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Haematological changes in male and female pharaoh quails (coturnix coturnix pharaoh) after ekatin intoxication
Camp. Eiochem. Physiol. Vol. 85C, No. I, pp. 4148, 1986 Printed in Great Britain
HAEMATOLOGICAL PHARAOH QUAILS AFTER K. Department
of Animal
03064492186 $3.00 + 0.00 Pergamon Journals Ltd
CHANGES IN MALE AND FEMALE (COTURNIX COTURNIX PHARAOH) EKATIN INTOXICATION
GROMYSZ-KAEKOWSKA Physiology,
and E.
Institute of Biology, Lublin, Poland
SZUBARTOWSKA Maria Curie-Sklodowska
University,
(Receiwd 16 December 1986) The purpose of this study was to determine changes in the blood of pharaoh quails after Ekatin intoxication, to define the duration of disturbances caused by intoxication and to examine possible
Abstract-l.
sex differences in the birds’ reaction to intoxication. 2. It was found that Ekatin reduced the number of erythrocytes, haemoglobin level and haematocrit value, increased erythroblast and reticulocyte numbers and increased the osmotic resistance of blood cells. 3. It was shown that this pesticide caused neutrophilic leucocytosis with lymphopaenia and eosinopaenia. 4. In males changes in the blood appeared far earlier than in females and they underwent compensation earlier, that is, 3 weeks after intoxication the majority of the haemotological parameters reached values similar to the control.
INTRODUCTION
We have found no data in the literature dealing with blood parameters after intoxication by organophosphorus compounds depending on the sex of animals (Giermaziak, 1973; Kossakowski and Patyra, 1973; Kurski, 1975; Smigielska, 1977; Ali and Shakon, 1981; Gromysz-Kalkowska et al., 1981; Szubartowska, 1983). It is however possible to state, on the basis of examination of LD,, values for these compounds, that females are more susceptible to the effect of a considerable number of organophosphorus compounds (Frawley et al., 1952; Gaines, 1969; Rusiecki, 1973; Benke and Murphy, 1975). These data agree with the haematological studies made by Gromysz-Kalkowska and Szubartowska (1983), who report the absence of sex differences in the reaction of Rana temporaria to small amounts of trichlorfon, and a greater response of females than males to a dose of 300 mg/kg body wt. In view of the data presented and the fact that Ekatin causes considerable changes in blood morphology (Hirnlowa et al., 1965; Giermaziak, 1973; Giermaziak and Andrzejewski, 1976) and the fact that relatively little attention has been given to studies of these changes, it appeared useful to trace the effect of this preparation on the peripheral blood of this quail. Another question taken into consideration in this study was the definition of the duration of disturbances caused by intoxication, while a further problem was the investigation of possible sex differences in the birds’ reaction to Ekatin intoxication. MATERIALS
The experiments were carried out on 18 male and 18 female pharaoh quails. The birds were 4 months old, weighed from 180 to 2 10 g, and were kept on a uniform diet and under the same maintenance conditions. The birds were divided at random into two groups: control (eight quails) and experimental (10 quails). The experimental groups were given water solvent Ekatin in doses of 50mg/kg body wt. The individuals in the control groups were given water only at the same time as the experimental groups. Blood was taken from the brachial vein and the number of erythrocytes (RBC) and leucocytes (WBC) was determined by the chamber method in Natt-Herrick diluting fluid (Natt and Herrick, 1952) haemoglobin level (Hb) by the cyanmethaemoglobin method and haematocrit value (Ht) by the micromethod. Mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were calculated in accordance with the models given by Wintrobe (1956). The percentage composition of leucocytes was calculated in preparations stained by the Pappenheim method. The number of erythroblasts per 1000 erythrocytes was determined in the same preparations, The number of reticulocytes per 1000 erythrocytes was determined in preparations stained in uiuo with brilliant cresyl blue (Pinkiewicz, 1971). The first determination was carried out 3 hr after administering Ekatin or water, and then after the 1st and 3rd weeks of the experiment. The results obtained were analysed statistically using Student’s f-test for independent data. A value of P < 0.05 was taken as statistically significant (Oktaba and Niedokos, 1980).
RESULTS The
studies
causes
certain
quail,
AND METHODS
place
while
showed changes
the direction
that in
administration the
in which
erythrocytes
of Ekatin of
these changes
the take
and their intensity point to some degree of dependence on the birds’ sex. The number of erythrocytes in males fell by 23.2% 3 hr after intoxication (P < 0.05). After 1 week this number was still 20.8% lower than the control value
Ekatin, the pesticide used for these studies, was obtained from Sandoz AG, Switzerland. It contains 25% of the active substance thiometon-O,O-dimethyldithiophosphate 2-(ethylothio)ethyl and is classified as belonging to toxicity class II. 41
42
K.
GROMYSZ-KMKOWSKA
Table
Parameters
Sex
I. Ervtbrocvte Control (X f SE)
and E. SZUBARTOWSKA
values in Doisoninp. bv Ekatin Time after intoxication of Ekatin I week 3 hr (R ? SE) (R + SE)
3 weeks (R k SE)
Erythrocytes (mln/mm’)
;
4.789kO.170 4.135*0.112
3.680 f 0.451’ 4.030 f 0.238
3.792 f 0.297t 4.082 i 0.263
4.741 & 0.423 3.540 * 0.149f
Haemoglobin (%)
$ 0*
16.36 20.461 12.72 + 0.376
12.27 f 0.26% 12.63 _+0.296
13.70 f 0.3805 12.29 f 0.294
15.17 kO.640 12.23 i 0.273
Haematocrit (%)
c? F
50.81 i I .375 43.00 i 0.924
41.60* 1.6075 47.90 f I .277f
42.30 f 1.37% 48. IO k 1.293f
52.80 f 1.097 44.50 & 0.997
MCV (p3)
c? 0
106.43 + 2.026 104.15 + I.417
123.04 f 6.850 121.85 k6.197,
116.92 & 8.913 120.88 f 7.480
105.97 + 5.558 128.59 i 8.1 IOf
MCH
s E
34.23 ? 0.632 30.79 + 0.698
37.30 & 4.222 32.27 + I .924
37.93 f 2.925 31.18 f 2.008
35.16f4.517 35.07 + 1.640’
(%)
s
32.07 i 0.447 29.55 f 0.462
29.90 f I.414 26.48 f 0.6831
32.54 & 1.054 25.77 k 1.027f
28.76 + 1.278* 27.63 + 0.983
Erythroblasts (%d
d 0
12.12 * 0.680 14.50 i 0.965
18.90 + 1.3705 14.90 k 0.876
23.70 & 1.76% 2l.lOk 1.346f
16.40 + 1.301§ 21.30+ I.3982
Reticulocytes (%)
$ 0
94.37 + 3.918 84.60 + 2.209
188.50 rf: 6.9806 192.60 + 3.6485
172.40 + 2.43% 164.70 i 4.43@
204.50 f 6.8039
(pg)
MCHC
*P tP $P $P
< < < <
0.05. 0.02. 0.01. 0.001.
