Sequential study of the pathology of prudhoe bay crude oil in chicken embryos

ECOTOXICOLOGY

AND

ENVIRONMENTAL

Sequential

SAFETY

19,17-23 ( 1990)

Study of the Pathology of Prudhoe Bay Crude Oil in Chicken Embryos

C. M. COUILLARD AND F. A. LEIGHTON Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0 WO Received May 5, I989

Ten microlitersof Prudhoe Bay crude oil was applied to the shell of fertile leghorn chicken eggs on Day 9 of incubation. Gross and microscopic pathological changes were examined in embryos surviving 1,2,4, and 9 days after treatment. Liver necrosis, renal lesions, and extensive edema appeared 2 days after treatment and reached maximal prevalence 4 days after treatment. There was minimal repair of the lesions from Day 4 to Day 9 after treatment. Pathological changes, including liver necrosis, mineralization in the kidney, infiltration by a large number of heterophils in the liver and spleen, subcutaneous edema with formation of large blisters, and reduction in body weight and length were still present on Day 18 ofincubation, close to hatching titlle.

0 1990 Academic

Press. Inc.

Petroleum oils are spilled frequently into the aquatic environment and are a serious hazard for marine birds. Large-scale mortality and a variety of physical and chemical toxic effects have been reported in seabirds exposed to oil (Holmes, 1984; Leighton et al., 1985; National Research Council, 1985). One well-documented toxic effect of petroleum oil for birds is embryotoxicity (Albers, 1980; Ring and Lefever, 1979; Albers and Szaro, 1978). Less than 10 ~1 of different petroleum oils, applied to the eggshell, can cause mortality and teratogenicity in embryos of various avian species (Hoffman and Albers, 1984; Macko and King, 1980; Hoffman, 1979). Transfer of petroleum hydrocarbons from the eggshell to the tissues ofthe embryo has been documented (Hoffman and Gay, 198 1). Recently, liver necrosis, renal lesions, and extensive edema have been described in 13-day-old chicken embryos exposed to Prudhoe Bay crude oil (PBCO) on Day 9 of incubation. Little is known about the mechanism of this embryotoxicity. Oil does not act simply as a physical sealant of the eggshell. The prominent edema and the distribution of the liver lesions suggest a primary vascular disturbance (Couillard and Leighton, 1989a and 1989b). The study reported here was done to assessthe early development and further evolution of lesions in embryos suffering toxic effects from Prudhoe Bay crude oil. Such information has important implications for the diagnosis of embryotoxicity in the field, for the long-term significance of exposure to oil, and for the potential use of avian embryos in bioassays for the relative toxicity of different oils. MATERIALS

AND

METHODS

Animal egg and incubation. Fertile white leghorn chicken (Gallus gallus) eggs were shipped by air within 2 days of collection at a commercial hatchery.’ Upon arrival, ’ Keystone hatchery, Niverville, Manitoba, Canada. 17

0147-6513/90$3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

18

COUILLARD

AND

TABLE

LEIGHTON

1

MORTALITYANDGROSSLESIONSINCHICKENEMBRYOSATDIFFERENTTIMES AFTER TREATMENTWITH~OF~PRUDHOEBAYCRUDEOIL' Days after treatment 1 2 4 9

% Mortality

(n)

% Edema/ascites in live embryos (n)

% Gross liver lesions in live embryos (n)

0 (20) 4 (22) 41(17) 18 (38)

0 (20) 32 (22) 59(17) 66 (38)

0 (20) 12 (25) 51(35) 37 (60)

’ Oil was applied to the eggshell on Day 9 of incubation.

