Mutation Research, 116 (1983) 21-34 21 Elsevier Biomedical Press

Mutagenic activity of airborne particulates at non-industrial locations

G . M . A l i n k , H . A . Smi t , J .J . v a n H o u d t , J .R . K o l k m a n a n d J .S .M. Bolei j l

Department of Toxicology, and ~ Department of Air Pollution, Agricultural University, Wageningen (The Netherlands)

(Received 16 October 1981) (Revision received 27 May 1982)

(Accepted 22 June 1982)

Summary

Many studies indicate that aerosols in the ambient atmosphere may have muta- genic properties. Most of these investigations were conducted in industrial or urban areas or near specific sources. The purpose of our studies was to assess the variation in mutagenesis at non-industrial locations over a prolonged period of time using a bacterial test system, the SalmoneUa-microsome test (TA98) and to compare these mutagenic effects with those obtained in a mammalian test system, the sister-chro- matid exchange (SCE) test. Samples were taken every 4th day over a period of 4 months at a background location (Terschelling) and 12 months at a rural location (Wageningen). Particulate matter was collected on glass-fiber filters during 24 h from about 2400 m 3 of air with the standard high-volume technique. After Soxhlet extraction with methanol for 8 h and evaporation to dryness, the residue was dissolved in DMSO. In the Salmonella-microsome test, particulate matter derived from 10-60 m 3 air was tested per plate with and without the addition of liver homogenate.

At both the background and rural locations, mutagenicity varied markedly with time. Mutagenicity at both locations was positively correlated ( r = 0.80, n = 22, P < 0.01). On days with northern and western winds no or only a slight increase in the number of mutants was found, whereas on days with a wind from southern or eastern directions the mutagenicity generally was much higher at both locations. In the 1-year-period study in Wageningen the relation between mutagenicity and wind direction in segments of 60 ° was established. Mutagenicity was strongly correlated with wind direction but not with other meteorological factors such as wind speed,

Mailing address: G.M. Alink, Department of Toxicology, Agricultural University, De Dreijen 12, 6703 BC Wageningen (The Netherlands).

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22

relative humidity and rainfall. In winter, mutagenic effects were higher than in summer. Furthermore, mutagenicity of airborne particulates was correlated with SO 2 concentration and to a lesser extent with the amount of particulate matter. Extracts of airborne particulates showed a direct mutagenicity in the Salmonella-microsome test. Addition of a metabolic system ($9 fraction) led to either decrease or increase in the mutagenic potential, not related to any of the factors under study. In the summer period, 4 samples at eastern and 2 at northern winds were taken for testing both in the Salmonella-microsome test and in the SCE test. Samples taken at eastern wind showed a dose-related increase in the number of SCEs. Dose-response curves for both tests correlated very well ( r between 0.95 and 0.99).

The SCE test appears to be a sensitive assay system for airborne particulates.

Many known mutagens and carcinogens have been detected in the ambient atmosphere (Fishbein, 1976; Broddin et al., 1980). The atmosphere also contains many compounds of which the mutagenic effect is unknown. Risk assessment by testing individual compounds has serious drawbacks because of the large number of chemicals involved and the complexity of the mixture in which various forms of interaction may occur, such as synergism and antagonism. Therefore, the assessment of the overall mutagenic activity in air samples may provide a more realistic basis for the evaluation of the possible risks for man and environment than the former approach. Short-term mutagenicity test systems with a certain predictive value for carcinogenicity have been developed, and can be used to assess mutagenicity of extracts of complex mixtures of particulate matter (Harnden, 1978).

Shortly after the development of a sensitive microbiological assay for mutagenic- ity by Ames (1971), Pitts and co-workers (1977) reported preliminary data on mutagenic activity in the organic fraction of ambient airborne particulates using the Ames assay system. In later years many other studies, using predominantly the microbial assay system, were undertaken (Chrisp and Fisher, 1980; Hughes et al., 1980). Mutagenic activity was observed in ambient particulate samples from in- dustrial or urban areas (Dehnen et al., 1977, 1981; Talcott and Wei, 1977; Tokiwa et al., 1977, 1980; Commoner et al., 1978; Pitts, 1978; Teranishi et al., 1978; Alfheim and MeCller, 1979; Daisey et al., 1980; Hoffmann et al., 1980; MeCller and Alfheim, 1980; Wang et al., 1980) and in particulate matter from special sources such as automobile and diesel exhausts (Huisingh et al., 1978; Wang et al., 1978; Ohnishi et al., 1980; Rappaport et al., 1980; Wei et al., 1980) and fly ash from various combustion processes (LSfroth, 1978; Kubitschek and Venta, 1979; Kubitschek and Williams, 1980).