(P < 0.02). Three weeks after Ekatin intoxication this parameter was observed to return to the control level. In the case of females the number of erythrocytes was maintained at the control level both after 3 h and 1 week after intoxication and it was not until after 3 weeks after intoxication that it was found to fall by 14.4% (P < 0.01; Table 1, Fig. 1). The haematocrit and males fell by 18.1% after 3 hr and by 16.8% 1 week after Ekatin intoxication (in both cases P < O.OOl), but after 3 weeks approached the control value. In females an increase of 11.4% in haematocrit value took place after 3 hr and 11.9% after 1 week (in both cases p < 0.01). After 3 weeks the haematocrit value, as in the males, returned to the control level (Table 1, Fig. 1). In males and females the mean corpuscular volume increased by 15.6 and 17.0%, respectively, 3 hr after intoxication, although these changes were not statistically significant. The next determination in males showed that the value of this index was 9.9% higher, but after 3 weeks it returned to the control value. In the case of females the mean corpuscular volume was 16.1% higher 1 week and 23.5% higher 3 weeks after Ekatin intoxication (P < 0.05, 0.02; Table 1, Fig. 1). The haemoglobin level in males fell distinctly in successive determinations by 25.0, 16.3 (in both cases P < 0.001) and 7.3% (NS). In females the haemoglobin level was maintained at the control level for the whole of the experimental period (Table 1, Fig. 2). Mean corpuscular haemoglobin in males increased 3 hr and 1 week after intoxication by 8.9 and 10.8%, respectively, but after 3 weeks it was only 2.7% higher than the control level. In females the value of this index increased in all determinations by 4.8, 1.3 and 13.9%, respectively (P < 0.05; Table 1, Fig. 2). In the males the mean corpuscular haemoglobin concentration fell in successive determinations by 6.8, 1.5 and 10.3%. In females the value of this index is 10.4% lower 3 hr, 12.8% lower 1 week (in both cases P c 0.01) and 6.5% lower 3 weeks after intoxication (Table 1, Fig. 2).
During Ekatin intoxication the erythroblast content in males increased in all determinations by 55.9, 95.5 and 35.5%, respectively. These changes were statistically significant with P < 0.001; 0.001; 0.02. In females a significant increase in erythroblast content of 45.5% was observed until 1 week after intoxication, and an increase of 46.9% 3 weeks after intoxication (in both cases P < 0.01; Table 1, Fig. 3).
The reticulocyte content in males was 99.7% higher than the control 3 hr, 82.7% 1 week and
..
-1
C
3h.
;,.
3w.
oftcr intoxication Fig. 1. The number of erythrocytes (RBC), haematocrit value (Ht) and mean corpuscular volume (MCV) in the control (C) and after Ekatin intoxication. tinn
Blood changes in quails after Ekatin
1
C
3h.
3w.
1 W.
C
3h.
1W.
3w.
260
PQ
-C
3h.
3w.
IW.
time after intoxication Fig. 2. The level of haemoglobin (Hb), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) in the control (C) and after Ekatin intoxication.
37.7% 3 weeks after intoxication (with P < 0.001; 0.001; 0.01, respectively). In females the increase in reticulocyte content was higher than in males, 127.6, 94.7 and 141.7%, respectively (in all cases P < 0.001; Table I, Fig. 3).
The increase in the osmotic resistance of erythrocytes was accompanied by an increased content of the young forms of blood cells. In males maximum resistance was observed 3 hr after intoxication, while
C
3h.
Iw.
3w.
time after intoxication Fig. 3. The number of erythroblasts and reticulocytes in the control (C) and after Ekatin intoxication.
after 1 and 3 weeks it was observed to decrease. In females the osmotic resistance of blood cells increased in successive determinations, reaching a maximum value 3 weeks after intoxication (Fig. 4). Changes were observed in leucocytes of quails intoxicated by Ekatin which, as in the case of erythrocytes, exhibited a certain relation to the sex of the birds. The number of leucocytes increased by 24.5% in males and 21.3% in females 3 hr after intoxication.
.I.
‘I.
‘I.
100
100
100
80
80
00
60
60
60
40
40
40
ZJ 20 5 “I 0
20
20
0
0
v)
w
2 I
100 80 60 40
20 0
Fig. 4. The osmotic resistance of erythrocytes in poisoning by Ekatin
.I. NaCl
K.
GROMYSZ-KAEKOWSKAand
E.
SZIJBARTOWSKA
Table 2. Leucocytevaluesin poisoningby Ekatin Time afterintoxication of Ekatin 3 hr I week (R*SE) (X +SE)
3 weeks (B+SE)
Sex
Control (r *SE)
Leucocytes(1000/mm~)
c? 0
16.404k 0.682 16.748iO.318
20.419& 1.843 20.320f 1.830
18.064F0.767 21.287k 0.4335
18.554k I.107 16.509kO.767
Neutrophils (lOOO/mm')
s ?
