

tetrahydrochlorid© and 0.003 M hydrogen peroxide for 15 min. The reaction was 

terminated with a 15 min rinse in running water and the sections were 

counterstained with Harris hematoxylin. 

Dot-blot assay of c? -globulin mRNA in liver 

- Total hepatic RNA, extracted with phenol and sodium dodecyl sulfate was used 

as the starting material to isolate poly (A)-containing RNA by oligo(dt)-

cellulose chromatography. Serially diluted poly(M-containing RNA was spotted 

on nitro-cellulose filters and hybridized with P-labeled a« -globulin cDNA 

(6). After hybridization, quantitative binding of the probe was determined by 

autoradiography and soft-laser scanning densitometry. 

-3-

ineiiati M^atMamMi 
P.61 

Radioimmunoassay of a,, -globulin in kidney and liver 

Homogenates of whole kidney and liver were prepared and centrifuged at 

100,000 x g; the supernatant fluid was used for double antibody RIA of a„ -

globulin as described by Roy (7). The concentration of a« -globulin in tHe 

supernatant fraction was expressed as fig a„ -globulin per mg total protein. 

-4-
RESULTS 

Figure 1. Serial sections of renal cortical tissue from a saline-treated rat 

(2.0 mL/kg, p.o., 9 days) prepared with Mallory's Heidenhain stain to 

demonstrate hyaline droplets (Panel A; • • ) or the immuno-peroxidase 

method for localization of a« -globulin (Panel B;^-) . Note degree of 

correlation between areas which are positive for both stains. 50X. 

Figure 2. Renal serial sections from a gasoline-treated rat (2.0 mL/kg, p.o., 9 

days). An increase in hyaline droplet number and size is evident in 

comparison to Fig. 1. Compare the localization of Mallory's stainpositive hyaline droplets (Panel A; • • ) with areas which stain 

intensely for a- -globulin (Panel B;^») . 50 X. 

Figure 3. Accumulation of a„ -globulin in the kidney as a function of gasoline 

dose. Gasoline or corn oil vehicle were administered by gavage once 

daily for nine consecutive days. 18 hr after the final administration 

of gasoline, kidney (*f)) and liver (O) tissues were sampled for RIA 

determination of a„ -globulin. Data are plotted as mean * SD a„ -

globulin content, n = 3 - 7 rats per treatment group. p < 0.01 

• • 

compared to control (Dunnett's test), p < 0.01 compared to next 

lowest dose (Duncans's test). 

Figure 4. Dot-blot hybridization of hepatic mRNA with cDNA for a„ -globulin. 

Lane A; poly (A)-containing RNA from a saline-treated rat, lane B; 

RNA from a rat treated with 2.0 mL/kg gasoline, p.o. for 9 days. Row 

1 a 1.0 /ig RNA, rows 2- 4 = serial 1:1 dilutions of RNA from row 1. 

Original densitometry traces of lanes A and B are shown. B/A is the 

ratio of areas under the curve for corresponding dots in lanes B and 

A. 

• 5 -
P.63 

*/ - - .*. •. 

.*£ 

* -, 

* a 

at 

* m> • 

1 

^3> 

Figure 1 

- 6 -
P.64 

^T1>mv. ktmWmMr . 7r> raMOT

 rk.-..;\ ;'..•. •"/ ' • 7 4 

• m ^ if 

• tm~T e > 

Figure 2 

- 7-
150 

4 x 10"4 4 x 10-3 4 x 10-2 4 x 10"1 

Gasoline Dose (mL/kg) 

Figure 3 

- 8 -
1 2 3 4 

A 

B 

IOO, 

0' 

lOOi 

B/A (%) no *i i n 101 

Figure 4 

- 9 -
Table 1 

Time Course of Renal and Hepatic a« -Globulin Accumulation During Oral 

Dosing WitK Gasoline 

Treatment Renal a„ -Globulin Hepatic a„ -Globulin 

(jig/mg total protein) 

Saline x9d 20.7 * 0.9

 l 0.8*0. 2 

Gasoline x ld 44.2 * 4.0 **'** 0.8 * 0.1 

" x 3d 66.6 * 11.1 **'** 0.8 * 0.2 

" x 5d 69.0 * 14.1 ** 1.1 * 0.1 

" x 7d 82.7 * 34.4 ** 1.0 * 0.2 

" x 9d 63.8 * 13.9 ** 0.8 * 0.0 

mean * S.E.M., n = 3 rats per treatment group. different from salinetreated group, p < 0.01 (Dunnett's test). different from preceding value, 

p < 0.01 (Duncan's test). Gasoline (2.0 mL/kg) or an equivalent volume of 

saline were administered by gavage once daily. 

-10-
P.68 

Table 2 

Effect of Recovery Time on Renal a„ -Globulin Content After 

Oral Gasoline Administration 

** 

Treatment Renal a„ -Globulin 

2u 

(/ig/mg total protein) 

Saline, 2.0 mL/kg x 9 d 24.2 * 4.2

 X (3) 

Gasoline, 2.0 mL/kg x 9 d 127.0 * 11.0 (6) 

Gasoline x 9 d • 3 d recovery 33.0 * 5.1 (3) 

Gasoline x 9 d • 6 d recovery 31.9 (2) 

Gasoline x 9 d • 9 d recovery 26.2 * 4.1 (3) 

Gasoline x 9 d • 12 d recovery 34.0 * 5.2 (3) 

mean * S.E.M., n in parentheses. different from all other group means, 

p < 0.01 (Duncan's test). Rats were administered saline or 2.0 mL/kg gasoline 

by gavage once daily for nine consecutive days followed by the recovery periods 

shown. 

•11-
P.69 

SUMMARY AND CONCLUSIONS 

We have demonstrated that short-term oral administration of unleaded 

gasoline to male rats causes hyaline droplet nephropathy, the characteristic 

initial renal lesion observed during chronic inhalation of wholly volatilized 

gasoline by rats. Microscopically the renal pathology was confined to the PCT 

and was characterized by accumulation of hyaline (protein resorption) droplets. 

Morphological and biochemical characterization of the kidneys of gasolineintoxicated rats showed that: 

1. Oral administration of unleaded gasoline caused simultaneous increases in 

the number of renal hyaline droplets and a_ -globulin; doses as small as 

0.04 mL/kg induced significant renal accumulation of a„ -globulin. 

2. Hyaline droplet nephropathy was induced within one day of initiating oral 

administration of unleaded gasoline; three days of treatment induced maximal 

accumulation of a„ -globulin and hyaline droplets. 

3. Localization of a„ -globulin, by a specific immunochemical reaction, was 

demonstrated in PCT which were most severely involved in gasoline-induced 

hyaIine droplet accumulation. However, not all Mallory's stain-positive 

hyaline droplets were recognized as containing <x« --globulin. 

4. The hepatic content of a_ -globulin and expression of its specific mRNA in 

the liver were not significantly affected by gasoline administration at 

times when renal a. -globulin was increased. 

5. Hyaline droplet nephropathy and the renal accumulation of a« -globulin 

induced by gasoline were rapidly reversible after subacute administration of 

gasoline. 

Experiments are in progress to define the sequence of events preceding 

accumulation of a« -globulin in the kidney. The goal of these experiments is to 

reach a mechanistic explanation for hydrocarbon-induced hyaline droplet 

nephropathy. 

•12-
P.70 

REFERENCES 

1. Renal Effects of Petroleum Hydrocarbons, M.A. Mehlman, G.P. Hemstreet, III, 

J.J. Thorpe and N.K. Weaver, eds., Princeton Scientific Publishers, 

Princeton, NJ, 1984. 

2. MacFarland, H.N., C.E. Ulrich, C.E. Holdsworth, D.N. Kitchen, N.H. 

Ha 11iweII and S.C. Blum, J. Am. Coll. Toxicol. 3: 231-248, 1984. 

3. Loury, D.J., T. Smith-Oliver, S. Strom, R. Jirtle, G. Michalopoulos and 

B.E. Butterworth, Toxicol. Appl. Pharmacol. 85: 11-23, 1986. 

4. Roy, A.K. and O.W. Neuhaus, Proc. Soc. Exp. Biol. Med. 1: 894-899, 1984. 

5. Hsu, S.M., L. Raine and H. Fanger, Am. J. Clin. Path. 75: 734-738, 1981. 

6. Roy, A.K., B. Chatterjee, W.F. Demyan, T.S. Nath and N.M. Motwani, J. Biol. 

Chem. 257: 7834-7838, 1982. 

7. Roy, A.K., Eur. J. Biochem. 73: 537-543, 1977. 

-13-
P.71 

UNLEADED GASOLINE-INDUCED PROTEIN DROPLET NEPHROPATHY: 

RELEVANCE OF A MALE RAT SPECIFIC SYNDROME TO HUMANS 

Michel Charbonneau, Brian G. Short, Edward A. Lock 

and James A. Swenberg 

Chemical Industry Institute of Toxicology 

Department of Biochemical Toxicology 

and Pathobiology 

P. 0. Box 12137, Research Triangle Park 

North Carolina 27709 

Tn* Auoelollon 

Dmdlcolmd to 

Al> Pollution Control and 

Moiardout Wotlm Manogmmmnt 

For Presentation at the 

80th Annual Meeting of APCA 

New York, New York 

June 21-26,1987 
represents a possibl e oourco of human asposuro vi a th e consumption of 

drinking wate r containin g water-oolublo chemicals from UG. 

RENAL EFFECTS OF UNLEADED GASOLINE IN ANIMALS 

UG carcinogenicit y i n aaisal o wao tooted i n a 2-yoar otudy{ osposure 

t o vaporo of OG caused a a ineroaoo d iaeidoacG of roaa l adaaoaa s and 

eareiaomao i a ©alo FischGr-344 rato , but aot i a female rat o @£ oitha r sex 

of mice (2,3) . Subchroai e inhalatie a Gzspeouro t o UG vapor o produced a 

©ale rat-spacifi e nephropath y characterise d by a a iaeroaoo d protei n 

(hyaline ) dropla t fersatie a i a opitholia l coll o of tho prosisa l tubules, 

with accompanying degenerative changes of tho proximal tubul e i a the rena l 

cortea , aad tubula r dilatatio n and eaoto a t th e juactioa s of tho iane r and 

oute r otrip o of tho oute r ©odulla(4)o 

Evaluation of tho aophrotoxie preportioo ©f ocvora l eo©penoato of UG 

do©oaotratod that , ao a elaao , braaehod aliphati c alkaaoo (iooparaffiao ) 

aro primaril y raoponoiblo for tho aophrotoxie actio a ©f UG (4) . Among tho 

aophrotesi e hydroearboao typicall y fouad i a OG0 2 9 2 9 4°tei@othylpQataa e 

(TKF)D a choaica l added as aa oati-kaoekiag agoat, woo ©boosted t© bo very 

aetiv q (5). Thorafera , mW hao booa uood oo a pretetyp o compound t o otudy 

t h e aechanio© of OG=iadueed aephreteaieit y i a rat o (5,7,8,9,10) 0 

Aooesomeat of th e geaotosi c propertie s of ualeade d gasolin e by a 

batter y of tests has shown tha t OG io a aoa-genotoxic chemical (11,12,13). 

Xt ha s been postulate d ( 9,14 ) tha t tumor productio n i o cause d by a 

chroni c regenerativ e proceoo . Loury £ t a^. (13) reporte d tha t ©ale rats 

trGatod (inhalatio n or gavage) wit h UG ohowed a ©arkod iaeroao o i a th o 

perceatag o of kidne y cell o i a S-phaoG, i.o . coll o undergoin g DNA 

syntheoio , whoraao col l proliforatio n wao aot oigaificaatl y enhanced i n 

female rato . Doing a ©ulti-otag o careinogeneoio modol (15,IS,17 ) i t was 

propoood tha t th o productio n of ©aligaan t tumors rosult o fro© repeate d 

geneti c changeo i a th o cell o of th e hoot tiooue . An adaptatio n of thi s 

mode l fo r aoa-gaaotosi c chemical o producin g tumor o afte r chroni c 

administratio n t o aaimal o coul d pootulat o thats 1° Cello , i a whic h 

spoataaoou s DNA altGration o naturall y occur , replicat e followin g 

chemically-iaduce d coll.proliferatio a cauoia g a mutate d coll . 2 - The 

mutate d cel l uadergoe o clona l expaaoio a du e t o iacrease d cel l 

proliferation . 3° This greatl y increase s th e likelihoo d of a secon d 

spontaneous mutatio n occurring , leadin g an increase d incidenc e of rena l 

cancer. CIIT' a approach t o study UG-iaduced rena l tumors has therefor e 

been focusec^on UG-induced col l proliferation . 

ONLEADED GASOLINE-INDUCED CELL PROLIFERATION 

A 21-day hiotoautoradiographi c stud y was undertake n t o assess th e 

intra-rena l localisatio n of UG-induced cel l proliferation . Thre e male 

Fischer-34 4 rat o pe r group were exposed t o 0, 2 , 20, 200 or 2000 ppm UG 

(6 hr/day , 5 days/week fo r 3 wooko). Oomotic pumps fillo d wit h [ H]-

thymidine ( H-TdR) were implanted oubcutaneouoly on day 12 t o aoour e a 

constant releao e of H-TdR during the last week of oxpooura. Tho animals 

were kille d 1 day afte r th e last exposure , and perfuoioa-fiso d kidney s 

were removed and procesoo d fo r ligh t and dar k fiol d autoradiographi c 

examination of rena l cel l proliferation . Percentages of labolod colls (Sphase) over tota l cells were determined for the Pl , P2 and P3 segments of 

the proximal, tubule . Figur e 1 shows tha t the percentag e of labelo d cells 

over tota l cell o was significantl y (p < 0.05) increased for tho P2 segment 

- 2 -
rena l DNA measured i n kidneys of female Fiecher-344 rats treate d wit h 500 

mg/kg TMP 4 os 6 dayo previousl y were not significantl y differen t from 

contro l value . Therefore , TMP-induced rGnal DNA synthesis is incroaoed i n 

a dose-dependent manner afte r the administratio n of a oingl e ora l dose of 

TMP t o male but not female rato . 

