Induced recessive lethals in the second and third chromosomes of Drosophila fed on fly ash

ENVIKONMENTAL

KESEAKCH

35,

327-332

Induced Recessive Chromosomes

(1984)

Lethals in the Second and Third of Drosophila Fed on Fly Ash

ALY H. MOHAMED Department

of Biology

and

School

S. STERN

AND MKHELE of Medicine,

Uniwrsifv

of Missouri.

Kunsus City. Missouri 64110 Received April 20, 1983 INTRODUCTION

In recent years, there has been a growing concern regarding the “energy crisis.” By the year 2000, 75% of the total energy and SO% of electrical energy needs in the United States will be met by using coal (Gangoli and Thodos, 1976). With this expanded use of coal for electric power generation will come the problem of disposing of massive amounts of solid wastes, which include fly ash. Huge amounts of fly ash are produced from coal combustion for energy generation, According to Fisher and Natusch (1979) a total of 0.5-2.5 million metric tons of respirable fly ash was released to the atmosphere from all coal-burning facilities in the United States. It is also predicted that by the year 2000, 99 million tons of fly ash will be produced per year (Theis and DePinto, 1976). Mutagenicity has been reported in the finest fly ash fraction collected from power plants by utilizing the Ames bacterial assay (Chrisp et cd., 1978; Wei ef ni., 1982). Since a high positive correlation has been found between carcinogenicity of substances for animals and humans and the mutagenic activity in the bacterial system (Ames et al., 1973, the positive mutagenic activity of fly ash particles raises concern over potential genetic and carcinogenic risks (Fisher of ai., 1980). In spite of the advantage of the Ames test, some technical and even theoretical limitations may occur. While agents identified as mutagenics in the Ames test may be presumed to be potentially carcinogenic substances, negative results do not necessarily imply there is no health risk. A false-negative response can be corrected by substituting other tester bacteria or using other systems (Devoret, 1979: Wei et al., 1982). Smith-Sonneborn et nl. (1981a, b) showed that fly ash particles had mutagenic effects on Parumecium. One of the common tests for the detection of mutations is the utilization of Drosophilu melanogaster in the detection of natural or induced autosomal and X-linked recessive lethals (Dawood, 1961; Allen, 1969; Mohamed, 1971; Foltz and Fuerst, 1974; Valencia, 1981). Auerbach and Kilbey (1971) stated that Drosophila lethals are the best tool for the detection of mutagenic ability of chemical compounds. Many persons have been hesitant to accept results with an insect as being relevant to humans, since it was thought that their metabolism was probably too different. These concerns have been reduced by the discovery that Drosophilu has enzymes that carry out metabolic activities similar to those effects produced by mammalian enzymes (Vogel and Sobels, 1976). 321 0013-9351/84

$3.00

CopyrIght Q 1984 by Academic Pras. Inc All rights of reproduction m any form rc,cned

328

MOHAMED

AND

STERN

The objective of the present study was to determine on D. melanogaster. MATERIALS