7.481f 0.898 7.108kO.216
16.196+ 2.35@ 11.439f l.OOO$
ll.310?0.647f 15.589f 0.3405
10.333* 0.943' 8.381? 0.520
Eosinophils (lOOO/mm')
s 0
0.536f 0.114 0.524+0.121
0.061f 0.0315 0.156+0.046$
0.197+0.083* 0.149kO.O57$
0.155+ 0.067f 0.052+ 0.027s
Basophils(lOOO/mm')
$ 0
0.090+ 0.073 0.107f 0.056
0.04020.027 0.073kO.040
0.018kO.018 0.040f 0.027
0.053kO.036 0.030*0.020
Lymphocytes (1000/mm3)
$ 0
7.501f 0.375 8.310f0.207
3.369f 0.2625 7.816kO.865
6.350kO.538 4.881kO.2735
7.586kO.907 7.522kO.572
Monocytes (1000/mm')
d 0
0.792k 0.112 0.699iO.138
0.753f 0.166 0.837kO.167
0.189+0.0575 0.62750.088
0.427~0.086t 0.524+O.l40
WBCI
6 P
0.996f 0.095 0.869+ 0.038
4.000f 0.3435 1.490+0.233*
l.880+0.177~ 2.970+ 0.237i
1.460+0.187 1.120+0.113
Parameters
'P < 0.05. tP <0.02. $P < 0.01. §P
At the next determination the increase in the number of leucocytes was 10.1% in males and 27.1% in females (P < 0.001). After 3 weeks the leucocyte level in males was still 13.1% higher than in the control, whereas in females it fell to slightly below this value (Table 2, Fig. 5). In males the number of neutrophils after Ekatin intoxication increased in successive determinations by 116.5, 51.2 and 38.1%, respectively (with
C
3h.
C
lw. 3h. time after intoxication
1w.
3 w.
3 w.
Fig. 5. The number of leucocytes (WBC) and white blood cells index value (WBCI) in the control (C) and after Ekatin intoxication.
P c 0.001; 0.01; 0.05). In females the increase in the number of neutrophils was 60.9% after 3 hr, 119.3% after 1 week and 17.9% after 3 weeks (P < 0.01; 0.001; NS, respectively; Table 2, Fig. 6). The proportion of neutrophils in the leucogram increased in all determinations in both males and females (Fig. 9). The number of eosinophils in males fell by 88.6% after 3 hr, by 63.3% after 1 week and by 71% after 3 weeks (P c 0.001; 0.05; 0.01). In females the number of eosinophils fell in successive determinations by 70.2, 71.6 and 90.1%, respectively (P < 0.01; 0.01 and 0.001, respectively; Table 2, Fig. 7). The percentage of eosinophils was lower in both sexes after Ekatin intoxication (Fig. 9). The number of basophils in males fell in successive determinations by 55.6, 80.0 and 41.1%, respectively, and in females by 31.8, 62.6 and 72.0% (Table 2, Fig. 7), but these changes were not statistically significant. No significant changes were found in the percentage of basophils in the two experimental groups (Fig. 9). A significant reduction in the number of lymphocytes was found in males only 3 hr after intoxication,
7-
C
iW. jh. .’ time after intoxkotion
4,
Fig. 6. The number of neutrophils in the control (C) and after Ekatin intoxication.
45
Blood changes in quails after Ekatin
80.
C
IW.
3h.
3w
80,
40,
80v) =! I
60-
-
4 2
40-
20,
0 20s ew
-
c
3w.
time after intoxication
C
lw
3h.
3w.
Fig. 7. The number of eosinophils and basophils in the control (C) and after Ekatin intoxication.
m
neutrophils
0
eosinophils
m
basophils
m
limphocytes
m
monocytes
Fig. 9. The percentage composition of leucocytes. the number decreasing by 55.1% (P < 0.001). After 1 week there was a 15.4% decrease in number, not statistically significant. Three weeks after intoxication the number of lymphocytes did not differ from the
% 110-l
;
go-
:
-
”
z
70-
I” 5
50-
i
--XT.’ C
120 1
/
A
lw.
3w.
\
loo-
;
80-
> u 0 z
60-
0 I
-
control value. In females a significant reduction of 41.3% in the number of lymphocytes was observed only 1 week after intoxication (P < 0.001; Table 2, Fig. 8). The percentage of lymphocytes in the leucogram was lower in both males and females (Fig. 9). The number of monocytes in males fell in all determinations by 4.9, 76.1 (P < 0.001) and 46.1% (P < 0.02), respectively. In females the number of monocytes was 19.7% higher after 3 hr and after 1 and 3 weeks 10.3 and 25.0% lower. These changes were not statistically significant (Table 2, Fig. 8). The proportion of monocytes in the leucogram in males was slightly lower than in females (Fig. 9). The value of the leucocyte index increased by 301.6% in males 3 hr after Ekatin intoxication (P < 0.001) and by 71.5% in females (P < 0.05). One week after intoxication the value of this index was 88.7% higher in males (P < 0.001) and 241.8% higher in females (P < 0.01). Three weeks after intoxication the leucocyte index was 46.6% higher in males and 28.9% higher in females, but the changes were not statistically significant (Table 2, Fig. 5).
DISCUSSION
40-
20% v- C time after
1w. intoxication
3w.
Fig. 8. The number of lymphocytes and monocytes in the control (C) and after Ekatin intoxication.