PROTEIN DROPLET ACCUMULATION 

ThG P2 oogaeat corrospoado t© tho rGgiea of tho aophroa whoro th e low 

©oloeula r weigh t protoiao , filtoro d iat o th o tubul o luso a a t th e 

gle©oruluo , ar e reabsorbe d by ondoeyteoi o iat© coll o ©f th o proxima l 

tubtsloo (18) o' Tho oadeeytetie . vooieloo (oadoooaeo) fuoo wit h lyoeoomes, 

aad th e protoia o coataiae d i a tho rooultia g ondolyoooemes ar e hydrolysed 

t o .thoir constituen t aaiao acido by lyo©o©mal Gagyaeo (19). @2u=Gl©bulin, 

a low ©oloeula r woight protei a produeod by ©alo bu t ae t fosal o rat o is 

roaboerbod by thi o precooo (20). Haldor o^ al . (4) reporte d aa iaereaood 

pretoi a (hyaliao ) drople t foraatie a i a th o opitholia l coll o @f th o 

prosiaa l fcubuloo afto r UG oxpoaura . Tho rol o of @2u=globulin i n th o 

pretoi a dreplo t nephropathy wil l bo dioeuoood i a tho following paragraphs. 

Short oji cAo (9 ) reperto d tha t th o ostoa t (au©bor of tubulo o 

affoetod ) oad/e r th o oevorit y (ois o @r ohapo) ©f £ho jpsetai a dreplot o 

iaereaoe d afte r a 21-day gavago regiao a ©f 50 t© SO© ©g/kg TMP5 th e 

reopoaoe wao P2 OGgmoat-opGcifie, aad a© dooe-reopoaoo rolatieaohi p was 

obocrvGd. Pretoi a droplo t ovaluatio a i a kidaeyo of ©alo rat o d©oed with 0 

t o 50 ©g/kg TMP for 21 dayo ohewod, however, a dooe-relatGd iacroas e i a 

t h e sea a ocoro for droplot o (Piguro 3? I* = ©ild , 2* •= ©odorate , 3+ ° 

OGvaro aad 4* <=> ©arkodly oovoro). ©vor tho raago ©f doooo otudiod , th o 

iaerooooo i a tho ©oaa ocoro fe r dreplot o parallolo d th o iacroaooo i a tho 

porcoatag o of labele d coll o ©vor th o tota l cell o (Figur a 3) . Thuo, 

protoi n drople t foraatio n and col l proliferatio n appoa r t o bo rolato d 

phenomena. ° 

MalG rat o treate d with a ainglo dooo of 500 mg/kg TMP ohowed a highe r 

©eaa ocoro fo r protoi n droplet o tha a coatrol o (3. 4 a 0. 1 vo 1.4 & 0.1 , 

respectively). Kidney heaogenateo obtained frc© treate d animals were used 

t o ©eaoure th e rena l concentratio n of @2u-globulin usin g aa ELISA assa y 

with a EOUOG ©onoclonal antibody raise d toward purifio d ra t urinar y e2uglobulin . Renal coacGatratio a of @2u-globulin wao ©arkGdly iaereaoe d i n 

t h e TMP-treatod group coapared t o tha t of corn oil-treate d rat o (28. 1 & 

2.7 vo 9.2 a O.S. ag @2u-globulin/g wot weight). Moaa ocoroo for droplet o 

aad rena l @2u^-globulia coaceatratio n wGro ao t sigaificaatl y (p > 0.05) 

iacroaoG d i a fosal o rat o troato d wit h 50 0 ©g/k g TMP. laauao -

histochemistr y studio s performed uoing th e mouse monoclona l antibod y 

ohowed tha t i a wel l porfuood kidaoyo ebtoiao d fro© 50 ag/k g TMP°treated 

rat o tho roaa l @2u-glebulia woo localise d withi a th e protoi a droploto . 

BIOCHEMICAL EVALUATION OF THE MECHANISM OF TMP-INDUCED NEPHROPATHY 

The results discusse d i a tho prGviouo ooctiono indicate d tha t rena l 

tumor formation may bo relate d t o aa iacreaood col l proliforatio a of th e 

cell s from a specifi c aro a of th o kidaoy , aad tha t thi o rogGnorativ e 

process &aj be rolato d t e tho protei a droplGt nephropathy ©ceuriag a t this 

samo oit o i n th o aephroa . @2u=globulia appoaro t o coatribut o i n th e 

nephropathy oinco thG protei a droploto coatai a thi o aajo r ©alo ra t urinar y 

protein . Howovor, tho aechanio® of it o rolo i n th e nephropathy hao yot t o 

be detcrminod. The presen t oectio n wil l sussaarige tho reoult o of recen t 

- 4 -
P.74. 

fraction was associated with s2u-globulinj gel filtration (Sephadex G-75 

coluaa oluted with 67 ©M phosphatG buffor, pH 7.2) wao used to separate 

tho protGino. Chromatography of aalo rat kidney cytosol separated ®2uglobulin fro© other high and low molecular weight proteins. Radiolabel 

derived fro© [ HJ=TMP-troated ©alG rat kidney cytosol eluted into two 

distinct poako, ©no ia tho molecular weight range of e2u-globulia, which 

constituted abeut 262 ©f tho total radiolabel ia tho eyteoelie fraction or 

152 ef tho tetal sadielabal in tho kidney? material ia tho froctioas of 

tho firot poak e?@oo=roaetod with tho ©onocloaal aatibedy raiood toward 

purified rat urinary ®2u=globulia. The ooeead poak of radielabal satorial 

olutod ia tho opall ©oloeular weight raago aad io thought t© repreoeat TMP 

aotabelitooo Fosalo sat kidnoy eyteoelie fraetieno proparod fro© animals 

troatod 24 hr previously with [ H]°TMP rooelvod into oao diotiact peak ia 

tho osall ©olocular weight rango. ThorGfero, eo-olutioa of TMP-derived 

radielabol with ©^ -globulin io a ©alo rat-opocific phGaeaoaoa. 

Appreaiaotofy 202 ®f tho radielabolod ©atGrial proooat ia aalo rat 

kidaoy ©yteool wao aoa-dialysabla fellewiag 18 hr oquiliferiua dialysis 

(1000 d cutoff) agaiaot 10 mM phoopha^ buffer, pH 7.0. Chromatography of 

tho dialysod eyteool @a the Sophadoa ©°75 coluaa ohowed that the aoadialysablo radielabolod ©atGrial ee=olutod with tho fraetieno eeataiaiag 

tho <32u°glebulia. Equilibria© dialyoio ef ©alo rat kidaoy cytosol agaiast 

buffor eeataiaiag 0.12 oodiua dodocyl oulfato (SDS) lod te looo of thio 

biadiag. SDS io a dGtorgent which deotroyo tho oeeondary and tertiary 

otructureo ©f protoiao (22)8 ehomicalo bouad te tho protGia via weak 

forcGo (hydrogea boado, ionic beads, Vaa dor Uaal°o foreos) will be 

diooeciatod from it and released iato the dialyoio buffor upoa addition of 

SDS. T&s&o, ©ur fiadiago iadieatod a rovoroiblo aooeciatioa botwooa TMPderived radiolabel and s2u=globulino 

Ethyl aeotato attraction of tho pooled @2u-globulia-coataiaiag 

fraetieno of ©alo rat kidney cytosol from [ H]=TMP-treatod rato recovered 

©oro than ©12 ©f tho total radiolabel. Gas chroaatography-eass 

spectrometry ©f tho acidified ethyl acetate oatract ohowed that 2,4,4-

trimothyl-2-pentaaol woo the metabolite associated with the 82u°globulia. 

Goo chromatographic aaalyoio of acidified ethyl acetate eatract prepared 

fro© kidaeyo of aalo rato expooed by iahalatioa to 50 ppa TMP or 300 ppa 

UG (6 hr/day, 5 dayo/woek, 3 weoko) aloo ideatified 2,4,4-tri©othyl-2-

pentanol ao tho eho@ieal bound to @2u-globulin§ two othor minor 

components (yet to be identified; their retention times suggest other 

branched alcohols) wero aloo extracted fro© the samples prepared from UGexpoood rato. Gol.filtration of kidney cytoool prepared from 500 ag/kg 

[ H]=TMP=treStod rato and determination of tho radiolabeled material in 

"the @2u-globulia-eeataining fractions showed that 8, 24, 48 and 72 hr 

after dooing, tho coaeoatratioa of bouad 2,4,4=trimethyl-2°pentaaol was 

78.9 o 7.2, 118.0 * 4.3, 101.3 a 8.5 and 85.5 * 13.8 nmol/©l of kidney 

cytoool, roopoetivoly. Thio oboorvation indicates that the 2,4,4= 

trimethyl=2°pontanol=@2u-globulin cosplGa remains in the kidney of male 

rats for a long period of time suggesting a fairly stable association in 

vivo. 

Cell proliferation has boon aoooooed biochemically, 4 dayo after a 

singlo oral dooo (4.4 saol/kgs equivalent to 500 mg/kg TMP), for each of 

the known TMP metabolite©. No male rat-opecific increase wao oboorved in 

theoe conditions. Cell proliferation hao also been measured. 1 day after 

tho laot of 7 doooo of 0.44 mmol/kg 2,4,4-trimethyl-2-pentanol or 2,4,4-

trimethyl-l-pontaaoic acid, the major urinary metaboliteos a oignificant 

increaoG above control value wao observed for the 2,4,4 = trimethyl-2-

pentanol-treated group, but not for the 2,4,4-trimethyl-l-pentanoic acidtreated group. Therefore, the metabolite of TMP revoroibly bound to 62u-

-6-
exposure to UG via contaminated drinking water, Coleman ejt aJL. (25) 

reported that 97Z by weight of the components in the water-solubl e 

fraction of unleaded gasoline were aromatic compounds. Results presented 

in this paper showed that the aromatics in UG do not appear to be 

implicated in the nephrotoxicity, and that renal tumor formation may only 

be a male rat-specifi c response. Therefore, renal tumor formation in 

humans following consumption of drinking water contaminated with UG 

appears unlikely to occur. 

ACKNOWLEDGEMENT 

Ve gratefully acknowledge Dr. James S. Bus for his contribution in 

the creation of this research program, and Ms. Vieki L. Burnette and Mr. 

Josef Strasser, Jr. for their excellent technical work. 

- 8-
13. D.J. Loury, T. Smith-Oliver and I.E. Butterworth. 1987. Assessment 

of unscheduled and roplicativ o DNA oynthooio in ra t kidney colls 

exposed in vitr o or in vivo to unloaded gaooline . Toxicol. Aool. 

Pharmacol., 87s 127-140. 

14. B.F. Trump, T.H. Jones ond M.M. Lipoky. 1985. Light hydrocarbon 

nephropathy. Ins Renal heterogeneity and targe t cel l toxicity . P.H. 

lae h aad S.A. Leek, odo. , Joha Hiley and Sons,^Chiehootor, Gr. 

Britain, pp. 493-504. 

15. H. Honaiago, 1. Shoroo, M.L. Uoak, E.F. Spaaglor, R. Tar©ao aad S.H. 

Yuspa. 1983. Maligaaat conversion ©f aouse okia tusouso io iacreased 

by tumour iaitiater o and uaaffoetod by tuaour preaotoro. Katuro, 304s 

67-69. 

16. S.H. Moolgavkar. 1986. Carciaogeaeoio aodoliags Frea aolecula r 

biology to epidemiology. Ana. Rev. Publie Health, 78 151=169. 

17." J.A. SwenbGrg and S.G. Short. 1987. Influeaeo of cytotoaicity oa the 

iaductioa of tussoro. Banbury Roport 25 s Noagoaetoaie ©ochaaisas in 

carcinogoneoio. (ia proso) 

18. U. Strauo. 1964. Occurrence of phagoooaeo aad phago-lyooooaeo i a 

difforGnt oogaonto of the nephron i a relatie a t e tho roaboorptioa , 

traaopert , digootioa , aad oatruoie a ©f iatravoaouol y iajoete d 

horoeradioh poroaidooe. J. Coll Biol.o 21s 295-308. 

19. T. Maack, CH . Park and M.J. Caaarge. 1985. Ronal filtration , 

transport, and ©etabolism of protoiao. las The kidneys Physiology 

and pathophysiology, D.H. Seldin and G. Giobioch, odo.,Raven Press, 

New fork, pp. 1773-1803. 

20. O.H. Neuhauo. 1986. Renal roaboorptioa of lew aolocula r weight 

protoiao i a odult raale rato8 @2u°globulia. Proc. Soc. gap. Biol. 

Med. 182s 531-539. 

21. J.D. Sua aad Doat, J. C 1980. A aew aothod for aoaouriag eevaloa t 

biadiag of chesiealo to cellula r aacrcaolQCuleo. Che©.-Biol. Xntorac. 

328 41-61. 

22. A.L. Lohninger. 1975. Biochesiotry. Tho aolocula r baoio ©f col l 

otructure and function. Second odition. Uorth PubliohGro, Now York, 

p.180. 

23. A.K. Roy and O.H. Neuhauo. 1966. Proof of the hepatic oynthooio of a 

oea-dependent protein in the rat. Biochem. Biophys. Acta 127s 82=87. 

24. CT. Oloon, K.O. Yu and M.P. Servo. 1986. Motabolioa of nophrotoaic 

cio-and trano-decali n in Fischer-344 rata . J. Toxieol. Environ. 

Health, 18s 285-292. 

25. H.E. Coleman, J.H. Munch, R.P. Streicher, H.P. Ringhand and F.C. 

Kopfler. "1984. The identificatio n and measurement of componento in 

gaooline, kerooene, and No. 2 fuel oil that partitio n into the aqueous 

phase after mixing. Arch. Envi ron. Contam. Toxicol. 3s 171—178. 