the mutagenicity

of fly ash

AND METHODS

Plastic teaspoons were tilled with an agar solution (3 g of agar dissolved in 100 ml water by boiling plus 3 ml of 3 parts ethanol:1 part glacial acetic acid, by volume). After cooling and solidifying, two drops of thick yeast suspension were placed on the top of the agar. Each spoon was inserted in an empty breeding bottle (‘h-pint milk bottle) and three or four unetherized Oregon-R well-fed adult females were introduced into the bottle which was then covered with a cotton plug. The females were left to lay eggs for 2 days. Each spoon with its eggs was placed either in the control standard cornmeal medium or in medium treated with fly ash. The fly ash was added to the standard cornmeal medium just before the end of the cooking time. Three concentrations of fly ash were used in relation to the volume of the dry material in the cornmeal medium, namely the cornmeal, agar, and the dry yeast. These concentrations were 4: 1, 2: 1, and 1: 1 of medium to fly ash. The hatched larvae were left to feed on the untreated or treated media for their complete cycle. The fly ash used in this study was collected in 1979 from the ash hoppers of electrostatic precipitators of a commercial electric power plant located in western Missouri. This plant burned an Illinois coal with a 3.47% sulfur and a 10.68% ash content. MgO was added to the ash during processing to regulate its acidity. The ash was composed primarily of 84% silica, alumina, ferric oxide, and lime. The trace element composition of this fly ash was determined by Stern and Stern (1980). The elemental concentrations (kg/g) analyzed by atomic absorption spectrophotometry were Cd (15), Cr (161), Cu (60), Fe (127,000), Mn (730) Ni (94), Pb (202), and Zn (235). Males from media treated with the fly ash, as well as from the control, were mated singly with two virgin females of the tester shock, ZnUL)Cy, Cy sp2/1n (2LR) bw”; In(3LR) DcxF, DISb. This tester shock was provided kindly by the Mid-Amerira Drosophila Center, Bowling Green State University, Bowling Green, Ohio. Only one each of the second and third chromosomes from each male, either untreated (50 males) or treated (30 males from each treatment), was tested. Fi males of the phenotype Cy;D were mated singly to two virgin females of the tester shock. In order to obtain progeny homozygous for a specific second chromosome and a specific third chromosome, males and virgin females of the Cy;D phenotype from the F, generation were mated. This mating was expected to produce 419 Cy;D : 219 Cy;+ : 2/9 + ;D : l/9 + ;+ individuals. All cultures were maintained at a temperature of 22°C. Care was exercised in all experiments not to classify only first emerging flies. If less than 40 flies were produced in a given culture vial in F3, the data from this vial were omitted. RESULTS

The relative viability of the second and third chromosomes was estimated from data collected in F, for the three treatments as well as for the control (Table 1). The actual numbers of chromosomes tested and F, counted are given in Table 2.

LETHALS

TABLE RELATIVE

VIAIULITY

1

OF HOMOZYGOUS CHROMOSOMES 2 AND 3 FROM DIFFERENT INCLUDING AND EXCLUDING LETHALS AND SEMILE’THALS

-.

No. chromosomes

No. chromosomes tested

%(’

%”

tested

41

34.45

+ 1.04

t 1.73 -c 1.99

26 21

33.10 38.07

+ 2.42 z!z 2.89

28.22 30.84

2 2.15 2 2.51

24

18

29.88 38.67

t 3.43 z!c 2.91

26.93 31.21

k 1.90 + 2.50

24 18

29.88 36.71

L? 2.78 rfr 2.18

Control

47

31.16

t

4:l

26 22

28.36 31.25

‘:I

24 22 24 17

1:l

TREATMENTS

Chromosome 3

Chromosome 2 Population

329

IN L)rosophila FED FLY ASH

.39

Treatments with fly ash were able to reduce the viability of the homozygotes for both chromosomes (Table 1). The t test indicated that the mean difference of 4.23% between control (3.6) and the 1: 1 treatment (26.93) for Chromosome 2 was significant (t = 2.18 with 96 df, P value 0.05-0.02). On the other hand, t tests showed that the remaining treatments for either Chromosome 2 or 3 did not differ significantly from the control. The different treatments, except for the 1:1 concentration for Chromosome 2, had no effect in reducing significantly the viability for either chromosome. There was, however, a positive relationship between the number of chromosomes showing drastic (lethal and semilethal) mutants and the concentration of fly ash (Table 3). This finding holds for all treatments except for the 2: 1 concentration for Chromosome 2. Since the total data indicated a difference in number of drastics on Chromosomes 2 and 3, a consideration of joint distribution of drastics was made for the chromosomes from different treatments

TABLE NUMBER

OF FL.IES

IN GENERATION

3, INCLUDING

ESTHER

OR BOTH

2 AND

EXCLUDING

COUNTS

WITH

DRASTIC

ON

CHROMOSOMES .-

Chromosome 2

Chromosome 3

No. chromosomes Population

tested

No. chromosomes c,

+

tested

D

+

Total

Control

47

3880

1763

47

3663

1980

5643

4:l

26 22

1872 1509

774 695

26 21

1797 1327

849 783

7646

2:l

24 22

1100 1030

467 459

24 18

1110

457 444

1567

739

24 17

1877 1301

740 611

24 18

176s 1353

852 780

2617

1:I

.--

330

MOHAMED

AND STERN

TABLE VIABILITY

3

CLASSES IN DIFFERENT

TREATMENTS

Chromosome 2 Population

Quasi-normal

Control 4:1 2:l 1:l

100 84.6 91.7 70.9

Chromosome 3 Drastic

Quasi-normal

Drastic

15.3 8.3 29.1

100 80.7 75.0 70.9

19.1 25.0 29.1

under the hypothesis that the number of drastics on Chromosome of Chromosome 3. Viability classes are divided into four classes 1213,