the eggs were stored for 36 hr at 4°C and then were placed in an incubator (Humidair-e, New Madison, OH) maintained at 37.X and 50-55% relative humidity. After 9 days of incubation, all the eggs were candled and infertile or cracked eggs were removed. Dosage regimen. All eggs were assigned randomly to 8 treatment groups and were treated on Day 9 of incubation. Four groups (A, C, E, G) were treated with 10 ~1 Prudhoe Bay crude oi1.2 PBCO was applied with a microliter pipet to the shell surface of upright eggsjust below the airspace and was allowed to spread freely (Albers, 1977). Four groups (B, D, F, H) were used as untreated controls. Groups A, B, D, and F had 20 eggs each, groups E and H had 35 eggs each, and groups C and G had 25 and 60 eggs, respectively. A higher number of eggs was assigned to treated groups to assure the survival of enough treated embryos for pathological examination. Only live embryos were examined because the tissues of dead embryos were autolysed. All eggs were distributed randomly in the incubator immediately after treatment. Embryos from one control and one treated group were examined at four successive times after treatment. Groups A and B were examined on Day 10 of incubation (1 day after treatment), groups C and D on Day 11 (2 days after treatment), groups E and F on Day 13 (4 days after treatment), and groups G and H on Day 18 (9 days after treatment, 3 days before expected hatching). Gross and histologic methods. All embryos were removed from the eggs, weight and crown-rump length were recorded, and the embryos were examined. Embryos without evident heartbeat were considered dead. Live embryos were killed by decapitation and were fixed in 10% phosphate-buffered formalin (pH 7.0). For each embryo, the fixed liver, heart, and spleen were blotted dry and weighed. Tissue samples were embedded in paraffin and sections 5 pm thick were stained with hematoxylin and eosin. A few livers from control and treated embryos were lost during processing and could not be examined histologically. The number of livers examined in each group is specified in the tables presented under Results. Histological sections of liver, kidneys, and spleen were examined without knowledge as to treatment group. Prevalences of necrosis, mineralization, and accumulation of heterophils in the liver and of mineralization and cellular casts in the kidney were determined as described previously (Couillard and Leighton, 1989a). ’ PBCO was obtained from the American Petroleum Institute through the Environmental Agency (U.S.A.) and was kept at 4°C in amher glass bottles.

Protection

PRUDHOE

BAY CRUDE OIL AND CHICKEN TABLE

HISTOLCXXCAL

19

EMBRYOS

2

CHANGES IN THE LIVERS OF CHICKEN EMBRYOS AT DIFFERENT TIMES AFTER TREATMENT WITH PRUDHOE BAY CRUDE OIL Necrosis

Mineralization

Heterophils

Days after treatment

Control

Treated”

Control

Treated

Control

Treated

1 2 4 9

O/20b o/20 O/24 o/20

o/20 8/22C 1 l/17’ 24/33C

o/20 O/20 O/24 O/29

O/20 o/22 5117’ 23133’

o/20 o/20 O/24 4129

o/20 o/22 1 l/17’ 33/33c

’ Ten microliters Prudhoe Bay crude oil was applied to the eggshell on Day 9 of incubation. h Results are expressed as number of embryos with lesion/total number of embryos examined. ’ Significantly different from control, Fisher, P G 0.05.

Statistical analyses.Statistical analyses were done with the SAS statistical computing system (SAS Institute, Inc., 1985). Body weight, crown-rump length, and organ weights were compared among groups using one-way analysis of variance with Fisher’s least significant difference test. The prevalence of histological changes in the liver, kidneys, and spleen among groups was compared with Fisher’s exact test. RESULTS One day after treatment, no mortality or other changes were found in exposed or control embryos. Two days after treatment, there was 12% mortality in embryos exposed to PBCO and 32% of the surviving embryos had liver necrosis (Tables 1 and 2). Only one embryo had slight ascites and none had subcutaneous edema. One third of the exposed embryos had large cellular casts in a small number of renal tubules in the mesonephros. Mineralization was more frequent in the kidneys of treated embryos than in those of controls (Table 3). Prominent cords of hematopoietic tissue were found in the spleens of 11 of the 2 1 exposed embryos and in none of the controls.

TABLE

3

HISTOLOGICAL CHANGES IN THE KIDNEYS OF CHICKEN EMBRYOS AT DIFFERENT TIMES AFTER TREATMENT WITH PRUDHOE BAY CRUDE OIL Mineralization in renal tubules Days after treatment :

Cellular casts in renal tubules

Control

Treated”

Control

Treated

2/20b 5119 2124 2129

2120 13/21’ 1 l/17< 18139’

o/20 o/20 O/24 O/29

o/20 7121’ 7117’ 2139

” Ten milliliters Prudhoe Bay crude oil was applied to the eggshell on Day 9 of incubation. b Results expressed as number of embryos with lesion/total number of embryos examined. ’ Significantly different from control, Fisher, P d 0.05.