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P.79 

Control TMP TMP TMP TMP 

SmgAg 10mg/Vg SOmgAg SOOmgAg 
Breathing Zone Levels Of Gasoline Vapors 

During Automobile Fueling 

Ronald L. Williams 

General Motors Research Laboratories 

Environmental Science Department 

Warren, MI 48090-9055 

P.80 

87-31.1 
P.81 

87-31.1 

INTRODUCTION 

Humans are exposured to gasoline vapors in refineries, at bulk terminals, 

and in service stations. Health concerns about gasoline have historically 

focused on certain constituents such as polycyclic aromatic hydrocarbons, 

benzene, and lead additives. Potential human health effects from these 

components have been extrapolated from animal exposure studies and have been 

evaluated in occupational groups. A recent study sponsored by the American 

Petroleum Institute (API) showed that exposure to vaporised whole gasoline had 

induced kidney tumors in male rats,^ although the specific cause was unknown. 

This study prompted the automobile industry and EPA to ask the Health Effects 

Institute (HEI) to review the scientific literature relating gasoline vapor 

exposure to human, cancer. The HEI report,3 issued in September of 1985 

concluded that although wholly vaporised unleaded gasoline is an animal 

carcinogen, the vapor composition in the animal studies was sufficiently 

different from that to which humans are exposed that "use of the API data to 

develop dose-response relationships suitable for human risk assessment is 

questionable. In addition, adequate data are not currently available to 

characterise accurately the nature and extent of human exposures to gasoline 

vapors.a 

This paper summarises an earlier General Motors study that was undertaken 

to measure the concentrations of hydrocarbon (HC) vapors in the breathing zone 

of a person fueling a vehicle.1* In addition, a review is presented of other 

publications.that address gasoline vapor exposures during fueling. 

Differences among all these publications in the sampling and analysis 

techniques and in the units used to report exposures are reconciled. Finally, 

a best value for the range of exposures is derived from the total data set. 

EXPERIMENTAL 

In 1985, fifteen vapor samples were collected during February and 18 

samples during August and September near a gasoline pump at the General Motors 

Technical Center in Warren, Michigan used for vehicle fueling. A small Tedlar 

bag was filled with air taken from the breathing zone of a person standing 

adjacent to the car's filler pipe during only the time that the gasoline pump 

was running. Wind speed, wind direction, and ambient temperature were noted 

before collecting each sample. The concentrations of individual HC were 

determined by gas chromatography (GC) and the total hydrocarbon (THC) was 

measured by summing the areas of all the GC peaks. Two background air samples 

were collected during the sampling program to verify that background levels of 

HC did not contribute measurably to the levels of the gasoline-vapor 

compounds. 

The gasolines used for fueling were commercial unleaded gasolines. One 

sample each of the winter and summer fuels was collected from the gasoline 

pump for GC analysis and Reid vapor pressure measurements. The fueled cars 

were all 1985 General Motors models from a corporate fleet. The tank fuel and 

the dispensed fuel were generally the same, except for changes in the 

composition of the tank fuel due to weathering. The use of a single fuel is 

an important consideration because it permits an unconfounded comparison 

between the composition of the refueling vapors and the composition of the 

dispensed gasoline. Furey and Nagel recently showed that fueling vapors are 

composed of about two-thirds dispensed-fuel vapor and about one-third tankfuel vapor, which would complicate the comparison if the two fuels were 

different. The fuel-tank filler pipe location was documented for each vehicle 

because this parameter affected the orientation of the sampling point relative 

to compass wind direction, as indicated in.Figure 1. 
P.82 

87-31.1 

Procedure for Collecting Vapor Samples 

The objective was to collect an air sample representative of what a 

vehicle fueler would breath, in a container that could be taken to the 

laboratory for GC analysis. Tedlar sampling bags (3-liter size, SKC, Inc.) 

were partially inflated by air drawn by a Gillian personal air-sampling pump 

(Model HFS-113, Wayne, N J). The sampling line was clipped to the lapel of the 

fueler's coat, essentially in his breathing sons. The fueler stood adjacent 

to the filler pipe into which the fuel-pump nozzle was inserted, a position 

common for all who fuel vehicles. The distance between the saaple line inlet 

and the filler pipe varied from two to four feet, depending upon exactly where 

and how erect the fueler stood. The sampling pump was turned on and off 

concurrently with the gasoline pump. The bag thus contained vapors with a 

composition averaged over the filling period. Since the gasoline pump was 

equipped with an automatic shut-off valve, the fueling rate (about 8 gal/min) 

was the same for all the sampling periods, and the length of the sampling 

period was proportional to the fuel volume added to the tank. The sampling 

period ranged from 30 seconds to two minutes, corresponding to fill volumes of 

4 to 16 gallons. The pump-and-bag unit was returned to the laboratory for GC 

analysis on the same day. 

Analysis of Vapor Samples 

The vapor samples were analyzed for individual hydrocarbons with a Perkin 

Elmer Model 3920 gas chromatograph equipped with a flame ionization detector 

(FID), a single capillary column, a sample loop and 8-port Carle sampling 

valve for gas samples, and a syringe injection port for liquid samples. The 

operating conditions are given in Table I. 

Vapor samples and calibrating gases were pumped or flowed into the 25 c@^ 

sample loop and injected onto the column with the Carle valve. The detector 

output was connected to a Hewlett-Packard Model 3390A integrator, which 

printed a chromatogram and a table of peak areas and retention times. The 

chromatograph was calibrated twice daily with two cylinder-gas mixtures (Scott 

Speciality Gases), one containing 5 ppmV of benzene and the other containing 

about 15 ppmV each of propane, n-butane, n-pentane, n-hexane, and n-heptane. 

The cylinder gases were precision-diluted about 10- and 20- fold with HC-free 

air. The calibration factor was taken as the average of the ratios of the 

area counts divided by the diluted concentration of each hydrocarbon and 

varied between 21900 and 23900 area counts per ppaC from day to day. The 

individual hydrocarbon concentrations were calculated from the daily 

calibration factor, and the individual peak areas and were summed to obtain 

the total hydrocarbon concentration. 

The individual hydrocarbon peaks were identified by their retention times, 

by analyzing many cylinder-gas mixtures containing known hydrocarbons and by 

preparing other mixtures from pure hydrocarbons and hydrocarbon-free air. Not 

all the hydrocarbons in the fueling samples have been identified, but all the 

main peaks have been classified. That is, the principal hydrocarbon or 

hydrocarbons in each peak have been identified, although some minor ones may 

also be present. The identification is reasonably complete and definite 

through the Cg compounds; the Cg olefins are not individually identified since 

the retention times of all 17 isomers have not been determined. Only the 

major C*? and Cg paraffins have been identified; the Cy and Cg olefins are 

relatively minor constituents. Four naphthenes (cyclopentane, 

methylcyclopentane, cyclohexane, and methylcyclohexane) and six aromatics 

(benzene, toluene, ethylbenzene, and the xylenes) complete the list of 

identified hydrocarbons. Since no peaks were seen beyond o-xylene at 
P.83 

87-31.1 

51 minutes retention time, we believe that essentially all the hydrocarbons in 

the fueling vapors were detected. 

Analysis of Dispensed Fuels 

A sample of the winter-grade gasoline was taken from the gasoline pump on 

February 15 and was analyzed for individual hydrocarbons using the Perkin 

Elmer chromatograph. A sample of the summer-grade gasoline was taken from the 

pump on August 22 and was analyzed using a different chromatograph, which 

provided better separations of the higher molecular weight hydrocarbons. In 

both cases, the fuel analyses extended through Q*n anc^ included essentially 

all the constituents in the gasolines. The gasoline compositions were deduced 

from the chromatograms and the integrator counts for each peak. The 

percentage of each constituent on a carbon basis was calculated as the ratio 

of the integrator counts for that peak to the summation of counts for all the 

integrated peaks. Because of the use of different chromatographs, some HC 

pairs (notably p-xylene and m-xylene) were separated in the summer gasoline 

that were not separated in the winter gasoline or in any of the vapor samples. 

Thus, when the summer gasoline and refueling vapor compositions are compared, 

some groupings of the gasoline data will be necessary. 

The Reid vapor pressure of the winter-grade gasoline was 13.7 psi, while 

that of the summer-grade gasoline was 10.4 psi. 

RESULTS AND DISCUSSION 

Both fuel volatility and weather conditions were suspected as factors that 

might influence the exposure of a vehicle fueler to HC vapors. In most U.S. 

locations, fuel composition is adjusted seasonally to match volatility with 

the expected ambient temperature. Consequently, we designed our sampling 

program with both winter and summer segments to include the widest range of 

possible fuel and weather effects. At the outset, we were not certain which 

factors would be most important to monitor, and therefore, made some 

procedural changes during the program. Since the sampling time was 

proportional to the amount of fuel added to the car, fills of less than five 

gallons did not provide enough sample to adequately flush the GC loop before 

injection. In those cases, one or two additional cars were sampled to 

increase the sample volume. This modification should have no effect on the 

results, since the fuel and weather were identical. Also, the individuals 

collecting the samples were different in the two segments, which caused other 

differences that will be discussed. As a result, the two data sets will be 

treated in parallel fashion and intercomparisons will be made only when they 

can be done witji confidence. 

One of the important factors affecting the THC concentration of the 

samples was the exact position of the fueler; his body orientation and how 

close the sampling probe was to the filler pipe. This was reflected in the 

last four samples of the summer study, that were collected while the fueler 

leaned over and grasped the fuel pump nozzle with his right hand, just as 

though the pump nozzle did not have a trigger-latching mechanism. This 

position required the fueler to turn his body perpendicular to the car, 

creating a human shield to a westerly wind. The average THC for these four 

samples was 890 ppmC, whereas the average THC for the first eight samples of 

the summer study, with similar wind speeds and directions, was 71 ppmC. All 

the samples in the winter study were collected in the shielded position, while 

the distance-to-filler-pipe variation was within the ergonomic range in the 

general population. For safety purposes, Michigan and some other states 

require that the trigger-latching mechanism on the pump nozzle be removed at 
P.84 

— n 

87-31.1 

self-serve stations, thus forcing the motorist to keep his hand on the nozzle 

to keep fuel flowing. 

The effect of wind speed and direction on THC is examined in Figure 2. 

Most of the samples were collected with the wind direction from the quadrant 

centered on the west, but those samples spanned the entire concentration 

range. Similarly, wind speed, which ranged from less than one to four meters 

per second, did not systematically affect the THC values. Ia fact, some of 

the highest THC samples were collected when the refueler was standing upwind 

of the filler pipe with a measured wind velocity of 1.5 s/s. Apparently, eddy 

currents were created by the sampling person's body snd the vehicle, which 

were sufficient to cause these high concentrations at the sample-line inlet. 

Similar effects have been observed in laboratory situations in which fume 

hoods fail to protect workers from point sources in front of them.0"

8 For 

subtle differences in body orientation to have such large effects on the THC 

exposures, the concentration gradient in the vicinity of the filler pipe must 

be very great. Strong gradients are also indicated by the comparison of the 

THC concentrations of the refueling vapors, a few hundred parts per million, 

with those in the fuel-tank head space, about 50 percent by volume,® or 

almost 1000 times the values observed in the breathing zone samples. 

Composition of Fueling Vapors 

The hydrocarbon composition of the fueling vapors did not change greatly 

from sample to sample within each seasonal study and was dominated by the 

light paraffins. Table II lists the percent by mass (carbon) of 12 of the 

most abundant hydrocarbons in the fueling vapors. The greatest difference 

between the winter and summer samples was the higher n-butane content in 

winter, which is consistent with the higher vapor pressure of the winter-grade 

gasoline. N-butane, isopentane, isobutane, n-pentane, and propane ranked 1 to 

5 in abundance, and combined constituted about 82 percent of the total in 

winter and about 73 percent of the total in summer. The other seven 

hydrocarbons shown in Table II were all present in larger amounts in the 

summer samples than in the winter samples. The representative light olefins, 

trans-2-butene and 2-methyl-2-butene, were each present in amounts of 1 to 

2 percent. The dominant Cg, C^, and Cg paraffins were 2-methylpentane, 2-

methylhexahe, and 2,2,4-trimetnylpentane. The dominant aromatics were benzene 

and toluene; they were present at about 0.5 percent in the winter saaples and 

slightly over 1 percent in the summer samples. Together, these 

12 hydrocarbons constituted about 84 percent of the fueling vapors by mass 

(carbon) in summer and 88 percent in winter. 

Propane was,, the most variable hydrocarbon present in amounts greater than 

I. percent, as judged by its coefficient of variation (Table II). Propane is 

lost from gasoline more readily than are other hydrocarbons because of its 

extreme volatility. This phenomenon is called weathering and affects the 

gasoline in both automobile fuel tanks and, to a lesser extent, in underground 

storage tanks. The other light paraffins and light olefins are also affected 

by weathering, but to a lesser degree than propane. 

Relationship Between Fueling Vapor and Gasoline Composition 

It would be expensive to determine the exposure of fuelers to vapors from 

gasolines of many different compositions. Therefore, it is important to 

establish relationships between fueling vapor composition and gasoline 

composition. Table III provides a compound-by-compound comparison in mass 

(carbon) percent units of the fuels and vapors from the present study. The 

resultant vapor-to-liquid ratio (V/L) will be greater than 1.0 for the more 
P.85 

Pl 

87-31.1 

volatile hydrocarbons, which constitute a larger percentage of the THC in the 

vapor than they constitute in the liquid gasoline. Thus, V/L expresses the 

vapor enrichment factor for each compound. In Figure 3, the V/L enrichment 

factors are plotted against the normal boiling point for each hydrocarbon. In 

the winter study, the fueling vapor was enriched for all components with 

boiling points below 100°F. In the summer study, the fueling vapor was 

enriched for all components with boiling points below about 125°F. This 

difference is apparently due to the lower ambient temperature in winter, since 

the temperature of the dispensed fuel (stored underground) is relatively 

constant year-round. The magnitude of the enrichment factors - from less than 

0.01 to over 10 - illustrates the dramatic difference in composition between 

the fueling vapors and the liquid gasoline. Fuel components heavier than the 

xylenes were not found in measurable concentrations in the fueling vapors. 

Comparison with Other Studies 

Several studies of gasoline vapor exposure have now been published. 