12n3,

n213,

2 equals that

n2n3,

where 12 or 13 = lethal-bearing second or third chromosomes, respectively, and n2 or n3 = nonlethal second or third chromosomes, respectively. For calculating the expected frequency for each of the above classes, the method of Allen (1969) was used. Based on chance alone, the frequencies of induced drastics are expected to be the same for both chromosomes since these are of approximately the same length. This was found to be the case, indicating no difference in the number of lethals induced in either chromosome by each treatment (Table 4). DISCUSSION Mutagenicity of fly ash has been demonstrated both in the Ames Salmonella system (Chrisp et al., 1978; Fisher and Wilson, 1980) and in Paramecium (SmithSonneborn et al., 1981a, b). Both systems provided evidence for particles associated mutagens. While coal fly ash collected from the electrostatic precipitator hopper was nonmutagenic in the Ames assay, fly ash collected from smoke stack breeching was mutagenic (Crisp et al., 1978; Crowley et al., 1979; Kutischeck and Venta, 1979; Fisher and Wilson, 1980). Natusch and Tompkins (1978) suggested that this could be due to the PAHs (polycyclic aromatic hydrocarbons) and probably other organics that are often gases at high temperatures in the power plants, but absorb to the fly ash surface on cooling when they leave the stack. According to Smith-Sonneborn et al. (1981a) these absorbed organics are responsible probably for the heat-labile promutagens detected in fly ash by the Ames and Paramecium tests. TABLE CONTRIBUTION

4

TO x2 UNDER THE HYPOTHESIS THAT THE NUMBER CHROMOSOME 2 EQUALS THAT ON CHROMOSOME

OF DRASTICS ON

3

Population

1213

12n3

n213

n2n3

df

P value

4:l 2:l 1:l

0 0 2

4 2 5

5 6 4

17 16 13

2 2 2

so-.30 .70-so .98-.95

LETHALS

IN

Drmophiln

FED FLY .4SH

!?I

The fly ash used in our study was collected from the electrostatic precipitator hopper; nevertheless, it was able to induce recessive lethal mutants in our Drosophila assay particularly in high concentrations. This could be due to the biologically active surface-associated trace elements that were present in the fly ash. These inorganic compounds have been pointed out by Smith-Sonneborn et ctl. (1981a) to have a mutagenic effect in Pnrumecicrm. Organic compounds present in the fly ash may have also contributed to the induction of such lethals (Fisher et cd.. 1980). The present study shows that Drosophila can be used as a prescreen for the particle mutagenicity test by feeding larvae on fly ash. According to Vogel (197 1) and Clark (1982), larvae fed on mutagenic substances are often more sensitive to these substances than adult flies for the detection of induced mutations. REFERENCES Allen. A. C. ( 1969). Lethal frequencies on second and third chromosomes in populations of ~),-os(Jphi/u

melonogrrster-.

Genetics

63, 619-637.