Only few data on mutagenicity of airborne particulates are available of air samples taken at rural or background stations (Pitts et al., 1977; Alfheim and MeCller, 1978; Dehnen et al., 1981) or obtained with mammalian test systems in vitro (Seemayer, 1980). Therefore, the purpose of this study was dual. Firstly, we wanted to assess systematically the variation in mutagenesis of airborne particulates at non-industrial locations over a prolonged period, sampling eacla 4th day and

23

correlating the data with meteorological and air-pollution parameters. Secondly, we wanted to compare mutagenicity as found in a bacterial system with mutagenicity observed in a mammalian test system in vitro.

Materials and methods

Sampling Sampling of particulate matter was done at 2 measuring sites: Wageningen and

Terschelling (Fig. 4). The station at Terschelling was also used for background measurements of air pollution in The Netherlands. The rural sampling site near Wageningen was located at the meteorological station of the Agricultural University, which also functioned as a station of the Dutch Air Pollution Monitoring Network and a synoptic station of the Royal Dutch Meteorological Institute. Over a period of 4 months at Terschelling and 12 months at Wageningen, samples were taken every 4th day. With the standard high-volume technique, particulate matter was collected during 24 h from about 2400 m 3 of air, by using glass-fiber filters (Gelman G F / A ) . Filters were equilibrated and weighed before and after sampling at room temper- ature and relative humidity of 45%. They were stored at room temperature for maximally a few weeks. In a test series, storage within this period appeared to have no effect.

Extraction The filters with collected material were extracted with 350 ml of methanol for 8 h

in a Soxhlet apparatus. The methanol extracts were evaporated to dryness with a vacuum film rotator. Then the residue was dissolved in about 12 ml DMSO so that 1 ml of the solution corresponded to 200 m 3 of air. Extracts in DMSO were kept at + 4 ° C no longer than a few weeks before testing.

Mutagenicity testing Salmonella-microsome test. The extracts were tested in the Salmonella-micro-

some test as described by Ames et al. (1975). Because literature data and preliminary experiments indicated that strain TA98 is the most sensitive of tester strains for air particulate matter, this strain was used in all experiments. Experiments were performed with and without the addition of Aroclor-induced liver homogenate. Per filter, 2 experiments were done, each in triplicate. Extracts derived from different amounts of air (maximally 60 m 3) were tested. For comparison between different samples, in most cases single dose (30 m 3) data were used. Ethyl methanesulfonate and dimethyl nitrosamine were used as positive controls. All data give the net number of revertants. Background mutagenicity varied between 18 and 52 ( - $ 9 ) and between 30 and 62 ( + $9) revertants per dish.

Sister-chromatid exchange test. Exponentially growing hamster cells (V79) were trypsinized. Amounts of 1 ml of a suspension containing 105 ceUs/ml were plated on object glasses placed in sterile 10-cm petri dishes. After 2 h, 9 ml of culture medium (Dulbecco's MEM) supplemented with 10% newborn calf serum (Gibco), penicillin

24

(Gist-Brocades, 200 uni ts /ml) and streptomycin (Gist-Brocades, 100 / lg /ml) was added. 24 h later, 0.1, 0.2, or 0.3 ml DMSO, containing extracts from 20, 40 and 60 m 3 air, respectively, was added to the plates. The culture medium was replaced by medium containing bromodeoxyuridine (BrdU, 10 #M) after 2h of exposure to airborne particulate matter. The cell-cycle rate is 12 h, therefore the exposure to BrdU was continued for 24 h. Then the cells were treated according to the procedure of Perry and Wolff (1974) with slight modifications (Jongen et al., 1981) to make the sister chromatid differential staining visible. Each sample was tested in 2 indepen- dent experiments. In each experiment, cells were plated on 2 object glasses. Per experiment and per concentration, 50 metaphases were scored. 6 samples were tested, namely 4 samples at eastern, 2 at northern wind. The samples were taken during the summer.

Results

Background versus rural location

In Fig. 1 the number of revertants per 30 m 3 air is given for the background location (Terschelling) and the rural location (Wageningen) during the months July-October . Although the difference between the geometric mean at Terschelling (24 rev. /30 m 3) and Wageningen (104 rev. /30 m 3) was significant ( t = - 3 . 4 7 6 , n - - 2 2 , P < 0.005), the mutagenicity (log rev. /30 m 3) at both locations was corre- lated ( r -- 0.80, n = 22, P < 0.01). On days with a higher mutagenicity the values at Terschelling were generally higher than at Wageningen.