Although each of the studies has a unique combination of sampling procedure, 

analysis method, reporting method, and test variables, careful examination and 

interpretation will show that the results do not differ greatly. In our own 

study, we observed that the concentrations of total HC ranged from 4 to 

3210 ppmC, even though the only identifiable variables were the wind speed and 

direction and the body orientation of the fueler. The aean value from the 

combined winter and summer programs was 110 ppmC, whereas the 90th percentile 

value was 1980 ppmC because a few samples had vary high concentrations. If a 

single value must be selected to represent a distribution of this kind, 

Haider, et al. ® suggested using the geometric mean, which we will adopt. In 

our study, we elected to use ppmC as our reporting units because the flame 

ionization detector effectively measures the carbon content of the gas stream 

being analyzed. Other units, such as volume concentration, can be computed 

for individual compounds or for known mixtures of HC. However, mass 

concentration units of mg/m^ meet the same criteria, and, in addition, are 

common exposure units for the worker environment. Usage of the term ppmV is 

most useful for expressing the vapor phase concentration of individual HC, but 

it is an ill-defined term for gasoline vapor mixtures whose composition is not 

fixed. 

The reports of six independent studies*'10-14 yjjj be critically reviewed. 

Only samples collected at the breathing zone of persons filling vehicles will 

be considered. The collection and analysis methods will be adjusted to a 

common basis wherever that is possible. Since the monitoring time varied from 

2 min to 8 h, all the results will be adjusted to actual filling time, to 

eliminate minu€es and hours for which the person monitored was distant from 

the filler pipe. All the reported values will be converted to mg/m* units, 

but the individual values will be preserved to permit the calculation of 

geometric means. 

The reported values are displayed in Table TV, along with brief 

descriptors of the parameters that will be adjusted. Kearney and Dunham" 

provided the best data for determining the actual filling time during an 

8-hour work shift. Pump island values would depend on the size of the service 

station, so they can be subtracted as part of the background. Since 

full-service attendants spend most of their time distant from the filler pipe, 

they are exposed to filling vapors for only about 26 min in an 8-hour shift. 

Haider, et al. " found that C^ and Cc HC account for about 75% of the total 

fueling vapor, so their Cg+ results must be multiplied by 4 to estimate the 

total exposures. Their direct comparison of diffusion badges with charcoal 
87-31.1 

tubes indicated that the diffusion badges gave incorrect values, about twice 

those of the charcoal tubes. Clayton Environmental's** 13-station study 

showed that the average self-service fill is 10 gallons, while 8 gal/min is a 

good average value for the fueling rate. Bond, et al." designed a sampling 

rack that could be reproducibly positioned adjacent to a car so that the 

breathing-zone sampling point was unobstructed. Unfortunately, the results of 

Tironi, et al.4 indicated that shielding by the body of the sampling person 

significantly affected the breathing zone concentration of vapors, obscuring 

the expected effects of wind speed and wind direction. 

CONCLUSIONS 

Much of the disparity among the reported results has been removed by the 

adjustments described above. The exposure ranges from each publication have 

been preserved through the adjustments (see Table IV). The wide ranges 

represent the real variability induced by fuel composition, temperature, and 

wind. The geometric mean values, however, all fall between 47 mg/nr and 

148 mg/m^, both reported by Tironi, et al. The close agreement among the 

mean values strongly suggests that the studies to date have reliably 

quantified the breathing zone levels of gasoline vapors during automobile 

fueling. Using a mean value of 100 mg/m' and the average composition values 

from Table II, the best estimate for the fueling exposure to twelve of the 

major individual HC in ppmV is shown in Table V. These exposure levels occur 

during the filling period only, and have a duration of one minute per eight 

gallons of gasoline pumped. Considering the 0.20 ppm winter value for 

benzene, an eight-gallon fill would have to be repeated 120,000 times to 

produce a benzene exposure equivalent to the occupational-exposure limit, 

40 hours at 10 ppm. 

The digested results of six independent studies provide the health effects 

community with a reliable characterization of the nature and extent of human 

exposures to gasoline fueling vapors. 
- P87 

87-31.1 

REFERENCES 

1. "Estimation of the public health risk from exposure to gasoline via the 

gasoline marketing system," U.S. Environmental Protection Agency, Office 

of Health and Environmental Assessment, Office of Air Quality Planning 

and Standards, June 1984. 

2. H. N. MacFarland, C. E. Ulrich, C. E. Holdsworth, D. N. Kitchen, 

W. H. Halliwell, and S. C. Blum, "A chronic inhalation study with 

unleaded gasoline vapor," J. Am. College Tox.. 3, 231 (1984). 

3. "Gasoline vapor exposure and human cancer: evaluation of existing 

scientific information and recommendations for future research," Health 

Effects Institute, Report of the Institute's Health Review Committee, 

September 1985. 

4. G. Tironi, G. J. Nebel, and R. L. Williams, "Measurement of vapor 

exposure during gasoline refueling," Society of Automotive Engineers, 

Paper No. 860087, February 1986. 

5. R. L. Furey and B. E. Nagel, "Composition of vapor emitted from a vehicle 

gasoline tank during refueling," Society of Automotive Engineers, Paper 

No. 860086, February 1986. 

6. K. J. Caplan and G. W. Knutson, "The effect of room air challenge on the 

efficiency of laboratory fume hoods," ASHRAE Transactions, 83, 141 

(1977). 

7. K. J. Caplan and G. W. Knutson, "Laboratory fume hoods: a performance 

test," ASHRAE Transactions, 84, 511 (1978). 

8. K. J. Caplan and G. W. Knutson, "Laboratory fume hoods: influence of room 

air supply," ASHRAE Transactions, 84, 522 (1978). 

9. D. E. Seizinger, W. F. Marshall, F. W. Cox, and M. Boyd, "Vehicle 

evaporative and exhaust benzene emissions as influenced by benzene 

content of gasoline," Society of Automotive Engineers, Paper No. 860531, 

February 1986. 

10. C. A. Haider, G. S. VanGorp, N. S. Hatoum, and T. M. Warne, "Gasoline 

vapor exposures. Part I. Characterization of workplace exposures," 

Am. Ind. Hyg. Assoc. J.. 47,(3) 164(1986). 

11. C. A. Kearney and D. B. Dunham, "Gasoline vapor exposures at a high 

volume service station," Am. Ind. Hyg. Assoc. J., 47(8) 535(1986). 

12. Clayton Environmental Consultants, Inc., "Gasoline exposure study for the 

American Petroleum Institute," CEC Job No. 18629-15, August 1983. 

13. A. E. Bond, V. L. Thompson, G. C. Ortman, F. M. Black, and J. E. Sigsby, 

"Self-service station refueling exposure study," U.S. EPA, 1985. 

14. H. J. McDermott and G. A. Vos, "Service station attendant's exposure to 

benzene and gasoline Yapors," Am. Ind. Hyg. Assoc. J., 40(4) 315(1979). 
87-31.1 

TABLE I Operating conditions for the Perkin 

Elmer model 3920 gas chromatograph. 

Oal« 

letector 

SPB-1 •ethyl •illeoaecoated capillary, 0.76* : 

I.D. by 60 aatan lose. 

Plaae ioaisatioa 

n o * rata* 

Celvaa heliua 

Total halloa* 

Burner air 

Bursar hydrogen 

6 ec/ain 

SO cc/ain 

SOO cc/ain (50 

24 cc/ain (20 ps 3 

Ooluan ores teaperature 

0 - 4 ainutee -80*C. 

4-4 6 ainutee -SO to 100'C S 4* 

aiaute 

40-6 8 ainutee 100'C 

Injector teaperature 60*C 

Detector teaperature 800*C 

Attenuation *2 

Aaplifier ranee si 

P** 

* Makeup heliua added at coluan outlet. 

TABLE II Average composition of fueling 

vapors — selected hydrocarbons. 

Mass fCarbon) Percent of Total HC 

nVdroearDone 

Liarht paraffins 

a-Butaae 

i-Pentane 

i-Butane 

a-Pentana 

Propane 

Lia-iit Olefins 

traas-2-Buteae 

3-Methyl-2-butene 

Mediae Paraffins 

2-Methylpentane 

2-Methylhexane 

2,2,4-Triaethylpentane 

Aroatatics 

Bensene 

Toluene 

Subtotal 

Winter(») 

51.04 (2.6) e 

14.41 (3.7) 

8.03 (18) 

6.17 (5.8) 

8.74 (28) 

1.16 

1.46 

1.74 

0.32 

0.16 

0.63 

0.47 

88.36 

flO) 

(7.6) 

(7.5) 

88 

88 

(0.80) 

83.03 (5.8) 

18.14 (3.6) 

6.40 (10) 

6.60 (5.6) 

6.03 (25) 

1.85 (13) 

2.13 (15) 

2.60 (6.5) 

0.86 (6.3) 

0.43 (28) 

1.12 (8.0) 

1.25 (16) 

83.55 (1.3) 

• 13 saaples - exclude f's 6,12,14 (low THC) 

• 14 saaples - exclude #'s 6 (lo* THC) and 11,13 (preaiua fuel) 

e —"efficient of rariation (standard deriation as percent of the aean) 

9 
P.89 

87-31.1 

TABLE III Hydrocarbon composition of fueling Yapor and 

Propaaa 

Isobutaae 

Isobutylaae • 1-Butaaa 

a-Butaae 

traas-2-Butene 

cis-2-Butene 

8-Methyl-l-butene 

laepaataaa 

1-Peataae 

2-Methyl-l-butene 

a-Pentaae 

trans-2-Penteae 

cis-2-Pentene 

2-Methyl-2-butene 

2,2-Diaethylbutane 

Oydopentene 

4-Methjrl-l-peateae 

Cyclopentane 

2,3-Dlaethylbutaae 

C. Olefin 

2-Methylpaataae 

C. Olafia 

8-Methylpantaae 

C«Olefins 

a-Hexaae 

C- Olefins 

Methrlejcloaentane 

2,2-Diaethylpentane • 

2,4-Diaathylpentane 

Beasea* 

S, 3-Diaethylpentaae 

Cyclohexaae 

2, 3-Di»ethylpentaae 

2-Methylhexane 

Cyclohexane 

S-Methylhazana 

C. Olefin 

S-Ethylpentana 

2,2,4-Triaethy1pentane 

a-Heptaae 

C. Olefins 

Methyleydophexaae 

2,5-Diaethylhexane 

2,4-Diaethylhexane 

2,3,4-Triaethy1pentane 

Toluene 

Cg Paraffins 

<£ Paraffins 

3-Methylheptane 

2,2,5-Triaethylhexane 

a-Octaaa 

Bthylbaaaeae 

p-Xylaae 

a-Xylene 

o-Xylese 

Vaaor* 

4.12 

7.76 

1.35 

81.19 

1.26 

1.30 

0.16 

14.26 

0.71 

1.05 

4.90 

1.27 

0.63 

1.48 

0.16 

0.27 

0.63 

1.77 

1.00 

0.17 

0.82 

0.61 

0.66 

0.10 

0.37 

0.18 

0.20 

0.13 

0.07 

0.40 

0.01 

0.02 

0.01 

dispensed gaso 

Mass (Carbon) fereent of Total IC 

Winter Study 

Casoline 

0.14 

0.68 

0.14 

S.27 

0.2* 

0.29 

0.12 

7.S1 

0.51 

0.76 

4.51 

1.22 

0.66 

1.74 

0.32 

0.45 

1.16 

3.83 

2.86 

0.47 

3.20 

1.96 

3.16 

0.70 

2.74 

1.63 

1.88 

1.66 

1.17 

7.82 

1.13 

S.03 

1.44 

2S.6 

U . 2 

8.78 

6.19 

4.67 

4.S6 

1.2S 

1.95 

1.S8 

1.S8 

1.09 

1.04 

0.95 

0.S5 

0.60 

0.60 

0.6S 

0.48 

0.S5 

0.S7 

0.26 

0.26 

0.21 

0.15 

0.13 

0.10 

0.10 

0.078 

0.056 

0.052 

0.008 

0.007 

0.006 

6 

Yswrb 

6.S2 

9.83 

0.9S 

80.65 

2.03 

2.07 

0.S6 

18.45 

1.05 

1.66 

6.40 

1.89 

0.90 

2.44 

0.61 

0.S1 

0.26 

0.74 

2.53 

0.21 

1.46 

0.33 

1.07 

1.S4 

0.64 

0.31 

1.07 

0.77 

0.31 

0.28 

0.25 

0.16 

0.11 

1.23 

0.08 

0.16 

uaaer Study 

Casoline 

0.06 

0.7S 

0.27 

4.06 

0.45 

0.42 

0.17 

7.S6 

O.SS 

0.94 

3.41 

1.24 

0.69 

1.76 

0.60 

0.28 

0.20 

O.SS 

1.03 

0.12 

S.S8 

0.21 

2.25 

0.62 

2.11 

2.88 

i . a 

0.90 

S.18 

0.21 

0.12 

0.16 

S.46 

0.06 

1.67 

0.36 

0.22 

1.67 

1.01 . 

0.64 

0.66 

0.S4 

0.33 

1.06 

12.63 

0.46 

1.02 

0.81 

0.26 

0.52 

1.36 

2.70 

1.03 

1.56 

V/LC 

70.2 

1S.1 

S.6S 

7.62 

4.48 

4.93 

3.26 

2.44 

. 1.91 

1.80 

l.SS 

1.62 

1.80 

1.S9 

1.22 

1.11 

0.74 

0.72 

0.76 

1.00 

0.65 

0.53 

0.61 

0.47 

0.43 

0.S4 

0.S4 

0.21 

0.20 

0.16 

0.25 

0.24 

0.10 

0.065 

0.056 

0.043 

Total Percent 88.68 67.68 

* Winter aaaple f 7 

* Suaaer aaaple f 3 

e

 Calculated before rounding 

9T775 70 S 

10 
Kearney ft Dunham (11) 

Balder, et al. (10) 

Bond, et al. (13) 

Tironi, et al. (4) 

Clayton Environ. (12) 

McDermott ft Vos (14) 

TABLE 

Sampling 

Situation 

Self service 

Full service 

attendant 

Service plaza 

attendant 

Self service 

simulation 

Single car with 

saapling rack 

Single car 

(winter) 

Single car 

(summer) 

2-7 cars in 

self-service 

Attendant filling 

only 

Full service 

IV 

4 

4 

attendant during 

busy period 

Full service 

attendant 

Adjustment of reporte 

t 

io. Saaples 

12 

8 

21 

1 

1 

parallel 

perpendicular 

15 

18 

288 

6 

2 

84 

Samplinn Time 

10 min 

400 min 

27 min 

354 min 

1.5 min 

1.5 min 

0.5-2.0 min 

0.5-2.0 min 

8-10 nin 

15 Bin 

15 min 

480 min 

id exposures to 

Reported Exposure 

3.0-63.5 ag/a3 

1.0-14.3 ag/m3 

1.1-130.3 mg/m3 

24.3 mg/m3 

6.5 mg/m3 

84.0-157 ppaC 

0.0-18 ppaC 

4.0-3210 ppaC 

5.0-1220 ppmC 

2.4-690 ppmCg 

12.1-09.2 ppa 

gasoline 

1.81, 2.31 ppm 

gasoline 

0.42-40.4 ppm 

gasoline 

tank fillingActual 

Filling Time 

1.25 ain 

21.7 ain 

27.0 ain 

3.1 ain 

22.0 min 

1.5 ain 

1.5 ain 

0.5-2.0 ain 

0.5-2.0 ain 

8-10 ain 

15 ain 

1.5 ain 

26 ain 

•time only. 