Ames. B. N.. McCann. J.. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagen\ with .Sa/mon~/la/mammalian-microsome mutagenicity test. Murut. Res. 31, 347-364. Auerbach. C.. and Kilbey. B. J. (1971). Mutation in eukaryoteh. Ant777. Re\,. Genrt. 5. 163-218. Chrisp. C. E., Fisher, G. L.. and Lammert. J. E. (1978). Mutagenicity of respirable coal fly a\h. Science (Washington. D.C.) 199. 73-75. Clark, A. M. (1982). The use of larval stages of Drosopl7iln in screening for some naturally occurring mutagens. Mutat. Rrs. 2, 84-97. Crowley. J. P.. Dennis A. J.. Facklan, T. J., and Margard, W. L. (1979). Comparative microbial and mammalian cell in vitro bioassay of fossil fuel generated particulates. In “Proceedings of Third International Symposium on Polyaromatic Hydrocarbons” (P. N. Jones and P. Leber. Ed\.). pp. 603-620. Ann Arbor Science Pub.. Ann Arbor. Mich. Dawood. M. M. (1961). The genetic load in the second chromosomes of some populations of Drosnpizi/iu meluno,+7ster in Egypt. Genetics 46, 239-246. Devoret. R. (1979). Bacterial tests for potential carcinogens. In “Readings from Sci. Amer. Genetic\” pp. 304-313. Freeman. San Francisco. Fisher. G. L.. Chrisp. C. E.. and Wilson. F. D. (1980). Coal fly ash as a model complex mixture for short term bioassay. In “Second Symposium on Application of Short Term Bioassay in the Fractionation and Analysis of Complex Environmental Mixtures, Williamsburg.” Fisher. G. L.. and Natusch. D. F. S. (1979). Size dependence of the physical and chemical properties of fly ash. 111 “Analytical Methods of Coal and Coal Products” (C. Karr. Ed.), Vol. 3, pp. 489541. Academic Press. New York. Fisher, G. L.. and Wilson, F. D. (1980). The effects of coal fly ash and silica inhalation on macrophage function and progenitors. J. Reticu/oendotk/. SM. 27, 513-574. Foltz. G. L., and Fuerst. R. (1974). Mutation studies with Drosophila r77elonogc7ster. exposed to four fluorinated hydrocarbon gases. En~~iron. Res. 7, 775-285, Gangoli, N.. and Thodos. G. (1976). Fly ash-a potential asset in wastewater treatment. In “Third National Conference on Complete Water Reuse.” pp. 623-628. Cincinnati, Ohio. Kubitschek. H. E.. and Venta. L. (1979). Mutagenicity of coal fly ash from electric power plant precipitators. Environ. Mumgen. 1, 79-82. Mohamed. A. H. (1971). Induced recessive lethals in second chromosomes of Drosopl~i/r~ ~~I&UIOgu.ster by hydrogen fluoride. In “Proceedings of the Second International Clean Air Congres\” (H. M. Englund and W. T. Beery. Eds.). pp. 158-161. Academic Press. New York, Natusch, D. F. S.. and Tompkins, B. A. (1978). Theoretical consideration of the absorption of poly cyclic aromatic hydrocarbon vapor onto fly ash in a coal-fired power plant, in “Carcinogens” (R. 1. Freundenthal. Ed.), pp. 145-156. Raven Press. New York. Smith-Sonneborn, J., Palizzi. R. A., and Herr. C. (1981a). Mutagenicity of fly ash particles in ,“oft~tnecirtm. Sci~nr~ ( Wtrshington. D.C.) 211, 180- 182.

332

MOHAMED

AND STERN

Smith-Sonneborn, J., Fisher, G. L., Pahzzi, R. A., and Herr, C. (1981b). Mutagenicity of coal fly ash: A new bioassay for mutagenic potential in a particle feeding ciliate. Environ. Mutagen. 3, 239-252.

Stern, M. S., and Stern, D. H. (1980). “Effects of Fly Ash Heavy Metals on Daphnia magna.” Completion Report, A-120-MO. Missouri Water Resources Research Center, Columbia, MO. Theis, T. L., and DePinto, J. V.(1976). “Studies on the Reclamation of Stone Lake, Michigan.” Ecol. Res. Ser., EPA-60012-76-106. Valencia, R. (1981). “Mutagenesis Screening of Pesticides Using Drosophila.” EPA-600/51-81-017. Vogel, E. (1977). Identification of carcinogens by mutagen testing in Drosophila. The relative reliability for the kinds of genetic damage measured. In “Origins of Human Cancer, Book C” (H. H. Hiatt, J. D. Watson, and J. A. Winsten, Eds.), pp. 1483-1497, Cold Spring Harbor Laboratory. Vogel. E., and Sobels, F. H. (1976). The function of Drosophila in genetic toxicology testing. In “Chemical Mutagens” (A. Hollaender, Ed.), Vol. 4, pp. 93-412. Plenum, New York. Wei, C., Raabe, 0. G., and Rosenblatt, L. S. (1982). Microbial detection of mutagenic nitro-organic compounds in filtrates of coal fly ash. Environ. Mutagen. 4, 249-258.