Analysis of the results obtained justify the statement that the direction and intensity of haematological changes in quails intoxicated with Ekatin exhibit some degree of relation to the sex of the birds. In males changes in erythrocytes, that is, a considerable reduction in the number of erythrocytes, haemoglobin level and haematocrit, agree with the results of studies by a large number of authors using both the above and other organophosphorus compounds (Giermaziak, 1973; Patyra et al., 1974a;
46
K. GROMYSZ-KAXKOWSKA
Chaitov et al., 1975; Giermaziak et al., 1975; Giermaziak and Andrzejewski, 1976; Smigielska, 1977; Ali and Shakon, 1981; Gromysz-Kalkowska et al., 1981; Szubartowska, 1983). A decrease in the number of erythrocytes and the haemoglobin level, by only a small percentage, was observed in females, whereas the haematocrit value showed a distinct increase both 3 hr and 1 week after intoxication. Similar results were obtained by Kalinowska et al. (1972). These authors found a decrease in the number of erythrocytes in hens intoxicated with Dermafos, while the haemoglobin level and haematocrit value exhibited a slight tendency to increase. Sex differences in the reaction of erythrocytes have been found not only in the early period of intoxication, but also 3 weeks after intoxication. While the values of the parameters of this system were close to the control in the case of males, a distinct decrease in the number of erythrocytes was observed in females. Both erythroblastosis and reticulocytosis were observed in both sexes. It must be emphasized that a greater number of erythroblasts was observed in males during the initial period of intoxication, while a greater number of reticulocytes was found in females. The increase in the number of young elements in the blood was reflected in the mean corpuscular volume, which increased in males 3 hr and 1 week after intoxication, then returned to the control value. In females the greatest mean erythrocyte volume was observed 3 weeks after intoxication, that is, when the number of erythrocytes had been greatly reduced. The reduction in the number of erythrocytes in Ekatin intoxication may take place as a result of the occurrence of haemorrhages and clotting. As demonstrated by Ziolo et al. (1973) extensive haemorrhaging to the pulmonary parenchyma and also in the mucous membrane of the stomach occur in Ekatin intoxication and veiling clots form in the arterioles. Erythropaenia may also take place as a result of disintegration of blood cells due to the destructive action of this compound on their zona. This is suggested by the results of studies by Truchlinski and JabIonski (1976), who found cytolysis of human fibroblasts after adding Intration to the incubation fluid. Gromysz-Kalkowska et al. (198 1) have drawn attention to the haemolytic action of organophosphorus pesticides. The appearance in the peripheral blood of young forms of erythrocytes observed in the present studies, the increase in mean erythrocyte volume and also the increased osmotic resistance of blood cells suggest an intensified compensating activity of the haematopoietic system in response to the haemolytic action of Ekatin. Similar results were obtained by Smigielska (1977) in studies on rabbits intoxicated by foschlor and by Gromysz-Kalkowska et al. (1981, 1984) on quails intoxicated by trichlorfon and chlorfenvinfos. The effect of Ekatin on leucocytes in quails was a neutrophil leucocytosis with lymphopaenia and eosinopaenia. A similar reaction of this system has been observed in other species of animals intoxicated with different pesticides (Bieljajev, 1969; Vartic et al., 1972; Giermaziak, 1973; Patyra et al., 1974b; Chaitov
and E.
SZUBARTOWSKA
et al., 1975; Giermaziak et al., 1975; KlukowskaSiczek, 1980; Gromysz-Kalkowska et al., 1981, 1984; Szubartowska, 1983). It would seem that both leucocytosis and also changes in the leucogram should be attributed to the effect of Ekatin as a chemical stressor, since there have been reports that organophosphorus compounds activate the pituitary-adrenal axis (Vartic et al., 1972; Brzezinski, 1973; Wojcik, 1975). In leucocytes the intensity of change was also to some extent dependent on the animals’ sex. In males a considerable increase in the number of neutrophils and a decrease in the number of lymphocytes was observed as early as 3 hr after intoxication. Changes in the number of monocytes are interesting. In males the number of monocytes decreased 3 hr after intoxication and this decrease continued in successive determinations. In females the number of monocytes increased during the first determination and did not decrease until successive measurements were made, but even then to a far lesser degree than in males. The decrease in the number of monocytes observed in males in the present study points to a deficiency in the functioning of the reticula-endothelial system, even during the initial period of intoxication, and is confirmed by studies made by Kossakowski (1970) and Norkowski (1972), who recorded the inhibiting effect of organophosphorus compounds on this system. The monocytopaenia found agrees with the data given by Ziolo et al. (1973). In the histopathological studies made by these authors they did not find any defensive reaction round the foci of necrosis after Ekatin intoxication. Our suggestions on the inhibiting effect of Ekatin on the reticula-endothelial system also agree with the reduced level of phosphatases found by Kadziolka et al. (1976) in hens intoxicated by this preparation. The slight increase in the number of monocytes found in females 3 hr after intoxication in the present studies, followed by their decrease, leads to the assumption that Ekatin mobilizes the reticuloendothelial system in females only during the early period of intoxication and only to a slight degree. This assumption can be confirmed by the results of Giermaziak’s studies (1973) in which he found an increase in phosphatases in rabbits several hours after Intration intoxication, which is a pesticide in which the active substance, as in Ekatin, is thiometon. These data indicate that in males changes appear in the blood far earlier than in females. This would appear to be due to the more rapid rate of metabolising of this pesticide in males than in females. It is known that the metabolic rate of poisons is connected with the activity of certain microsomal enzymes of the liver (Przeidziecki, 1976) which in turn exhibits a dependence on the kind and level of sex hormones. It has been shown in studies on rats that the metabolic rate of poisons increases in females after administering testosterone, while in males administration of estradiol reduces metabolic activity (Dutkiewicz, 1974). Ekatin, like other thiophosphoric and dithiophosphoric pesticides which contain a sulphur atom, is metabolized to the corresponding oxygen analogues. The resulting compounds exhibit far greater
Blood changes in quails after Ekatin
toxicity than the initial compounds (Przeidziecki, 1976). When considering the differences in the time of appearance of changes in the blood in males and females it is aIso necessary to take into account the fact that in females not only desulphuration-a process conditioning the occurrence of toxic metabolites of Ekatin-but also detoxication of metabolites may take place more slowly than in males. As Benke and Murphy (1975) demonstrated in their studies the oxygen analogue paraoxon, a metabolite of parathion, undergoes dearylation to a far lesser degree in females than in males, and as already mentioned, Ekatin is also metabolized to the corresponding oxygen analogues. Thus single estimations of changes in the blood during intoxication by pesticides within a short time after their administration may Iead to the conclusion that females are less susceptible to intoxication. Wong and Terriere (1965) make this statement with respect to females intoxicated by such organochloride insecticides as aldrine and heptachlor, which also undergo rapid transformation into more toxic epoxides. It would seem that long-term examination of changes and their intensity is essential in order to assess the susceptibility of males and females. The results obtained in the present study show that Ekatin is a compound with marked toxic effects on quails. The different reaction of females from males to intoxication by this pesticide may be due to the different LD,, values of this compound to the two sexes, since it has been found that LD,, values of organophosphorus pesticides exhibit considerable differences depending on the sex of animals (Gaines, 1969; Rusiecki, 1973). To sum up it may be said that although Ekatin intoxication leads to profound changes in the blood of quails, they are capable of compensating for these changes, since 3 weeks after intoxication the majority of the haematological parameters examined return to the values of the control group. REFERENCES
Ali S. S. and Shakon A. R. (1981) Resistance to malathion toxicity in rabbits as revealed by studies on blood and liver. Puk. J. 2001. 13, 269-281. Benke G. M. and Murphy S. D. (1975) The influence of age on the toxicity and metabolism of methyl parathion and parathion in male and female rats. Toxicol. uppl. Pharmat. 31, 254269.