Other 

Adjustaent 

None 

Diffusion 

badge x 1/2 

Cg4 reported 

X 4 

Diffusion 

badge x 1/2 

Diffusion 

badge x 1/2 

Units x 0.56 

Units x 0.56 

Units x 0.56 

Units x 0.56 

Units x 3.6 

Units x 2.75 

Units x 2.75 

Units x 2.75 

BK/B 

Adjusted 

Exposure 

Range 

31.2-508 

35.0-265 

35.0-4170 

— 

— 

47-88 

0-10 

2.2-1750 

2.8-680 

8.7-2500 

33.2-273 

50-64 

21.0-2050 

3 

Geoa. Mean 

102 

85 

128 

106 

52 

50 

148 

47 

140 

112 

56 

not 

available 

-1 

• 

! 

. 

00 

^» 

1 

OJ 

t-A 

t-t 
P.91 

87-31.1 

TABLE V Best estimate of exposure to fueling vapors. 

Component 

n-Butane 

i-Pentane 

i-Butane 

n-Pentane 

Propane 

trans-2-Butene 

2-Methyl-2-butene 

2-kethylpentane 

2-Uethylhexane 

2,2,4-TrimethyIpentane 

Benzene 

Toluene 

Winter 

(pp»n 

21.5 

4.S 

3.4 

1.8 

2.1 

0.52 

0.64 

0.40 

0.08 

0.04 

0.20 

0.12 

Summer 

fonmV} 

13.0 

6.1 

4.0 

2.2 

3.3 

0.85 

0.03 

0.74 

0.21 

0.00 

0.35 

0.33 

12 
Attendant's 

Shelter 

Curb 

87-31.1 

N 

Sampling Locations Depending 

t\W- on Filler Neck Locations 

Roadway 

Gas Pump 

Fig. 1 Plan view of fueling area. 

13 
87-31.1 

P.93 

Winter Study 

N 

• 

-4 meters/sec 

Summer Study 

N 

4 

4 meters/sec 

+ E 

Fig. 2 Wind direction and velocity during sampling. Numbers indicate total 

hydrocarbon concentration in ppmC. 

14 
87-31.1 

15.00 

10.00 

8.00 (- \ o 

6.00 

4.00 

2.00 

Ratio Liquid Vapor 

1.00 

.80 

.60 

.40 

.20 

.10 

.08 

.06 

.04 

.02 

.01 

.007 

T r i 1 — i r 

Winter Study. \ # 

^ aN 

Summer 

Study 

\ 

\ 

\ 

\ 

I ± 

\ 

,_A 

50 100 150 200 250 300 

Normal Boiling Point (°F) 

Fig. 3 Vapor enrichment factors (V/L) for C^-Cg hydrocarbons in fueling 

emissions. 

15 
P.95 

§u&mission Fee Received 

SOT Abstract Number — 

SOCIETY ©F T0XD< 

ABSTRACT FORM FOR THE H9©S ANNUAL MEETING 

HH 7075 

[

Any corroopondonco regarding you.-

abatract muot rotoroneo thio number. 

Rood carefully tho abotract Inotructlono (attached). Follow format for heading. Original muot be on this form. Loavo a double space between 

hooding and abotract. Yost ef obotraet rauot bo olngto opaeod. Thoro will bo a chargo of 823 for any abotract that muot bo rotyped. Loove no 

marglno—typo muot bo within biuo roctanglo. Slngto opaco, oncopt for double space between body of test and outhoro. Use typo no omaller than 

12-plteh. Proof corofuity. 

Ooodllno tor rocolpt In Enoeufteo Soerotory'o efflco lo Octobor IS, 1SQ7. Submit original and ono copy'only, togethor with your submission check 

foa ef 82S (no ptfrchaoo ordoro cccoptod). 

POSSIBLE INHIBITION OF RENAL PHAGOLYSOSOMAL (PL) 

PROTEOLYSIS BY GASOLINE IN MALE RAT: EVIDENCE 

FROM IMMUNOELECTRON MICROSCOPIC LOCALIZATION OF 

a- -GLOBULIN. MJ Olson1., MA Mancini2, BD.Garg1, 

anrj AK Roy8. 'Bioraed. Sci. Dept., GM Res. Labs., 

Warren, MI,

 aDept. of Biol. Sci., Oakland U., 

Rochester, MI. Sponsor: E W Lee 

o_ -Globulin (aG) is a protein thought to be 

integral to the development of male rat-specific 

nephrotoxicity caused by unleaded gasoline. A 

gold-labelled second antibody was used to 

determine the distribution of aG within cells of 

the renal proximal convoluted tubules (PCT). aG 

was localized almost exclusively in PL of 

saline- or gasoline-treated (2.0 mL/kg/d, 9 d) 

male rat kidney; no aG was apparent at sites 

other than PL, microvilli or endocytotic 

vacuoles. Further, treatment of male rats with 

leupeptin, to inhibit cathepsin B, caused 

accumulation of PL identical to those observed 

following gasoline administration and rapidly 

increased the renal content of aG. aG in PL of 

control rats was distributed uniformly. However, 

the enlarged, angular PL characteristic of 

gasoline and leupeptin intoxication contained 

deposits of anti-aG located preferentially over 

crystalline PL inclusions. In addition, numerous 

PL, unreactive with anti-aG, were observed in 

PCT of gasoline- or leupeptln-treated rats. 

Thus, it is concluded that a defect of lysosomal 

proteolysis is induSed by gasoline and results 

In sequestration of large amounts of protein, 

including aG, in PL of PCT. 

t. Noma and addrooo of firot (prooontlng) author: 

Sontef (flrot) outhoro con oubmtt only ono obotroct tot too mooting. 

Memo: Michael J. Olson 

Organisation: General Motors Research Labs 

Addrooo: Biomedical Science Department 

Warren MI 48090 

City Stafo Zip Coda 

USA 

Country 

Tolophono: < ^13 • 986-1667 

2. Cflemberohlp otatus: SOT member

 A Non-membe 

Abotrocto muot novo on SOT member (not o otudont member) oo on author or b 

oponoorod by a mombor. N aponoonrt. tfto SOT mornbof oponoer muot otgn botoo: 

Signature of SOT mombor oponso 

Eun Woo Lee 

Typed namo of SOT mombor oponoo 

3. Consider this abstract for selection for the Ptonary Session: 

.Yes .No 

4. Undergraduate and graduate students and post-doctoral fe' 

lows are also requested fo check ono of tho opaceo below: 

Undergraduate Graduato 

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5. The Program Committee reserves the right to assign papers t 

either a platform or poofor soooion. Ploaoo Indicato your prefei 

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your choice cannot be met: 

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cannot be met 

S. Sofcct from tho Hot bolow tho oooolon topic that boot oulto your paper. Specify your firot and second choice or specify another possible session 

topic QTOQ undor "ofhor." Specify only two choices. 

SESSION TOPICS 

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1 General/ Mathods 

2 Aquatic/Environmantal 

3. Molecular/Cellular 

4. Biotransformation/Disposition 

5. Reactive Intermediates 

6. Genotox/Mutagenesis 

7. Carcinogenesis 

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10 

i i 

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20. Metals/ Inorganic 

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21 

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(One topic only) 

Return thio form by Octobor 16.1987, to: Program Committee, % Enecutive Secretary, Society of Tomcology, 1133 Fifteenth Street. N.W., Suite 1000. 

Waohlngton, O.C. 20009 (202/293-5335). 

Submit abotract form, orto copy, and abotract oubmission fee of S25 per abstract. If more than one abstract lo submitted In the same envelope. 

Indicato number enclosed on the lower right hand corner of envelope. Abstracts submitted without fees will be returned. 
Submission Fee Received 

SOT Abstract Number 

SOCIETY OF T©XIC©LO<QV 

ABSTRACT FORM FOR THE 198© ANNUAL MEETtNG 

HH 7074 

[

Any corroopondonco rogardlng your] 

obotraei muot roferonco thio number. J 

Kaad carofulry the abotract Instructions (attached). Follow format for heading. Original muot bo on thio form. Loove a double space between 

hooding and abotract. Tont of abotract muot bo oingte opaeod. Thoro will be a chargo of S25 for any abotract that muot be rotyped. Leave no 

margins—typo muot bo within Wuo roctanglo. Single opace, oncopt for double space between body of tent and authors. Use type no smaller than 

12-pifch. Proof carefully. 

Ooodlirto tor rocoipt in Eaocutlvo Socrotory'o off ico io October 16,1907. Submit original and ono copy only, together with your submission chectt 

too of 835 (no purchaoo ordoro occoptod). 

C 

Nomo: Michael J. Olson 

FUEL HYDROCARBON-INDUCED HYALINE DROPLET (HD) '• ^^t^con^SSS^SJ ^ 

NEPHROPATHY IN MALE RATS DURING AGING. 

CVR Murty1, MJ Olson*, BD Garg2, and AK Roy*. 

lDept. of Biol. Sci.„ Oakland U., Rochester, MI, 

aBiomed. Sci. Dept., GM Res. Labs., Warren, MI. 

Sponsor; E W Lee 

Orgonlzotlon: General Motors Research Labs 

Addrooo: Biomedical Science Department 

Young male rats treated with certain hydrocarbons, including gasoline, develop nephropathy 

characterized by accumulation of HD in cells of 

the renal proximal tubules (PCT). However, no 

information is presently available on possible 

age-dependent susceptibility to HD accumulation. 

PCT of young (3 mo.), untreated, male Fischer 

rats had numerous HD in the P1 and P2 segments. 

By 12 mo. of age, the number of droplets had 

declined markedly; at 21-26 mo., no HD were 

visible. Electron microscopy of PCT of aged male 

rats showed that phagolysosomes, equivalent to 

HD, were reduced in number. We have also shown 

that a major constituent of many HD is the male 

rat urinary protein, ou -globulin, for which the 

rate of hepatic synthesis declines during aging. 

Levels of both hepatic and renal a -globulin in 

26 mo. old male rats were aboutu 1.5$ of the 

Warren MI 48090 

City State Zip Cods 

USA 

Country 

Telephone: L 313 ) 986-1667 

2. Membership status: SOT member _*l _ Non-membe 

Abotrocto muot hovo on SOT mombor (not o otudont mombor) oo on outnor or t 

oponoorod by o mombor. H oponoorod. tho SOT mombor oponoor muot ofgn boiov»: 

Signature of SOT member sponso 

Eun Woo Lee 

young adult. Unleaded 

5 d) caused a 3-fold 

content of a_ -globulin 

the same treatment did 

globulin in old rats. We 

major determinant in 

hydrocarbon-induced" nephropathy in male rats and 

only-rats which produce large amounts of 

a2 -globulin are susceptible. 

gasoline (O.H mL/kg/d, 

Increase in the renal 

in young males whereas 

not increase renal a. -

conclude that age is a 

the development of 

Typed nomo of SOT member sponso 

3. Consider this abstract for selection for the Plenary Session: 

x

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topic at-o under "ofhor." Spocify only two choices. 

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Kood sarofulfy tho obotract. Inofructtano (attochod). Follow format tor hooding. Original muot bo on thio form. Loave a doublo opoce between 

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fco ®tf 02§ (ft® poigtoco eir^oro c©eopto«IJ. 

FUEL HYDROCARBON-INDUCED SUBCELLULAR ALTERATIONS^-

IN KIDNEY OF MALE RATS. B D Garg1, M J Olson "f 

L C~Lil and A K Roya„ 'Bioraed. Sci. Dept., GM 

.Res, Labs., Warren, MI and * Dept. Biol. Sci., 

Oakland U., Rochester, MI. Sponsor; E W Lee 

Oral administration of unleaded gasoline to male 

rats produces phagolysosome (hyaline droplet) 

accumulation in epithelial cells of the renal 

proximal convoluted tubules (PCT) similar to 

that observed with Decalin, isoparaffInic solvents and numerous other volatile hydrocarbons. 

Transmission electron microscopy was used to 

study dose- and time-dependent alterations of 

renal PCT phagolysosomes in male rats treated 

with gasoline (0.4-2.0 mL/kg, po, once daily, 1-

9 d) . A progressive increase in the number and 

size of phagolysosomes in cells of the PCT was 

observed. Many of the enlarged (0.05-9 ym), 

angular phagolysosomes in treated rats contained 

electron-dense, crystalline Inclusions and occupied a considerable area of the cytoplasm in 

contrast to the relatively small (0.05-2.5 wm) 

round phagolysosomes of controls. These morphological observations parallel biochemical determination of dose- and time-dependent accumulation of o2 -globulin after gasoline exposure. 

Furthermore, studies with gold-tagged antibody 

to <x_ -globulin have demonstrated a- -globulin 

localization exclusively in the phagolysosomes. 

These results suggest that a common defect in 

phagolysosome-turnover induced by hydrocarbons 

is responsible for the unique sensitivity of 

male rats to hydrocarbon-induced nephropathy. 