His*rev/30 m 3 TA 98 800 -$9

o W0geningen Terschelling

600

~00

200

lO it, 1B 22 26 30 I 3 15 19 23 27

july august B 28 ~2 6

sept 1~ 18 22 26 i

october

Fig. 1. Mutagenic activity of extracts of particulate matter from 30 m 3 of air at Wageningen (rural location) and Terschelling (background location) in S. typhimurium TA98 (--$9) over a period of 4 months (July-October 1979).

25

His*revertonfs 600-

tOO-

200-

6 0 0

too

200

600-

tOO-

200-

600-

tOO

200-

Terschelling 28 - g -'79 wind: north

Wogeningen

0 ~

18 - 10 "79 wind : west

~ _ _ . . ~ . ~ . @ ~ ~ ~ ~ e - - - - - -------------~

10-10-'79 / wind: south /

/ d "

10 2'0 )b A

/ ~ o /

i0 5b 600 1b z0 20 ~0 50 6'0 m 3 oir

Fig. 2. Mutagenic activity of extracts of airborne particulate matter at Wageningen and TerscheUing in S. typhimurium TA98, with (O . . . . . . O) and without ( 0 O) rat-liver $9 fraction. Curves represent typical dose-effect relations for days with different wind directions.

Typical dose-effect curves for the mutagenicity at both locations on days with different wind directions are given in Fig. 2. On days with northern and western winds no or only a slight increase in the number of mutants was found at the 2 locations. However, on days with a wind from southern or eastern directions the mutagenicity of aerosols was generally much higher at both locations.

Mutagenici ty over a one-year period The mutagenicity of extracts of particulate matter obtained frorrl 30 m 3 of air at

26

H is + r e v / ~ m 3 air TA98 Wageningen 6OO

500

t.00

300-

200-

100-

0 juty laugustlsept, oct Inov.I dec.

1979

jJl lrl j an I feb 1980

IIll I, II i,I ,,, march [q~'it I mQy june I

Fig. 3. Mutagen ic i ty of ext rac ts of suspended mat te r f rom 30 m 3 of a i r at Wagen ingen in S. typhimurium TA98 ( - $ 9 ) over a l -year per iod (July 1979-June 1980).

Wageningen on 78 days over a 1-year period is given in Fig. 3. Although mutagenic- ity varied markedly from day to day, in winter it was significantly higher than in summer (P < 0.01). In Table 1 and Fig. 4, mutagenicity is given for various wind directions in segments of 60 °. Samples were only attributed to a segment if the wind persisted for at least half the day in that segment. Samples collected on days with eastern and southern wind directions showed more mutagenic activity than samples obtained on other days.

Other meteorological parameters such as wind speed, relative humidity, and rainfall were not correlated with mutagenicity. As shown by linear regression curves, mutagenicity appeared to be significantly correlated with the SO 2 concentrations (average for 24 h, P < 0.001), for the winter as well as for the summer period (Fig. 5).

T A B L E 1

M U T A G E N I C I T Y O F E X T R A C T S O F A I R B O R N E P A R T I C U L A T E M A T T E R F R O M 30 m 3 O F A I R A T W A G E N I N G E N O N D A Y S W I T H D I F F E R E N T W I N D D I R E C T I O N S IN S E C T O R S O F 60 o

Sector no. Sector n m Range S.D.

1 3 1 5 - 15 ° 5 44 0 - 1 5 3 62

2 15- 75 ° 10 196 0 -471 199

3 75-135 ° 7 396 119-597 196 4 135-195 ° 13 270 69 -654 190 5 195-255 ° 19 143 1-316 95

6 255-315 ° 9 88 6 - 2 5 0 82

.+E S

NORTH SEA

TerscheLUng

\ \ \

Y /

/ I

I I

I i

5ERMANY

27

\

n: 9 ~ m=88

BELGIUM

n =19

I / /

/ /

\\\\\ 1=13 \ \

)m=270 \ \\

n=7 m= 396

t Fig. 4. Mutagenicity of extracts of airborne particulate matter from 30 m 3 of air at Wageningen in S.

typhimurium T A 9 8 ( - S9) on days with different wind directions. Arrows represent means in sectors of 60 ° .