Bieliajev V. I. (1969) Toksiczeskoje diejstvie na ivotnych t~chlo~etafosa-3, chtorofosa i trolena. Veterinaria 45, 58-60.
Brzezinski J. (1973) Wplyw zatrucia fenitrotionem na zmiany poziomu adrenaliny i noradrenaliny w moczu szszurow. Bromat. Chem. Toksykol. 6, 83-86. Chaitov V. Ch., Bajmuradov T. B. and Guliamov A. (1975) Toksicznost Anthio dla km. Veterinaria 24, 85-87. Dutkiewicz T. (1974) Chemia Tok~yko~og~cz~u. PZWL, Warzawa. Frawley J. P., Hagan E. C. and Fitzhungh 0. G. (1952) A comparative pharmacological and toxicological study of organic phosphate-anticholinesterase compounds. J. Pharm. exp. Ther. 15&165.
Gaines T. B. (1969) Acute toxicity of pesticles. Toxic. uppl. Pharmac. 14, 5 1S-534. Giermaziak H. (1973) Badania nad wplywem zwiazkbw
47
fosforoorganicznych na ustroj na modelu Intrationu. Med. Pracy 24, 481-494. Giermaziak H. and Andrzejewski S. (1976) Wplyw ostrego zatrucia Intrationem na aktywnoSC niektorych hydrolaz osocza i granulocytow oraz liczbe leukocytow i erytrocytbw krwi obwodowej krohkow. Med. Praey 27, 242-249. Giermaziak H., Filipowicz Z., Gebicki L. and Babinski S. (1975) Badania nad toksyczynym dziataniem zwigzkow fosforoorganicznych stosowanych w rolnictwie. Med. Wiejska 10, 255-262. Gromysz-KaIkowska K. and Szubartowska E. (1983) Zmiany w morfologii krwi pod wplywem pestycydbw fosforoorganicznych na tie niektorych egzo- i endogennych czynnikow. Przeglqd Zeol. 27, 295-312. Gromysz- Kalkowska K., Szubartowska E. and Sulikowska J. (1981) Effect of the pesticide trichlorfon on the picture of peripheral blood in two breeds of quail. Folia biol. 29, 185-200. Gromysz-KaIkows~~ K., Szubartowska E., Trocewicz K. and Zielinska B. (1984) Obraz krwi obwodowej przepiorki japonskiej (Coturnix coturnir japonica) po jednorazowych dawkach chlorfenwinfosu. Med. Wer. 40, 147-151. Hirnlowa L., Pater W. and Cola A. (1965) Dwa przypadki ostrego zatrucia inhibitorami esterazy cholinowej, insektycydami Metasystox i Ekatin. Pal. Tyg. Lek. 20, 1167-1169. Kalinowska Z., Przaia F. and Zasdowski A. (1972) Toksykodynamika Dermafosu (tionofosforan O,Odwumetylu i O-2,4,5 trojchlorofenylu). I. Oznaczanie pozostalosci oraz zmiany hematologiczne u km i krolikow po jednorazowych dawkach Dermafosu produkcji IPO. III Sympozjum Toksykologiczne, Lodi. Kadziotka A., Rucinski T. and ZioIo T. (1976) Wplyw jednorazowego i powtarzanego zatrucia suble~Inymi dawkami Ekatinu na poziom lipidow i obraz morfologiczny watroby kurczat brojlerow. Ann. UMCS, set DD 31, 129-138. Klukowska-Siczek L. (1980) Wplyw chlorfenwinfosu na niektore wskainiki hematologiczne krwi u szczurbw. Bromar. Chem. Toksykof. 13, 145-149.
Kossakowski S. (1970) Wplyw zatrui’ zwiazkami fosforoorganicznymi na aktywnoSC ukladu siateczkowoHrodblonkowego. Med. We!. 26, 370-372. Kossakowski S. and Paryra S. (1973) Badania hematologiczne i elektroforetyczne biaiek surowicy krwi krolikow przy ostrych satruciach Foschlorem. Pesr_vcydy 1, 127-128.
Kurski B. (1975) Wplyw preparatu “Foschlor 20” na zachowanie sie bialek i niekt6rych enzymow w surowicy krblikow i owiec. Praca doktorska, Gtsztyn. Natt M. P. and Herrick C. A. (1952) A new blood diluent for counting the erythrocytes and leucocytes of the chicken. PO&. Sci. 3i, 7351738. Norkowski S. (1972) Badania nad toksvkodvnamika ostrych zatruC zwierzat. III Sympozjum “Toksykologiczne, tbdt. Oktaba W. and Niedokos E. (1980) Mafema~yko i Pod~rawy Staatystyki Maremalycznej. PWN, Warszawa. Patyra S., Kossakowski S. and Stryczek J. (1974a) Badania hematologiczne u bydla rasy nizinnej czamo-biatej po naskbrnym stosowaniu Neguvonu. Med. Wef. 30, 10-13. Patyra S., Kurek A. and Kossakowski S. (1974b) Badania hematologiczne u p&w po ostrym zatruciu Foschlorem. Med. Wet. 30, 478481.