Nomo and addrooo of firot (prooonflng) author: 

Sontw (firot) outttoro con oubralt only ono obatroet tor tho mooting. 

Mowto: Bhagwan D. Garg 

OrgonboMon: Genera l Motors Researc h Labs 

Addrooo: Biomedica l Scienc e Department 

Warren MI 48090 

City Stoto Zip Coda 

USA 

Country 

Tolophono: < 31 3 • 986-1652 

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Afeoticeto muot novo an SOT mombor (not a otudont .mombor) oo on outttor or bo 

apanoorod by o mombor. H oponoorod, tho SOT mombor oponoor muot otgn botocr: 

Signature of SOT mombor oponoor 

Eun Woo Lee 

5. 

Typed nomo of SOT mombor oponoor 

Conoider this abotract for oelectlon for tho Plonary Sooolon: 

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topic croa undo? "otftor." Specify only two cholceo. 

T 

.1. General/ Methods 

2. Aquatic/Environmental 

3. Molecular/Cellular 

4. Biotransformation/Disposition 

5. Reactive Intermediates 

6. Genoton/Mutagenesis 

7. Carcinogenesis 

8. Reproductive/Teratology 

9. Cardiovascular/ Renal 

10. 

11. 

12. 

13. 

14. 

15. 

16. 

17. 

18. 

SSSSSON TOPICS 

Inhalation/Respiratory Systems 

Hematologic 

Dermal/ Ocular 

Hepatic/GI System 

Neurochemistry 

Neuropathology 

Electrophysiotogy 

Behavior 

Immune System 

19. Endocrine System 

20. Metals/ Inorganic 

21. Pesticides 

V> Rolv«nts 

23. Halogenated Hydrocarbons 

24. Food/Drugs 

25. Biomaterials/ Medical Devices 

26 Communication/Education 

27. OTHFR-

(Ono topic only) 

Rotttro thio torm by October 13,1987, to: Program Committee, % Eaocutlvo Secretary, Society of Tonlcology, 1133 Fifteenth Street, NMH.. Suite 1000, 

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Submit abotroct form, ono copy, and obotract oubmloolon feo of 825 por abotract. If more than ono abotract lo oubmlttod In the same envelope. 

Indicato niranbor oncloood on tho towoir right hand cornor of onvotopo. Abotrocto oubmlttod without feoo will bo rotumod. 
TOXICOLOGY AND APPLIED PHARMACOLOGY 90. 43-5 I (1987) 

Accumulation of a2u-Globulin in the Renal Proximal Tubules of Male Rats 

Exposed to Unleaded Gasoline 

MICHAEL J. OLSON.* BHAGWAN D. GARG,* C. V. RAMANA MuRTY.t AND ARUN K. RoYt 

• Biomedical Science Department. General Motors Research Laboratories. Warren. Michigan 48090. and 

t Department of Biological Sciences. Oakland University. Rochester. Michigan 48063 

Received December 9. 1986: accepted February 18. 1987 

Accumulation of a.u-Globulin in the Renal Proximal Tubules of Male Rats Exposed to Unleaded Gasoline. OLSON. M. J.. GARG. B. D.. MURTY. C. V. R.. AND ROY, A. K. (1987). Toxicol. 

Appl. Pharmacol. 90, 43-31. Saturated branched-chain aliphatic hydrocarbons. Tound in motor fuels, induce nephrotoxicity in male rats. Treatment of male rats with unleaded gasoline 

(0.04-2.0 ml/kg body wt. po> for 9 days increased markedly the number and size of hyaline 

(protein resorption) droplets in epithelial cells ofthe renal proximal convoluted tubules (PCT) 

and enhanced cellular exfoliation at high dose levels. No other treatment-related pathological 

effects were observed in the glomeruli, distal tubules, or medulla. The renal content of aZuglobulin, a major urinary protein of male rats, was increased maximally by about 4.4-fold after 

gasoline administration (1.0 ml/kg, po. 9 days): no further increase was observed at higher doses. . 

Immunoperoxidase staining of kidney tissue sections for a2u-globulin revealed large accumulations of antigen localized in many ofthe PCT epithelial cells which contained hyaline droplets. 

The hepatic content of a2ll-globulin and its mRNA were not altered by gasoline administration. 

These data show, for the first time, that a!u-globulin is accumulated in the kidneys of gasolineintoxicated male rats and sequestered specifically in some ofthe hyaline droplets characteristic 

of gasoline-induced nephropathy. A hydrocarbon-induced defect in the renal lysosomal degradation of low-molecular-weight urinary proteins, rather than increased synthesis of these proteins, appears to cause hyaline droplet accumulation, c mi Academic Pres. inc. 

Volatile petroleum hydrocarbon mixtures 

are produced and consumed in huge volumes 

as motor fuels, solvents, paint and varnish additives, degreasing agents, and industrial intermediates. Toxicological assessment has revealed dose-dependent alterations in the 

morphology and function of the renal cortex 

of male rats following acute and chronic exposure to many of these products (Alden et 

al.. 1984; Haider et at.. 1984; Phillips and 

Cockrell. 1984; Phillips and Egan. 1984; 

Garg etal.. 1986; Viau etal.. 1986). Recently, 

the importance of understanding hydrocarbon-mediated nephropathy was increased 

greatly by the observation that rats exposed 

for 18 months or longer to wholly volatilized 

unleaded gasoline exhibited an increased incidence of malignant renal neoplasia (Mac43 

Farland et al.. 1984). The occurrence of tumors was sex- and species-specific; male rats 

only exhibited treatment-related renal neoplasia (MacFarland et al.. 1984). Analysis of 

whole gasoline and 2.2.4-trimethylpentane. a 

potently nephrotoxic component of gasoline, 

in short-term assays for mutagenesis or DNA 

damage has produced equivocal or negative 

results (Farrow et al.. 1983: Loury et al.. 

1986: Richardson et al.. 1986). Thus, the 

mechanisms by which these agents induce 

overt nephrotoxicity are being studied in the 

hope that an epigenetic toxic event which 

stimulates tumor formation may be identified. 

The primary site of toxicity of unleaded 

gasoline, jet fuels, methyl isobutyl ketone. 

Cio-C,2 isoparaffinic solvent. Stoddard sol0041-008X/87 $3.00 

Copynght © 198? by Academic Presi. Inc. 

All nghu of reproduction in any form reserved. 
a2ll-GLOBULIN IN GASOLINE NEPHROPATHY 45 

1968) or immunostained for a2u-globulin with the avidin-biotin procedure (Hsu et at.. 1981). The Mallory's 

stain solution consisted of (w/v) 1% phosphotungstic 

acid. 2% orange G. 1% water soluble aniline blue, and 

3% acid fuchsin in distilled water. Tissues, hydrated with 

xylene and graded alcohols to water, were immersed for 

2 to 4 min in the staining solution, rinsed in water, and 

dehydrated in graded alcohols and xylene. Localization 

of a2u-globulin in deparaffinized tissue sections was accomplished after rehydration and incubation in a solution of 3% normal goat serum. 3% bovine serum, and 

2% gelatin. Polyclonal anti-cr2u-globulin (rabbit IgG) was 

applied for 30 rain, and the tissues were washed with 

phosphate-buffered saline (PBS; 0.14 M NaCl 0.01 M 

Na-phosphate. pH 7.4). further incubated for 30 min 

with biotinylated anti-rabbit IgG (goat IgG). and washed 

with PBS again. A final 30-min incubation with freshly 

prepared avidin-biotin reagent (Vector Laboratories. 

Burlingame. CA) was followed by extensive washing in 

PBS. The sections were then placed in 0.05 M Tris-HCl, 

pH 7.0. containing 0.0014 M 3.3'-diaminobenzidine tetrahydrochloride and 0.003 M hydrogen peroxide for 15 

min. The reaction was terminated with a 15-min rinse in 

running water and the sections were counterstained with 

Harris hematoxylin. 

Dot-blot assay ofa.a-globlin mRNA in liver. Total hepatic RNA, extracted with phenol and sodium dodecyl 

sulfate, was used as the starting material to isolate. 

poly(Af-containing RNA by oligo(dT><elluIose chromatography (Aviv and Leder. 1972). Serially diluted 

poly(A)*-containing RNA was spotted on nitrocellulose 

filters (Thomas. 1980), dried, and hybridized with "Plabeled a2u-globulin cDNA (Roy et al.. 1982). Binding 

of the probe was determined by autoradiography using 

Kodak X-ray film and quantitaied with soft-laser scanning densitometry (Biomed Instruments. Fullerton, CA) 

of the developed film. 

Radioimmunoassay of a,u-globulin in kidney and 

liver. The cytosolic fractions of kidney and liver homogenates were prepared and assayed for a2u-globulin by double-antibody RIA as described by Roy (1977). The quantity of a2u-globulin in the supernatant fraction was expressed relative to total protein (Lowry et al.. 1951). 

RESULTS 

Characterization of Test Material 

Gas chromatographic analysis of the gasoline administered to rats showed a composition by weight of about 51% paraffins. 5% 

naphthenes. 10% olefins, and 26% aromatic 

compounds (Table 1). The remaining 8% of 

the material was not identified by cochromatography with known standards. Ofthe parafTABLE 1 

SUMMARY OF HYDROCARBONS IN UNLEADED 

GASOLINE USED FOR ORAL ADMINISTRATION 

Hydrocarbon 

class 

/t-Paraffins 

Iso paraffins 

Naphthenes 

Olefins 

Aromatics 

Totals 

Carbon chain 

length detected 

Ct-C,. 

C-C,, 

Cj-Cu 

C4-C, 

Q-C,: 

Isomers 

identified 

14 

58 

40 

72 

53 

237 

Weight 

% 

16.4 

34.3 

5.4 

10.0 

26.4 

92.5 

Note. Gasoline samples used for individual experiments were analyzed by gas chromatography as described under Materials and Methods. Typical results 

were from analyses of three lots of gasoline. 

finic constituents about 68% were saturated 

branched carbon chain compounds (isoparaffins), such as 2,2.4- and 2,3.4-trimethylpentane, which have been identified as potent nephrotoxicants (Haider et al.. 1984). 

Light Microscopic Histology 

In kidneys of control rats, hyaline droplets 

were observed in the epithelial cells of a few 

scattered PCT (Fig. IA). The droplets were 

clearly visible only in tissue sections treated 

with Mallory's Heidenhain stain; in sections 

stained with hematoxylin and eosin. the hyaline material appeared as refractile. slightly 

acidophilic inclusions in the cytoplasm of 

PCT epithelial cells. Not all the epithelial cells 

of any single PCT contained hyaline droplets. 

Likewise, not all PCT contained epithelial 

cells with hyaline material (Fig. 1 A). Affected 

tubules were arranged in bands or rays oriented from the renal capsule to the outer zone 

of the medulla. The droplets were small (ca. 

0.5 um diameter), uniform in size, and exhibited a round or slightly oval cross-sectional 

profile. However, occasionally a single PCT 

epithelial cell was observed engorged with hyaline droplets which appeared coalescent. No 

hyaline material was noted in the cytoplasm 

of epithelial cells lining the distal tubules or 

collecting ducts. 
P.1 

o2ll-GLOBULIN IN GASOLINE NEPHROPATHY 47 

Treatment of rats with 0.04-2.0 ml gasoline/kg orally for 9 days produced a marked 

increase in the number of hyaline droplets in 

the PCT epithelium (Fig. IC). Both the number of tubules with hyaline droplets and the 

number of hyaline droplets per epithelial cell 

were increased in comparison to controls. 

The population of hyaline droplets induced 

by administration of gasoline was heterogeneous with both fine (ca. O.S Mtn diameter) 

and coarse (1-3 fim) droplets visible. The 

larger droplets frequently possessed irregular 

profiles. Occasionally, the continuity of the 

PCT epithelium was disrupted by focal loss 

of cells, which were shed into the tubule lumen. In many microscopic fields, lumina of 

some distal tubules were filled with granular 

or amorphous material which stained positively with the Mallory's Heidenhain 

method. As in the saline controls, glomeruli 

appeared nonnal with respect to size and cellularity of the vascular tuft and thickness of 

endothelial membranes. Cells lining distal 

portions of the nephron were morphologically normal. 

Histologic analysis of renal tissues suggested that doses from 0.04 to 1.0 ml gasoline/kg caused graded increases in the accumulation of hyaline droplets. However, signs 

of overt nephrotoxicity such as epithelial cell 

exfoliation were visible only at doses of 0.4 

ml/kg or more administered for 9 days. 

Renal and Hepatic a2u-Globulin Content 

Following 9 days of treatment, the kidneys 

of rats administered 0.04 ml gasoline/kg or 

more contained significantly more a2u-globulin than controls (Fig. 2). Administration of 

0.004 ml gasoline/kg or less did not alter the 

concentration of a-.u-globulin detected in the 

kidneys of treated rats compared to controls. 

The renal accumulation of a2u-globulin occurred in a dose-responsive manner only in 

the range of doses between 0.04 and 1.0 ml/ 

kg (Fig. 2). Maximal accumulation of a2uglobulin (440% of control) occurred following administration of 1.0 ml gasoline/kg. 

ISO 

8 i » 

"3 

o 

100 

75 

T?- M 

a 

-a 

c 

2S 

-fr-^- 1 

c 

S 

o 

a. 

o> 

E 

B> 

3 

c 

3 n o 

O 

3 

<M 

a 

2 S 

<s 

Q. 

<B 

X 

0 4 « 10-* 4 i KT3 4 « 10-2 « , io-< 

Gasoline Dose (mL/kg) 

FIG. 2. Accumulation of a!u-globulin in the kidney as 

a function of gasoline dose. Gasoline or corn oil vehicle 

were administered by gavage once daily for 9 consecutive 

days. Eighteen hours after the final administration of gasoline, kidney (•) and liver (O) tissues were sampled for 

RIA determination of a2u-globulin. Data are plotted as 

mean ± SD a2ll-globulin content, n •» 3-7 rats per treatment group. ••/» < 0.01 compared to control (Dunnett's 

test); ftp < 0.01 compared to next lowest dose (Duncans's test). 