In Fig. 6 the same curves are given for particle concentrations. In summer a lower correlation with mutagenicity was found than in winter. :

As none of the sampling days had 03 concentrations above 240 # g / m 3 the influence of photochemical air pollution could not be assessed. The effect of addition of metabolic enzymes to the assay plates is given in Fig. 7. Neither systematic change nor correlation with seasonal, meteorological or air pollution parameters was found.

Induction of sister-chromatid exchange, The results of the 4 samples at eastern and 2 samples at northern wind in the SCE

28

tog His+rev/30 m3oir TA 98 Wogenmgen

•

: .-. ..~..,'.':

• " :~y=O33+130x / / r : 0 70

. . ° ,.

WINTER • • ". ;be.

y=152+O59x r=060

Log HIs + rev/30 m3otr TA 98 Wogeningen

. < i •

WINTER

y= 0.85+085 x r=077

SUMMER

.'.:/ s • ~ / e

o. o~s/|" • y=O16+129x

/ / r=062

SUMMER

%

• .:" y/

"Z'~" .

~ y = ~ O 98 ll,9x • r=046

"'" ~" 2 3 , "" "2 Log ~g/m 3 SOz log l.tg/m 3 porficulote

Fig. 5. Linear regression curves, which give the correlation between the mutagenic activity of extracts of air particulate matter from 30 m 3 of air, and the SO 2 concentration in winter, summer and a whole-year period.

Fig. 6. Linear regression curves, which give the correlation between the mutagenic activity of extracts of air particulate matter from 30 m 3 of air, and the amount of particulate matter in winter, summer and a whole-year period.

test and in the Sa lmone l la -microsome test are presented in Table 2. In Fig. 8, typical d o s e - e f f e c t curves are given for each wind direction. Samples that were posi t ive in the A m e s test were also posit ive in the SCE test, while samples that gave no or a weak mutagenic response in the bacterial test also gave no or only a small effect in the SCE test. The correlation coeff ic ient between the d o s e - r e s p o n s e relations of both tests varied between r = 0.95 and r = 0.99.

TA

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ple

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0.

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±3

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-+

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-+

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19

5--

+

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263-

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6 28

0-+

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I'O

30

His + rev/30 m 3 His + rev/30 m3+Sg mLnus 300 His + rev/30 m 3-Sg

200

100-

-I00

-200

-30OJ july I aug Isptl oct. )novl dec I jan I feb I morch bp:l may june

1979 1980

Fig. 7. Effect of rat-liver $9 fraction on mutagenicity of extracts of air particulate matter at Wageningen from 30 m 3 of air. Data represent differences between revertants + $9 and - $ 9 .

SCE s/chromosome His* revertonts

1.25- -500

,t ° / /

/ /

100- / -~00 / /

/ /

/ 0.75- / -300

/ / /

050- / / __ - - ~ * ~ ----" -200

o I 2b do ~ 6'o m air/10 m[

Fig. 8. Mutagenicity of extracts of air particulate matter at Wageningen in S. typhimurium TA98 ( - $9) ( ~ 0 ) and Chinese hamster cells (V79 - - SCE test) (O . . . . . . Q) at eastern (e) and northern (n) wind directions. Curves represent typical dose-effect relations. Mean-- + S.E.M.

31

Discussion

Much evidence exists on the mutagenicity of airborne particulates at urban and industrial locations. In recent years the number of publications on this subject has increased substantially (Chrisp and Fisher, 1980; Hughes et al., 1980). Only a few studies deal with background or rural areas (Pitts et al., 1977; Alfheim and Moller, 1979; Dehnen et al., 1981). In this study, using the Ames assay and the SCE test, we collected data showing that, at background and rural locations, particulate matter may also bear mutagenic compounds. This was previously found by Alfheim and M_oller (1979) and Dehnen et al. (1981).

The correlation between mutagenicity of samples taken on the same day at different locations, a rural site and a background site, suggests that the mutagenic potential of suspended matter originates not from local sources but rather depends on a large-scale process. Our data provide some support for the suggestion of Alfheim and MOller (1979) that mutagenicity at non-urban locations may be due to long-range transport of mutagenic particulate matter.