20

COUILLARD

AND LEIGHTON

No differences were found in body weight, crown-rump length, or weights of liver and heart. Four days after treatment, there was 5 1% mortality in embryos exposed to PBCO (Table 1). There were 10 times more embryos with ascites or subcutaneous edema and 2 times more embryos with liver necrosis than there were 2 days after treatment (Table 1). Histological lesions typical of PBCO exposure were found in the liver, kidney, and spleen. Many necrotic areas in the liver were mineralized and an excessive number of immature heterophils were found around hepatic blood vessels (Table 2). Cellular casts in renal tubules were smaller and less cellular than those observed 2 days after treatment (Table 3). All of the exposed embryos and none of the controls had prominent cords of hematopoietic tissue in the spleen. Body weight and the ratio of body weight to crown-rump length were increased in the exposed embryos (Fig. 1). The weights of the liver, heart, and spleen of the exposed embryos were higher than those of controls (Fig. 1). Nine days after treatment, there was a cumulative 37% mortality in embryos (Table 1). Thirty-eight embryos survived and were examined. In edematous embryos, fluid was contained in large subcutaneous blebs on the neck, the back, or the rump. Grossly visible liver lesions similar to those observed 4 days after treatment were seen in 66% of the embryos (Table 1; Fig. 2). The livers of many of the exposed embryos were bright green while those of controls were yellowish or brown. Most of the necrotic areas were mineralized and half of them were infiltrated with a small number of heterophils (Table 2). In four livers, a distinct rim of heterophils surrounded the necrotic zones. These heterophils were not degenerated or degranulated and suggested an increase in hematopoietic activity rather than an inflammatory reaction. Perivascular heterophils were abundant in livers of all exposed embryos (Table 2). In 9 of the 24 necrotic livers, a rim of markedly vacuolated hepatocytes surrounded the necrotic areas. No fibrosis or scarring was seen. Only two exposed embryos had cellular casts in renal tubules but mineralization of tubules, when present, was extensive and involved a large number of tubules in the metanephros (Table 3). In the spleens of 18 of the 38 exposed embryos, there was massive accumulation of heterophils which masked the normal architecture. At this stage of incubation, spleens of control embryos contained many heterophils but never to the same extent as in exposed embryos. Body weight and crown-rump length were reduced in exposed embryos (Fig. 1). The weights of the liver, heart, and spleen were higher than those of the controls. DISCUSSION Two days after treatment, necrotic lesions appeared in the liver of exposed embryos. Prevalence of liver necrosis doubled from Day 2 to Day 4 after treatment and there was no change in prevalence and no resolution of the lesion from Day 4 to Day 9. No intermediate stage between normality and necrosis was found upon histological examination of the liver at anytime after treatment. This indicates that liver necrosis developed rapidly, within a few hours. On Days 2 and 4 after treatment, renal lesions were not extensive and were confined to the mesonephros, a part of the embryonic kidney that normally degenerates during the last week of incubation (Romanoff, 1960). On Day 9 after treatment, extensive mineralization was present in the metanephros, the definitive kidney. The moderate accumulation of heterophils in the liver and spleen could have resulted from an acceleration of the normal process of migration of the hematopoietic

PRUDHOE

* 1“in

BAY CRUDE OIL AND CHICKEN

Crown-Rump

Body Weight (g)

10

300 n

20-

R

Control lO$PBCO

21

EMBRYOS

Length (cm)

1

Cl Control n lO+PBCO

IT::

6 4

2 0

1

2 4 Day post-treatment

1

9

2

BW / C-R Length (g/cm)

"1

4

Day post-treatment

Liver Weight (mg) 600

IT

0 Control W lO$PBCO

600 400 200 0 1

2 4 Day post-treatment

9

Heart Weight (mg)

1

*

225

Spleen Weight (mg)

10 5

0

0 q

15

75

0 1

2 4 Day post-treatment

9

9

25 20

150

2 4 Day post-treatment

* 1 9

Control lOpIPBC0

*

-A

1

2 4 Day post-treatment

9

FIG. 1. Body weight and length and organ weights in chicken embryos treated with Prudhoe Bay crude oil (PBCO). Ten microliters PBCO was applied to the eggshell on Day 9 of incubation; surviving embryos were examined at four successive times after treatment. Results are expressed as means + 1 SD. *Significantly different from control (0 pl PBCO), ANOVA, PS 0.05.

tissue from the yolk sac to internal organs (Romanoff, 1960). However, the persistence and magnitude of heterophil accumulation in livers and spleens of 18-day-old embryos cannot be explained on this basis. The significance of this response is not known. Major morphological abnormalities persisted until Day 18 of incubation, the last day of the prenatal period in the chicken embryo (Rahn et al., 1979). Lesions found

22

COUILLARD

FIG. 2. Liver of chicken embryo examined on Day 18 of incubation;

treated with 10 ~1 PBCO on the eggshell on Day 9 of incubation pale zones of necrosis on the surface are indicated (arrows).