Higher doses of gasoline (i.e., 2.0 ml/kg) did 

not cause greater renal accumulation of this 

protein. The livers of control and gasolinetreated rats did not differ significantly in a2uglobulin content (Fig. 2). Further, dot-blot 

analysis of poly(A)+ hepatic RNA with

 32Plabeled a2u-globulin cDNA suggested that 

gasoline treatment did not alter significantly 

the content of hepatic mRNA coding for a2uglobulin (Fig. 3). 

Immunocytochemical Localization of a2uGlobulin 

In kidney sections from saline-treated rats, 

stained specifically for a2u-globulin, many 

PCT were stained positively (Fig. 1B). In general, many hyaline droplets which stained 

positively with Mallory's Heidenhain 

method also stained intensely for a2u-globulin (Figs. IA and IB). However, this correlation was not perfect and the obverse was not 
P.2 

a,u-GLOBULIN IN GASOLINE NEPHROPATHY 49 

1984). The single exception is that chronic inhalation of gasoline vapors is associated with 

the appearance of foci of cell regeneration 

(Busey and Cockrell, 1984; Haider et al.. 

1984), whereas tubular regeneration did not 

accompany the treatment reported here. 

Evidence of cytotoxicity, i.e.. exfoliated epithelial cells and proteinaceous casts, were, 

however, observed after 9 days of treatment. 

Time-course studies of the toxicity of 2,2.4-

trimethylpentane, Decalin, and isoparaffinic 

solvent show that the earliest alteration in 

kidney structure is accumulation of hyaline 

material in epithelial cells ofthe PCT, which 

occurs within 1-5 days (Alden et al.. 1984; 

Phillips and Cockrell. 1984; Short et al.. 

1986). Preliminary results from our studies 

indicate that gasoline produces hyaline droplet nephropathy in a similar period; maximal 

droplet accumulation occurred by 9 days of 

treatment. 

The distribution of gasoline-induced renal 

lesions and localized accumulation of hyaline 

material suggests strongly that the primary 

site of toxic action is the PCT epithelium. Information on urine chemistry gained from 

studies of other nephrotoxic hydrocarbon 

mixtures supports this conclusion. Specifically, subacute inhalation of isoparaffinic solvent increased urinary glucose and LDH concentrations and the number of epithelial cell 

casts (Phillips and Cockrell. 1984; Phillips 

and Egan, 1984; Viau et al.. 1986). ^-microglobulin excretion was unaffected by this 

treatment suggesting that the protein resorption function ofthe PCT was intact (Viau et 

al.. 1986). However, mild albuminuria, an 

indicator of glomerular damage, has been reported after inhalation of isoparaffinic solvent (Phillips and Cockrell, 1984; Viau et al.. 

1986). Thus, subtle alteration of glomerular 

function cannot be ruled out as contributory 

to the alteration of PCT morphology which 

accompanies volatile hydrocarbon exposure. 

Hyaline droplets are a constitutive feature 

of the renal PCT epithelium (Trump et al.. 

1984) and are most numerous in the S2 segment of the PCT, indicating the importance 

of this tubule segment in protein resorption 

(Straus, 1964). During 2.2.4-trimethylpentane intoxication of male rats the S2 segment 

accumulates hyaline material to a greater extent than other segments ofthe PCT (Short et 

al.. 1986). Accumulation of hyaline droplets 

occurs in circumstances other than hydrocarbon toxicity, e.g., during heterophagy resulting from administration of nontoxic low-molecular-weight proteins (Ericsson, 1965). 

Identification ofthe gasoline-induced hyaline 

droplets as secondary lysosomes was made 

upoti electron microscopic examination of 

the PCT epithelium of gasoline-treated male 

rats (Garg et al.. 1986). Qualitative and quantitative alterations of lysosomes have also 

been reported in studies of Decalin and isoparaffinic solvent toxicity (Alden et al.. 1984: 

Phillips and Cockrell, 1984). Positive identification ofthese subcellular structures as secondary lysosomes was made on the basis of 

acid phosphatase activity (Phillips and Cockrell, 1984). Whether lysosomal accumulation 

indicates a toxic effect of hydrocarbons specific to the lysosome or reflects a more generalized disturbance of cellular metabolism is 

unknown. 

The composition of renal hyaline droplets 

accumulated during volatile hydrocarbon exposure is of great interest. It has been reasoned that if a2u-globulin is the sole protein 

accumulated and if a specific defect in o2uglobulin metabolism leads to hyaline droplet 

nephropathy then hydrocarbon exposure 

presents little risk to other species (Alden et 

al., 1984). Our results, however, indicate that 

proteins which are not immunologically recognized as a2u-globulin compose some ofthe 

hyaline droplets accumulated after gasoline 

exposure. Two publications have indicated 

previously that male rats accumulate a2uglobulin and hyaline droplets in the renal cortex during intoxication with hydrocarbons 

other than gasoline (Alden et ai. 1984; Viau 

et ai. 1986). However, neither of these reports demonstrated the localization of a2uglobulin'to hyaline droplets. Further, the 

temporal relationship between development 

of hyaline droplet nephropathy and accumulation of a2u-globulin is presently uncertain. 
P.3 

a2u-GLOBULIN IN GASOLINE NEPHROPATHY 51 

inhalation of unleaded gasoiine. In Renal Effects of Petroleum Hydrocarbons (M. A. Mehlman. C. P. Hemstreet. III. J. J. Thorpe, and N. K. Weaver. Eds.), pp. 

57-64. Princeton Scientific Pub.. Inc.. Princeton. NJ. 

ERICSSON, J. L. E. (1965). Transport and digestion of hemoglobin in the proximal tubule. II. Electron microscopy. Lab. Invest. 14.16-25. 

FARROW, M.. MCCARROLL. N.. CORTINA. T.. DRAVS. 

M.. MUNSON, A.. STEINBERG. M.. KJRWIN. C . AND 

THOMAS. W. (1983). In vitro mutagenicity and genotoxicity of fuels and paraffinic hydrocarbons in the 

Ames', sister chromatid exchange and mouse lymphoma assays. Toxicologist 3,36. [Abstract] 

GARG. B. D.. OLSON. M. J.. SARKAR. F. H.. AND ROY, 

A. K. (1986). Renal hyalin droplet and a2ll-globulin 

accumulation after gasoline administration to male 

rats. Pharmacologist 28,210. [Abstract] 

HALDER. C. A.. WARNE. T. M.. AND HATOUM, N. S. 

(1984). Renal toxicity, of gasoline and related petroleum naphthas in male rats. In Renal Effects of Petroleum Hydrocarbons (M. A. Mehlman, C. P. Hemstreet, III. J. J. Thorpe, and N. K. Weaver. Eds.), pp. 

73-88. Princeton Scientific Pub.. Inc.. Princeton. NJ. 

Hsu, S. M., RAINE. L., AND FANGER. H. (1981). A comparative study of the peroxidase-anti-peroxidase 

method and an avidin-biotin complex method for 

studying polypeptide hormones with radioimmunoassay antibody. Amer. J. Clin. Pathol. 75, 734-738. 

KLOSS, M. W., AND BUS. J. S. (1985). Hydrocarbon-mediated nephrotoxicity. CUT Act. 5,1-8. 

KLOSS. M. W., COX. M. G.. NORTON, R. M.. AND BUS, 

J. S. (1986). Evidence of [,4C-4.5]-2.2.4-trimethylpentane binding in urine and serum of F-344 rats. Toxicologist 6,172. (Abstract] 

KLOSS. M. W.. COX, M. G.. NORTON. R. M.. SWENBERG, J. A., AND Bus. J. S. (1985). Sex-dependent 

differences in the disposition of (14C]-2,2.4-trimethylpentane in Fischer 344 rats. In Renal Heterogeneity 

and Target Cell Toxiciiy (P. Bach and E. Lock, Eds.), 

pp. 489-492. Wiley, Sussex. UK. 

LOURY, D. J., SMITH-OLIVER. T., STROM. S., JIRTLE. 

R.. MICHALOPOULOS. G., AND BUTTERWORTH. B. E. 

(1986). Assessment of unscheduled and replicative 

DNA synthesis in hepatocytes treated in vivo and in 

vitro with unleaded gasoline or 2.2.4-trimethylpentane. Toxicol. Appl. Pharmacol. 85. 11-23. 

LOWRY, O. H.. ROSEBROUGH. N. J.. FARR. A. L.. AND 

RANDALL. R. J. (1951). Protein measurements with 

the Folin phenol reagent. J. Biol. Chem. 193,265-275. 

MACFARLAND, H. N. (1984). Xenobiotic induced kidney lesions: Hydrocarbons. The 90-day and 2-year 

studies. In Renal Effects of Petroleum Hydrocarbons 

(M. A. Mehlman. C P. Hemstreet. III. J. J. Thorpe, 

and N. K. Weaver. Eds.), pp. 51-56. Princeton Scientific Pub., Inc.. Princeton. NJ. 

MACFARLAND. H. N„ ULRICH. C. E.. HOLDSWORTH. 

C. E.. KITCHEN, p. N.. HALLIWELL, N. H.. AND 

BLUM. S. C. (1984). A chronic inhalation study with 

unleaded gasoline. J. Amer. Coll. Toxicol. 3. 231-248. 

PHILLIPS. R. D.. AND COCKRELL. B. Y. (1984). Effects 

of certain light hydrocarbons on kidney function and 

structure in male rats. In Renal Effects of Petroleum 

Hydrocarbons (M. A. Mehlman. C P. Hemstreet. III. 

J. J. Thorpe, and N. K. Weaver. Eds.), pp. 89-105. 

Princeton Scientific Pub.. Inc.. Princeton. NJ. 

PHILLIPS, R. D*. AND EGAN. G. F. (1984). Effect of C, 0 -

C, i isoparaffinic solvent on kidney function in Fischer 

344 rats during eight weeks of inhalation. Toxicol. 

Appl. Pharmacol. 73,500-510. 

RICHARDSON, K. A.. WILMER, J. L . SMITH-SIMPSON. 

D., AND SKOPEK, T. R. (1986). Assessment of the genotoxic potential of unleaded gasoline and 2.2.4-tnmethylpentane in human lymphoblasts in vitro. Toxicol. Appi. Pharmacol. 82, 316-322. 

ROY. A. K. (1977). Early events in the steroidal regulation of a2u-globulin in the rat liver. Eur. J. Biochem. 

73,537-543. 

ROY, A. K.. CHATTERJEE. B.. DEMYAN. W. F.. NATH. 

T. S.. AND MOTWANI, N. M. (1982). Pretranslational 

regulation of a2„-globulin in rat liver by growth hormone. J. Biol. Chem. 257,7834-7838. 

ROY, A. K., AND NEUHAUS. O. W. (1966). Identification 

of rat urinary proteins by zone and immunoelectrophoresis. Proc. Soc. Exp. Biol. Med. 1,894-899. 

SARKAR. F. H.. MANCINI. M. A.. NAG, A.C, AND ROY. 

A. K. (1986). Cellular interactions in the hormonal induction of o2„-globulin in rat liver. /. Endocrinol. Ill , 

205-210. 

SHORT, B.. BURNETT, V.. COX. M.. BUS. J., AND SWENBERG. J. (1986). Dose response studies of 2.2.4-trimethylpentane(TMP)-induced nephrotoxicity and retention in the male rat. Toxicologist 6, 172. [Abstract] 

STRAUS, W. (1964). Cytochemical observations on the 

relationship between lysosomes and phagosomes in 

kidney and liver by combined staining for acid phosphatase and intravenously injected horseradish peroxidase. / Cell Biol. 20,497-507. . 

THOMAS. P. S. (1980). Hybridization of denatured RNA 

and small DNA fragments transferrred to nitrocellulose. Proc. Sail. Acad. Sci USA 77, 5201-5205. 

TRUMP, B. F., JONES, T. W.. AND HEATFIELD. B. M. 

(1984). The biology ofthe kidney. In Renal Effects of 

Petroleum Hydrocarbons (M. A. Mehlman. C. P. 

Hemstreet. III. J. J. Thorpe, and N. K. Weaver. Eds.), 

pp. 27-49. Princeton Scientific Pub.. Inc.. Princeton. 

NJ. 

VIAU. C. BERNARD, A.. GUERET. F.. MALDAUGLE, P.. 

GENGOUX. P., AND LAUWERYS. R. (1986). Isoparaffinic solvent-induced nephrotoxicity in the rat. Toxicology 38, 227-240. 
P.4 

t^NAL Hmi N DROPLET 

ACXWUlAfldN AFTER GASOLINE 

AD^N^TRATION TO MALE RATS 

"yx^y&'^J&jgy*-' 

.W^^qrr-^A-^-

^ej^ce Department 

•yy^'r "• 

MAY BE DISTRIBUTED OUTSIDE GENERAL MOTORS Not to be Reproduced Without Permission 
P.5 

Renal Hyalin Droplet and a„ -globulin Accumulation 

after Gasoline Administration to Male Rats 

B.D. Garg and M.J. Olson 

Biomedical Science Department 

General Motors Research Laboratories 

Warren, MI 48090 

F.H. Sarkar and A.K. Roy 

Biological Sciences Department 

Oakland University 

Rochester, MI 48063 
P.6 

Abstract 

Saturated branched-chain aliphatic hydrocarbons, such as those 

found in motor fuels, induce nephrotoxicity in male rats. 

Treatment of male rats with unleaded gasoline (0.2 - 2.0 mL/kg 

body weight, p.o.) for nine days caused a marked increase in the 

number and size of hyalin (protein resorption) droplets in 

epithelial cells of the proximal convoluted tubules (PCT) and 

increased cellular exfoliation at high dose levels. No other 

treatment-related pathological effects were observed in the 

glomeruli, distal tubules or medulla. Transmission electron 

microscopy of kidneys showed that gasoline caused a striking 

increase in the number and size of phagolysosomes in PCT 

epithelial cells. The renal content of ce_ -globulin, a major 

urinary protein of male rats, was increased 3-fold after gasoline 

administration (0.2 mL/kg, p.o., 9 days); no further increase was 

observed at higher doses. The accumulation of a„ -globulin, 

measured by radioimmunoassay (RIA) , was reversed within three 

days of terminating gasoline administration. Immunoperoxidase 

staining of kidney tissue sections for a„ -globulin revealed 

increased quantities of the protein in treated rats and large 

accumulations of antigen localized in PCT epithelial cells which 

contained hyalin droplets. The hepatic concentration of a. -

globulin and the quantity of a„ -globulin mRNA in liver were not 

altered by gasoline. These aata show that a~ -globulin is 

accumulated in the kidneys of gasoline-intoxicatea male rats and 

sequestered specifically in some of the hyalin droplets 

characteristic of gasoline-induced nephropathy. A hydrocarboninduced defect in the renal lysosomal degradation of low 

molecular weight proteins, rather than increased synthesis of 

these proteins, appears to cause hyalin droplet accumulation. 