Pinkiewicz E. (1971) Podstawowe Budaniu ~aboraforyj~e w Chorobach Zwierzaf. PWRiL. Warzawa. Przeidziecki Z. (1976) Biologiczne Przemiany Substancji Toksyczrlych. PWN, Warszawa. Rusiecki W. (1973) Toksykologia &-odkdw Ochrony RoSlin. PZWL, Warszawa. Smigielska J. (1977) WpIyw preparatu Foschfor R-20 na
48
K. GROMYSZ-KAZKOWSKA and E. SZUBARTOWSKA
zawartosc wolnych nukleotydow w krwinkach czerwonych i waksiniki hemogramu u krblikow. Med. Wet. 32, 534537. Szubartowska E. (1983) Peripheral blood picture in rabbits poisoned by the pesticide trichlorfon. Folia biol. Krakdw 31, 407418. Truchlinski J. and Jabionski L. (1976) Badania nad wpiywem wybranych pestycydow fosforoorganicznych na funkcje komorek fibroblastow ludzkich w hodowli in vitro. Med. DoSw. Mikrob. 28, 383-388. Vartic N., Jacob-Giurgea R. and Ivascu V. (1972) Schwankungen einiger Stoffwechselindikatoren beim Huhn nach Verabreichung von Bubulin. Arch. Exp. Vet. Med. 26. 207-2 14.
Wintrobe M. M. (1956) Clinical Hematology. Lea and Febiger, Philadelphia. Wong D. T. and Terriere L. C. (1965) Epoxidation of aldrin, isodrin and heptachlor by liver microsomes. Biochem. Pharmac. 14, 375. Wojcik J. (1975) Wplyw foschloru w wodzie do picia na narzady wewnetrzne trzech kolejnych pokolen szczurbw. Roczn. PZH 26, 383-391. Ziolo T., Rucinski T. and Kgdziolka A. (1973) Wplyw wielokrotnego podostrego zatrucia Ekatinem (Sandoz) na wybrane narzady wewnqtrzne szczurow. 1. Obraz zmian histologicznych. Ann. CJMCS, set DD 28, 199-206.
HAEMATOLOGICAL PHARAOH QUAILS AFTER K. Department
of Animal
03064492186 $3.00 + 0.00 Pergamon Journals Ltd
CHANGES IN MALE AND FEMALE (COTURNIX COTURNIX PHARAOH) EKATIN INTOXICATION
GROMYSZ-KAEKOWSKA Physiology,
and E.
Institute of Biology, Lublin, Poland
SZUBARTOWSKA Maria Curie-Sklodowska
University,
(Receiwd 16 December 1986) The purpose of this study was to determine changes in the blood of pharaoh quails after Ekatin intoxication, to define the duration of disturbances caused by intoxication and to examine possible
Abstract-l.
sex differences in the birds’ reaction to intoxication. 2. It was found that Ekatin reduced the number of erythrocytes, haemoglobin level and haematocrit value, increased erythroblast and reticulocyte numbers and increased the osmotic resistance of blood cells. 3. It was shown that this pesticide caused neutrophilic leucocytosis with lymphopaenia and eosinopaenia. 4. In males changes in the blood appeared far earlier than in females and they underwent compensation earlier, that is, 3 weeks after intoxication the majority of the haemotological parameters reached values similar to the control.
INTRODUCTION
We have found no data in the literature dealing with blood parameters after intoxication by organophosphorus compounds depending on the sex of animals (Giermaziak, 1973; Kossakowski and Patyra, 1973; Kurski, 1975; Smigielska, 1977; Ali and Shakon, 1981; Gromysz-Kalkowska et al., 1981; Szubartowska, 1983). It is however possible to state, on the basis of examination of LD,, values for these compounds, that females are more susceptible to the effect of a considerable number of organophosphorus compounds (Frawley et al., 1952; Gaines, 1969; Rusiecki, 1973; Benke and Murphy, 1975). These data agree with the haematological studies made by Gromysz-Kalkowska and Szubartowska (1983), who report the absence of sex differences in the reaction of Rana temporaria to small amounts of trichlorfon, and a greater response of females than males to a dose of 300 mg/kg body wt. In view of the data presented and the fact that Ekatin causes considerable changes in blood morphology (Hirnlowa et al., 1965; Giermaziak, 1973; Giermaziak and Andrzejewski, 1976) and the fact that relatively little attention has been given to studies of these changes, it appeared useful to trace the effect of this preparation on the peripheral blood of this quail. Another question taken into consideration in this study was the definition of the duration of disturbances caused by intoxication, while a further problem was the investigation of possible sex differences in the birds’ reaction to Ekatin intoxication. MATERIALS
The experiments were carried out on 18 male and 18 female pharaoh quails. The birds were 4 months old, weighed from 180 to 2 10 g, and were kept on a uniform diet and under the same maintenance conditions. The birds were divided at random into two groups: control (eight quails) and experimental (10 quails). The experimental groups were given water solvent Ekatin in doses of 50mg/kg body wt. The individuals in the control groups were given water only at the same time as the experimental groups. Blood was taken from the brachial vein and the number of erythrocytes (RBC) and leucocytes (WBC) was determined by the chamber method in Natt-Herrick diluting fluid (Natt and Herrick, 1952) haemoglobin level (Hb) by the cyanmethaemoglobin method and haematocrit value (Ht) by the micromethod. Mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were calculated in accordance with the models given by Wintrobe (1956). The percentage composition of leucocytes was calculated in preparations stained by the Pappenheim method. The number of erythroblasts per 1000 erythrocytes was determined in the same preparations, The number of reticulocytes per 1000 erythrocytes was determined in preparations stained in uiuo with brilliant cresyl blue (Pinkiewicz, 1971). The first determination was carried out 3 hr after administering Ekatin or water, and then after the 1st and 3rd weeks of the experiment. The results obtained were analysed statistically using Student’s f-test for independent data. A value of P < 0.05 was taken as statistically significant (Oktaba and Niedokos, 1980).
RESULTS The
studies
causes
certain
quail,
AND METHODS
place
while
showed changes
the direction
that in
administration the
in which
erythrocytes
of Ekatin of
these changes
the take
and their intensity point to some degree of dependence on the birds’ sex. The number of erythrocytes in males fell by 23.2% 3 hr after intoxication (P < 0.05). After 1 week this number was still 20.8% lower than the control value
Ekatin, the pesticide used for these studies, was obtained from Sandoz AG, Switzerland. It contains 25% of the active substance thiometon-O,O-dimethyldithiophosphate 2-(ethylothio)ethyl and is classified as belonging to toxicity class II. 41
42
K.