AND

LEIGHTON

and

at this late stage of incubation are likely to influence the survival of the chicks after hatching. Mallard ducklings hatched from eggs treated with fuel oil No. 2 had small weight gains in the first week after hatching (Albers, 1977). The survival and pathology of chickens hatching from eggs treated with petroleum oils has yet to be investigated. The percentage of mortality was higher in the group of embryos examined on Day 4 than in that examined on Day 9 after treatment, while the prevalence of liver necrosis was similar. Variation in the percentage of mortality between groups given the same dose of oil has been observed before and there is no clear explanation for this variation (Couillard and Leighton, 1989a). Liver necrosis was the most reliable index of toxicity. It can be recognized readily upon gross examination of embryos and, thus, it may be useful as an endpoint in assessment of the toxicity of petroleum oils. Prevalence of liver necrosis was maximal 4 days posttreatment; thus the fourth day after treatment is appropriate for examination of the embryos for assessment of toxicity. Liver necrosis also may be a useful lesion in the diagnosis of prehatching death due to exposure to oil. However, its pathogenesis and specificity for petroleum oil toxicity are not yet established. CONCLUSION In chicken embryos treated with petroleum oil on Day 9 of incubation, pathological changes appear on Day 11 and persist until Day 18 of incubation. changes could interfere with the survival of the chick after hatching.

severe These

ACKNOWLEDGMENTS This investigation was supported Wildlife Health Fund of the Western

by fellowships from College of Veterinary

the Medical Research Council of Canada, the Medicine, and the University of Saskatchewan,

PRUDHOE

BAY

CRUDE

OIL

AND

CHICKEN

EMBRYOS

23

and by grants from the Wildlife Toxicology Fund and the Natural Sciences and Engineering Council of Canada. We thank L. Pura for expert technical assistance.

REFERENCES ALBERS, P. H. (1977). Effects of external applications of fuel oil on hatchability of mallard eggs. In Fate and Eflects of Petroleum Hydrocarbons in Marine Ecosystems and Organisms (D. A. Wolfe, Ed.), pp. 15% 163. Pergamon, Elmsford, NY. ALBERS, P. H., AND SZARO. R. C. (1978). Effects of No. 2 fuel oil on common eider eggs. Mar. Pollut. Bull. 9, l38- 139. ALBERS, P. H. (1980). Transfer of crude oil from contaminated water to bird eggs. Environ. Res. 22, 307314. COUILLARD, C. M., AND LEIGHTON, F. A. (1989a). The toxicopathology of Prudhoe Bay crude oil in chicken embroys. Fundum. Appl. Toxical., in press. COUILLARD, C. M., AND LEIGHTON, F. A. (1989b). Comparative pathology of Prudhoe Bay crude oil and inert she11sealants in chicken embryo. Fund. Appl. Toxicol. 13, 165-173. HOFFMAN, D. J. (1979). Embryotoxic and teratogenic effects of crude oil on mallard embryos on Day one of development. Bull. Environm. Contam. Toxicol. 22,632-637. HOFFMAN, D. J., AND ALBERS, P. H. (1984). Evaluation ofpotential embryotoxicity and teratogenicity of 42 herbicides, insecticides, and petroleum contaminants to mallard eggs. Arch. Environ. Contam. Toxicol. 13, 15-27. HOFFMAN, D. J., AND GAY, M. L. (1981). Embryotoxic effects of benzo[a]pyrene, chrysene, and 7,12dimethylbenz[a] anthracene in petroleum hydrocarbon mixtures in mallard ducks. J. Toxicol. Environ. Health 7,775-787. HOLMES, W. N. (1984). Petroleum pollutants in the marine environment and their possible effects on seabirds. Rev. Environ. Toxicol. 1,25 l-3 17. KING, K. A., AND LEFEVER. C. A. (1979). Effects of oil transferred from incubating gulls to their eggs. Mar. Pollut. Bull. 10,3 19-32 I. LEIGHTON, F. A.. BUTLER, R. G., AND PEAKALL, D. B. (1985). Oil and arctic marine birds: An assessment of risk. In Petroleum Effects in the Arctic Environment (F. R. Engelhardt, Ed.), pp. 183-215. Elsevier Applied Science, New York. MACKO, S. A., AND KING, S. M. (1980). Weathered oil: Effect on hatchability of heron and gull eggs. Bull. Environ. Contam. Toxicol. 25,3 16-320. National Research Council (1985). Oil in the Sea, Inputs, Fates, and Effects. National Academy Press, Washington, DC. RAHN, H.. AR, A.. AND PAGANELLI, C. V. (1979). How bird eggs breathe. Sci. Amer. 240,297-309. ROMANOFF, A. L. (1960). The Avian Embryo-Structural and Functional Development. Macmillan Co., New York. SAS Institute, Inc. (1985). SAS User’s Guide. SAS Institute, Inc., Gary, NC.