Introduction 

Toxicological assessment of volatile petroleum hydrocarbon 

mixtures used as motor fuels, solvents, paint and varnish 

additives, degreasing agents and industrial intermediates has 

revealed dose-dependent mild alterations in the form and function 

of the renal cortex following subacute and chronic exposure (1). 

The importance of understanding hydrocarbon-mediated nephropathy 

was increased greatly by the observation that rats exposed for 18 

or more months to wholly volatilized unleaded gasoline exhibited 

an increased incidence of renal carcinoma (2). The occurrence of 

tumors was sex- and species-specific; male rats only exhibited 

treatment-related renal neoplasia. Analysis of gasoline and other 

complex hydrocarbon mixtures in short-term assays for mutagenesis 

has produced equivocal or negative results (3). Thus, research 

has been directed toward elucidating the mechanisms by which 

these agents induce overt nephrotoxicity in the hope that an 

epigenetic event related to toxicity, such as an increase in the 

rate of cell turnover, may explain the occurrence of renal 

tumors. 

- 1-
P.7 

collidine buffer (pH 7.4). 

Serial sections of kidney were stained with Mallory's 

Heidenhain method or immunostained for art-globulin with the 

avidin-biotin procedure (5). Briefly, the tissue was 

deparaffinized, rehydrated and incubated in a solution of 3% 

normal goat serum, 3% bovine serum and 2% gelatin. Sections were 

then incubated with polyclonal anti-o„ -globulin (rabbit IgG) for 

30 min and washed with phosphate buffered saline (PBS; 0.14 M 

NaCI, 0.01 M Na-phosphate, pH 7.4). The sections were further 

incubated for 30 min with biotinylated anti-rabbit IgG (goat IgG) 

and washed. A final 30 min incubation with freshly prepared 

avidin-biotin reagent (Vector Laboratories, Burlingame, CA) was 

followed by extensive washing in PBS. The tissues were then 

placed in 0.05 M Tris-HCl, pH 7.0, containing 0.0014 M 3,3'-

diamino-benzidine tetrahydrochloride and 0.003 M hydrogen 

peroxide for 15 min. The reaction was terminated with a 15 min 

rinse in running water and the sections were counterstained with 

Harris hematoxylin. 

Dot-blot assay of a„ -globulin mRNA in liver 

Total hepatic RNA, extracted with phenol and sodium dodecyl 

sulfate was used as the starting material to isolate poly (A)-

containing RNA by oligo(dt)-cellulose chromatography. Serially 

diluted poly(A)-containing RNAo was spotted on nitro-cellulose 

filters and hybridized .with P-labeled a„ -globulin cDNA (6). 

After hybridization, quantitative binding -

uof the probe was 

determined by autoradiography and densitometry. 

Radioimmunoassay of gp -globulin in kidney and liver 

Homogenates of whole kidney and liver were prepared and 

centrifuged at 100,000 x g; the supernatant fluid was used for 

double antibody RIA of a„ -globulin as described by Roy (7). The 

concentration of a. -globulin in the supernatant fraction was 

expressed as /ig a„ -globulin per mg total protein. 

-3-
P.8 

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- 7 -
P.11 

Table 1 

Summary of Hydrocarbon Types in Unleaded Gasoline 

Hydrocarbon Carbon skeleton Isomers Weight % 

Class Length Detected 

n-paraffins C„ - C.Q 8 13.0 

isoparaffins C - Cg 33 28.2 

napthenes Cfi - C„ 11 3.6 

olefins C3 - C.g 20 5.5 

aromatics Cg - C 1 0 33 36.6 

Totals 105 86.9 

Liquid gasoline was analyzed by capillary column gas 

chromatography (Hewlett-Packard model 5880A, F.I.D., 50 m x 0.2 

mm fused silica column coated with cross-linked dimethylsiloxane 

gum, oven temperature programmed from 0 to 200 C, injector anddetector at 200 and 250 C, respectively). Typical results from 

analyses of three lots of gasoline. 

-9-
P.12 

Table 3 

Effect of Recovery Time on Renal a„ -globulin 

Content After Gasoline Administration 

Treatment Renal a~ -globulin 

(/xg/mg total protein) 

Saline, 2.0 mL/kg x 9 d 

Gasoline, 2.0 mL/kg x 9 d 

Gasoline x 9 d + 3 d recovery 

Gasoline x 9 d + 6 d recovery 

Gasoline x 9 d + 9 d recovery 

Gasoline x 9 d + 12 d recovery 

24.2 ± 4.2 (3) 

127.0 ± 11.0 (6) 

33. 0 * 5. 1 

31. 9 

26. 2 ± 4. 1 

34. 0 ± 5. 2 

( 3 ) 

( 2 ) 

( 3 ) 

( 3 ) 

* * 

1 >tc ifc 

mean * S.E.M., n in parentheses. different from all other 

group means, p=0.01 (Duncan's test). Rats were administered 

saline or 2.0 mL/kg gasoline by gavage once daily for nine 

consecutive days followed by the recovery periods shown. 

-11-
References 

1. Renal Effects of Petroleum Hydrocarbons, M.A. Mehlman, G.P. 

Hemstreet, III, J.J. Thorpe and N.K. Weaver, eds., Princeton 

Scientific Publishers, Princeton, NJ, 1984. 

2. MacFarland, H.N., CE . Ulrich, CE . Holdsworth, D.N. Kitchen, 

N.H. Halliwell and S.C. Blum, J. Am. Coll. Toxicol. 3: 231-

248, 1984. 

3. Loury, D.J. and B.E. Butterworth, C.I.I.T. Activities 6: 1-7, 

1986. 

4. Roy, A.K. and O.W. Neuhaus, Proc. Soc. Exp. Biol. Med. 1: 

894-899, 1984. 

5. Hsu, S.M., L. Raine and H. Fanger, Am. J. Clin. Path. 75: 

734-738, 1981. 

6. Roy, A.K., B. Chatterjee, W.F. Demyan, T.S. Nath and N.M. 

Motwani, J. Biol. Chem. 257: 7834-7838, 1982. 

7. Roy, A.K., Eur. J. Biochem. 73: 537-543, 1977. 

-13-
P.14 

GMR-5716 

RAPID POST-EXPOSURE DECAY 

OF GASOLINE-INDUCED HYALIN 

DROPLETS AND ^-GLOBULIN 

ACCUMULATED IN THE MALE 

RAT KIDNEY 

Bhagwan D. Garg 

Michael J. Olson 

Biomedical Science Department 

Fl"Danyan 

land-Ohiversity 

February 11,1987 

iboratoii 

Warren, fclichigan 48090 

MAY BE DISTRIBUTED 0UTS10E GENERAL MOTORS Not to be Reproduced Without Permission 
P.15 

GMR-5716 

Rapid Post-Exposure Decay of Gasoline-Induced Hyalin Droplets 

and a„ -Globulin Accumulated in the Male Rat Kidney 

Bhagwan D. Garg and Michael J. Olson 

Biomedical Science Department 

General Motors Research Laboratories 

Warren, Michigan 48090-9058 

William F. Demyan and Arun K. Roy 

Department of Biological Sciences 

Oakland University 

Rochester, MI 48063 

Presented at the 26th Annual Meeting of the 

Society of Toxicology 

Washington, D.C. 

February 26, 1987 
which accumulates hyalin droplets subsequently shoos increased levels of DNA 

replication. 

Interest in the potentially adverse health effects of gasoline led us to 

develop a short-terra model for examining th© toxicity bf gasoline in rats (5). 

Work at GM Research Laboratories has shoem that a major component of gasolineindueod hyalin droplets is the androgen-dependent rat urinary protein e„ -

globulin. Accumulation of this protein due to defective renal (lysosomal 

degradation is proposed as an integral event on gasol5ne=5nduced cell death 

(5,8). H© have attempted to understand if and how gasoline alters the 

aetab©15s® of e* -globulin and whether such alteration does, in fact, 

contribute to the Nephropathy associated with fuel hydrocarbon, exposure. This 

report describes the relationships between duration of gasoline administration 

or dose of gasoline and the renal accumulation of e« -globulin. Further, the 

rate of decay of hyalin droplets and accumulated e„ -gfobulin after termination 

of gasoline exposure was determined. 

MATERIALS AND METHODS 

Experimental animals and chemicals 

D 

Male Fischer 344 rats (CDF (F-344)/Crl BR; Charles River Laboratories, 

Wilmington, MA) weighing approximately 250 g were used throughout these 

studies. Rats were maintained in conditions consistent with AAALAC 

recofftmendations with Purina 5012 chow and tap water freely available during the 

entire period. For dose-response and time-course experiments rats were 

administered physiological saline, corn oil or a commercially available 

unleaded gasoline (neat or mixed with corn oil) by intragastric intubation once 

daily. In hyalin droplet decay studies rats were treated with gasoline (2.0 

mL/kg, p.o., 9 days) or gasoline plus estradiol (1 mg/kg in a propylene glycol 

vehicle (7), s.e, 4 days, starting on the final day of gasoline treatment). 

Following conjoint gasoline and hormone treatment, animals were serially 

sacrificed at 3, 6 and 9 days after the last dose of gasoline. Controls 

receiving saline and rats treated with gasoline alone were also allowed to 

recover for 3 to 9 days after termination of treatment. 

Radioimmunoassay of a^ -globulin in kidney and liver 

Tho cytosolic fraction of kidney and liver homogenates were prepared from 

samples of the perfused organs (see below) and assayed for G_ -globulin by 

double-antibody radioimmunoassay as described by Roy (8). The quantity of fi- -

globulin in the cytosolic fraction was expressed relative to total protein 

which was determined by the method of Lowry et al. (9). 

Histological procedures 

Eighteen hours after the final dose of gasoline or after the period of 

recovery, rats were anesthetized with an intraperitoneal injection of 

pentobarbital, perfused via the heart with cold 0.9% sodium chloride solution 

and the kidneys and livers were removed, fixed by immersion in 10% neutral 

buffered formalin, processed routinely and embedded in paraffin. Sections of 

paraffin-embedded kidney (4 fim thickness) were stained with Mallory's 

Heidenhain method for visualization of hyalin droplets. 

-3-
P.17 

LU 

o 

cc 

Q. 

O) 

E 

*•»» 

O) 

3 

Z) 

CD 

O 

MOCD 70 -

I 

3 

\fo g 

KIDNEY 

12 15 

TIME (DAYS) 

18 

Figure1 . Effect of recovery time and estradiol treatment after termination OT 

gasoline exposure on renal a-, -globulin content. Rats were treated 

with vehicle alone (X) , 2.0 mLfkg gasoline, p.o. for nine days (# ) 

or gasoline plus estradiol (1 mg/kg, s.e.) for four days beginning on 

the final day of gasoline administration (O) • Data are expressed as 

mean * SEM a~ -globulin content in liver and kidney. Arrow indicates 

termination o¥ gasoline treatment of rats. Significantly different 

from all other group means, P < 0.01 (Duncan's test). Different 

from corresponding value for group not receiving estradiol, P < 0.05, 

** P.< 0.01 (Duncan's test). 

-5-
?

~i*f*»&t*L 

*'£V£y&&:' ... *v. iM^fi^><-'. 

Figure 2. Photomicrographs of renal tissue sections from saline-treated (2.0 

mL/kg, p.o., 9 days; Panel A) and gasoline-treated (2.0 mL/kg, p.o., 

9 days; Panel B) rats. Enhanced hyalin droplet (-^-.) accumulation, 

correlated to an increase in a? -globulin content, is obvious in the 

kidney of the gasoline-treated animal compared to control. Mallory's 

Heidenhain stain. 50X. 

-7-
P.19 

Figure4. Effect of 9 days post-gasoline exposure (2.0 mL/kg, p.o., 9 days) 

recovery on hyalin droplet number ( -^^- ) in renal PCT epithelial 

cells. Compare to Figs. 2 and 3. Mallory's stain. 50X. 

-9-
P.20 

SUMMARY AND CONCLUSIONS 

A short-term oral dosing paradigm was employed to study the rate df 

accumulation and disappearance of a„ -globulin, a protein intimately associated 

with the nephrotoxicity of fuel hydrocarbons in male rats, in the kidney during 

gasoline administration. The results of specific experiments show that; 

1. The hepatic content of a„ -globulin was not significantly affected by 

gasoline administration when evaluated at a variety of gasoline doses and 

durations of exposure. 

2. The renal accumulation of a_ -globulin induced by gasoline was rapidly 

reversible after terminating subacute administration of gasoline; complete 

reversal of renal a« -globulin accumulation occurred within three days of 

discontinuing gasoline treatment. However, resolution of hyalin droplet 

nephropathy required 6 to 9 days post-exposure recovery. 

3. Inhibition of the hepatic synthesis of a„ -globulin immediately prior to 

termination of gasoline administration enhanced the removal of a„ -globulin 

accumulated in the kidney. This suggests that hydrocarbon-induced renal 

accumulation of a« -globulin is directly dependent on the rate of hepatic 

synthesis of a„ -globulin. 

In summary, these data suggest that unleaded gasoline induces a nephrotoxic 

response in male rats characterized by renal accumulation of a« -globulin. The 

mechanism for such an effect is unknown. However, data presented are consistent 

with the hypothesis that gasoline produces a transient, readily reversible 

diminution of the renal catabolism (degradation) of low molecular weight 

proteins, such as a„ -globulin. 

-11-