GROMYSZ-KMKOWSKA
Table
Parameters
Sex
I. Ervtbrocvte Control (X f SE)
and E. SZUBARTOWSKA
values in Doisoninp. bv Ekatin Time after intoxication of Ekatin I week 3 hr (R ? SE) (R + SE)
3 weeks (R k SE)
Erythrocytes (mln/mm’)
;
4.789kO.170 4.135*0.112
3.680 f 0.451’ 4.030 f 0.238
3.792 f 0.297t 4.082 i 0.263
4.741 & 0.423 3.540 * 0.149f
Haemoglobin (%)
$ 0*
16.36 20.461 12.72 + 0.376
12.27 f 0.26% 12.63 _+0.296
13.70 f 0.3805 12.29 f 0.294
15.17 kO.640 12.23 i 0.273
Haematocrit (%)
c? F
50.81 i I .375 43.00 i 0.924
41.60* 1.6075 47.90 f I .277f
42.30 f 1.37% 48. IO k 1.293f
52.80 f 1.097 44.50 & 0.997
MCV (p3)
c? 0
106.43 + 2.026 104.15 + I.417
123.04 f 6.850 121.85 k6.197,
116.92 & 8.913 120.88 f 7.480
105.97 + 5.558 128.59 i 8.1 IOf
MCH
s E
34.23 ? 0.632 30.79 + 0.698
37.30 & 4.222 32.27 + I .924
37.93 f 2.925 31.18 f 2.008
35.16f4.517 35.07 + 1.640’
(%)
s
32.07 i 0.447 29.55 f 0.462
29.90 f I.414 26.48 f 0.6831
32.54 & 1.054 25.77 k 1.027f
28.76 + 1.278* 27.63 + 0.983
Erythroblasts (%d
d 0
12.12 * 0.680 14.50 i 0.965
18.90 + 1.3705 14.90 k 0.876
23.70 & 1.76% 2l.lOk 1.346f
16.40 + 1.301§ 21.30+ I.3982
Reticulocytes (%)
$ 0
94.37 + 3.918 84.60 + 2.209
188.50 rf: 6.9806 192.60 + 3.6485
172.40 + 2.43% 164.70 i 4.43@
204.50 f 6.8039
(pg)
MCHC
*P tP $P $P
< < < <
0.05. 0.02. 0.01. 0.001.
(P < 0.02). Three weeks after Ekatin intoxication this parameter was observed to return to the control level. In the case of females the number of erythrocytes was maintained at the control level both after 3 h and 1 week after intoxication and it was not until after 3 weeks after intoxication that it was found to fall by 14.4% (P < 0.01; Table 1, Fig. 1). The haematocrit and males fell by 18.1% after 3 hr and by 16.8% 1 week after Ekatin intoxication (in both cases P < O.OOl), but after 3 weeks approached the control value. In females an increase of 11.4% in haematocrit value took place after 3 hr and 11.9% after 1 week (in both cases p < 0.01). After 3 weeks the haematocrit value, as in the males, returned to the control level (Table 1, Fig. 1). In males and females the mean corpuscular volume increased by 15.6 and 17.0%, respectively, 3 hr after intoxication, although these changes were not statistically significant. The next determination in males showed that the value of this index was 9.9% higher, but after 3 weeks it returned to the control value. In the case of females the mean corpuscular volume was 16.1% higher 1 week and 23.5% higher 3 weeks after Ekatin intoxication (P < 0.05, 0.02; Table 1, Fig. 1). The haemoglobin level in males fell distinctly in successive determinations by 25.0, 16.3 (in both cases P < 0.001) and 7.3% (NS). In females the haemoglobin level was maintained at the control level for the whole of the experimental period (Table 1, Fig. 2). Mean corpuscular haemoglobin in males increased 3 hr and 1 week after intoxication by 8.9 and 10.8%, respectively, but after 3 weeks it was only 2.7% higher than the control level. In females the value of this index increased in all determinations by 4.8, 1.3 and 13.9%, respectively (P < 0.05; Table 1, Fig. 2). In the males the mean corpuscular haemoglobin concentration fell in successive determinations by 6.8, 1.5 and 10.3%. In females the value of this index is 10.4% lower 3 hr, 12.8% lower 1 week (in both cases P c 0.01) and 6.5% lower 3 weeks after intoxication (Table 1, Fig. 2).
During Ekatin intoxication the erythroblast content in males increased in all determinations by 55.9, 95.5 and 35.5%, respectively. These changes were statistically significant with P < 0.001; 0.001; 0.02. In females a significant increase in erythroblast content of 45.5% was observed until 1 week after intoxication, and an increase of 46.9% 3 weeks after intoxication (in both cases P < 0.01; Table 1, Fig. 3).
The reticulocyte content in males was 99.7% higher than the control 3 hr, 82.7% 1 week and
..
-1
C
3h.
;,.
3w.
oftcr intoxication Fig. 1. The number of erythrocytes (RBC), haematocrit value (Ht) and mean corpuscular volume (MCV) in the control (C) and after Ekatin intoxication. tinn
Blood changes in quails after Ekatin
1
C
3h.
3w.
1 W.
C
3h.
1W.
3w.
260
PQ
-C
3h.
3w.
IW.
time after intoxication Fig. 2. The level of haemoglobin (Hb), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) in the control (C) and after Ekatin intoxication.
37.7% 3 weeks after intoxication (with P < 0.001; 0.001; 0.01, respectively). In females the increase in reticulocyte content was higher than in males, 127.6, 94.7 and 141.7%, respectively (in all cases P < 0.001; Table I, Fig. 3).
The increase in the osmotic resistance of erythrocytes was accompanied by an increased content of the young forms of blood cells. In males maximum resistance was observed 3 hr after intoxication, while
C
3h.
Iw.
3w.
time after intoxication Fig. 3. The number of erythroblasts and reticulocytes in the control (C) and after Ekatin intoxication.
after 1 and 3 weeks it was observed to decrease. In females the osmotic resistance of blood cells increased in successive determinations, reaching a maximum value 3 weeks after intoxication (Fig. 4). Changes were observed in leucocytes of quails intoxicated by Ekatin which, as in the case of erythrocytes, exhibited a certain relation to the sex of the birds. The number of leucocytes increased by 24.5% in males and 21.3% in females 3 hr after intoxication.