The long-range transport in The Netherlands of air pollution in general was already known. Van Egmond et al. (1978) demonstrated the influence of industrial areas in Belgium and Germany. When mutagenicity is studied over a l-year period at a rural location the variation in mutagenicity between different days over the entire period is considerable. Our studies indicate that, in the winter period, mutagenicity is significantly higher than in the summer period. Probably this is due to seasonal differences in generating sources, such as combustion processes for space heating. Differences between summer and winter were also found at other locations by Alfheim and Mztller (1979), Daisey et al. (1980), Mefller and Alfheim (1980) and Dehnen et al. (1981). In Dehnen's study the mutagenicity varied with the benzo[a]pyrene (B[a]P) concentration. It was suggested that, particularly in a heavily polluted area, a larger part of the mutagens may originate from combustion processes. As in the studies of Moller and Alfheim (1980), in our study mutagenicity was correlated with SO 2, which in addition to the correlation with B[a]P, suggests that combustion processes play a role in initiating mutagenicity. Although the correlation with SO 2 in summer is the same as in winter, in the summer period non-mutagenic samples were obtained on days with a relatively high SO 2 concentra- tion. Because the correlation between mutagenicity and the amount of suspended matter is higher in winter than in summer, there are indications for different sources in the two seasons. This does not mean per se that the composition of the particulate matter may vary with respect to mutagens, because our data suggest no difference between the ratio of direct-acting and indirect-acting mutagens in winter and summer, which was observed by De/men et al. (1981).

This study has shown that meteorological factors such as wind direction are of importance for the mutagenic burden. Previously, Commoner et al. (1978) made some observations on the relationship between mutagenicity and wind direction. Alfheim and MOiler (1979), too, indicated the significance of wind direction for the mutagenic potential.

Ambient levels of photochemical smog (NO 2 or 03) may react with benzo[a]pyrene

32

and polycyclic hydrocarbons to form direct-acting mutagens that display strong activity in the Ames assay (Pitts, 1978; Pitts et al., 1980). In our study, too few sampling days with an enhanced 03 concentration were included in the experiments to permit a conclusion to be drawn on the correlation between ozone concentration and mutagenicity.

This study shows that, at non-industrial locations, too, suspended matter may contain direct-acting mutagens. This is in agreement with data published by others for urban and industrial areas (Dehnen et al., 1977, 1981; Pitts et al., 1977; Talcott and Wei, 1977; Tokiwa et al., 1977, 1980; Commoner et al., 1978; Teranishi et al., 1978; Daisey et al., 1980; Hoffmann et al., 1980; Mefller and Alfheim, 1980; Wang et al., 1980). The addition of metabolic enzymes leads to either an unchanged, a decreased or enhanced number of revertants. These effects were neither correlated with a season, nor with a meteorological or air pollution factor. The unpredictive effect of liver microsomes on the mutagenic potential of extracts may be due to the changing composition of the extracts with substances that are directly mutagenic and others that require the presence of microsomes, whereas other directly muta- genic substances or non-mutagenic toxic substances may be inactivated when microsomes are present. Commoner et al. (1978) found more inactivation in the first half of the year than in the second half.

Tokiwa et al. (1980) and Teranishi et al. (1978) observed that different fractions prepared from the crude extract react in a different way on the addition of microsomes. Acid fractions and substituted polycyclic aromatic hydrocarbons al- ways display direct mutagenicity whereas unsubstituted PAHs and the oxygenated fractions need $9. Wang et al. (1978) found a decrease of mutagenicity of some samples of automobile exhaust, containing a high number of direct-acting mutagens, after addition of microsomes. The same decrease was found for many samples associated with combustion processes (L6froth, 1978; Kubitschek and Venta, 1979). Wei et al. (1980) showed a possible non-enzymatic antimutagenic effect of $9 for diesel exhaust. Extracts of airborne particulates may display toxic effects (Dehnen et al., 1977; Seemayer and Manojlovic, 1980) which may be inhibited after addition of $9 (Dehnen et al., 1977).

Almost all mutagenicity testing of airborne particulate matter has been conducted by the Ames assay. For a better risk evaluation a battery approach has to be followed. In this study, we started with the SCE test, a mammalian test system in vitro, which is suggested as an indicator of mutagenicity although not all classes of mutagens have been detected so far by SCE analysis (Carrano et al., 1978; Holstein and McCann, 1979). Recently Schiarer et al. (1980) presented preliminary data on the induction of SCEs by city smog extracts. Our studies on extracts of airborne particulates from a rural area show that samples that are positive in the Ames test are also positive in the SCE test. The SCE test appears to be a very sensitive test system for extracts of airborne particulate matter. Preliminary experiments, however, indicate that airborne particulate matter does not induce point mutations in mam- malian cells. Since other studies suggest (Seemayer, 1980) that airborne particulates are positive in the cell transformation test, further studies are needed to assess whether or not these particles exhibit a mutagenic a n d / o r carcinogenic risk for man and environment.

33

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