.I.
‘I.
‘I.
100
100
100
80
80
00
60
60
60
40
40
40
ZJ 20 5 “I 0
20
20
0
0
v)
w
2 I
100 80 60 40
20 0
Fig. 4. The osmotic resistance of erythrocytes in poisoning by Ekatin
.I. NaCl
K.
GROMYSZ-KAEKOWSKAand
E.
SZIJBARTOWSKA
Table 2. Leucocytevaluesin poisoningby Ekatin Time afterintoxication of Ekatin 3 hr I week (R*SE) (X +SE)
3 weeks (B+SE)
Sex
Control (r *SE)
Leucocytes(1000/mm~)
c? 0
16.404k 0.682 16.748iO.318
20.419& 1.843 20.320f 1.830
18.064F0.767 21.287k 0.4335
18.554k I.107 16.509kO.767
Neutrophils (lOOO/mm')
s ?
7.481f 0.898 7.108kO.216
16.196+ 2.35@ 11.439f l.OOO$
ll.310?0.647f 15.589f 0.3405
10.333* 0.943' 8.381? 0.520
Eosinophils (lOOO/mm')
s 0
0.536f 0.114 0.524+0.121
0.061f 0.0315 0.156+0.046$
0.197+0.083* 0.149kO.O57$
0.155+ 0.067f 0.052+ 0.027s
Basophils(lOOO/mm')
$ 0
0.090+ 0.073 0.107f 0.056
0.04020.027 0.073kO.040
0.018kO.018 0.040f 0.027
0.053kO.036 0.030*0.020
Lymphocytes (1000/mm3)
$ 0
7.501f 0.375 8.310f0.207
3.369f 0.2625 7.816kO.865
6.350kO.538 4.881kO.2735
7.586kO.907 7.522kO.572
Monocytes (1000/mm')
d 0
0.792k 0.112 0.699iO.138
0.753f 0.166 0.837kO.167
0.189+0.0575 0.62750.088
0.427~0.086t 0.524+O.l40
WBCI
6 P
0.996f 0.095 0.869+ 0.038
4.000f 0.3435 1.490+0.233*
l.880+0.177~ 2.970+ 0.237i
1.460+0.187 1.120+0.113
Parameters
'P < 0.05. tP <0.02. $P < 0.01. §P
At the next determination the increase in the number of leucocytes was 10.1% in males and 27.1% in females (P < 0.001). After 3 weeks the leucocyte level in males was still 13.1% higher than in the control, whereas in females it fell to slightly below this value (Table 2, Fig. 5). In males the number of neutrophils after Ekatin intoxication increased in successive determinations by 116.5, 51.2 and 38.1%, respectively (with
C
3h.
C
lw. 3h. time after intoxication
1w.
3 w.
3 w.
Fig. 5. The number of leucocytes (WBC) and white blood cells index value (WBCI) in the control (C) and after Ekatin intoxication.
P c 0.001; 0.01; 0.05). In females the increase in the number of neutrophils was 60.9% after 3 hr, 119.3% after 1 week and 17.9% after 3 weeks (P < 0.01; 0.001; NS, respectively; Table 2, Fig. 6). The proportion of neutrophils in the leucogram increased in all determinations in both males and females (Fig. 9). The number of eosinophils in males fell by 88.6% after 3 hr, by 63.3% after 1 week and by 71% after 3 weeks (P c 0.001; 0.05; 0.01). In females the number of eosinophils fell in successive determinations by 70.2, 71.6 and 90.1%, respectively (P < 0.01; 0.01 and 0.001, respectively; Table 2, Fig. 7). The percentage of eosinophils was lower in both sexes after Ekatin intoxication (Fig. 9). The number of basophils in males fell in successive determinations by 55.6, 80.0 and 41.1%, respectively, and in females by 31.8, 62.6 and 72.0% (Table 2, Fig. 7), but these changes were not statistically significant. No significant changes were found in the percentage of basophils in the two experimental groups (Fig. 9). A significant reduction in the number of lymphocytes was found in males only 3 hr after intoxication,
7-
C
iW. jh. .’ time after intoxkotion
4,
Fig. 6. The number of neutrophils in the control (C) and after Ekatin intoxication.
45
Blood changes in quails after Ekatin
80.
C
IW.
3h.
3w
80,
40,
80v) =! I
60-
-
4 2
40-
20,
0 20s ew
-
c
3w.
time after intoxication
C
lw
3h.
3w.
Fig. 7. The number of eosinophils and basophils in the control (C) and after Ekatin intoxication.
m
neutrophils
0
eosinophils
m
basophils
m
limphocytes
m
monocytes
Fig. 9. The percentage composition of leucocytes. the number decreasing by 55.1% (P < 0.001). After 1 week there was a 15.4% decrease in number, not statistically significant. Three weeks after intoxication the number of lymphocytes did not differ from the
% 110-l
;
go-
:
-
”
z
70-
I” 5
50-
i
--XT.’ C
120 1
/
A
lw.
3w.
\
loo-
;
80-
> u 0 z
60-
0 I
-
control value. In females a significant reduction of 41.3% in the number of lymphocytes was observed only 1 week after intoxication (P < 0.001; Table 2, Fig. 8). The percentage of lymphocytes in the leucogram was lower in both males and females (Fig. 9). The number of monocytes in males fell in all determinations by 4.9, 76.1 (P < 0.001) and 46.1% (P < 0.02), respectively. In females the number of monocytes was 19.7% higher after 3 hr and after 1 and 3 weeks 10.3 and 25.0% lower. These changes were not statistically significant (Table 2, Fig. 8). The proportion of monocytes in the leucogram in males was slightly lower than in females (Fig. 9). The value of the leucocyte index increased by 301.6% in males 3 hr after Ekatin intoxication (P < 0.001) and by 71.5% in females (P < 0.05). One week after intoxication the value of this index was 88.7% higher in males (P < 0.001) and 241.8% higher in females (P < 0.01). Three weeks after intoxication the leucocyte index was 46.6% higher in males and 28.9% higher in females, but the changes were not statistically significant (Table 2, Fig. 5).
DISCUSSION
40-
20% v- C time after
1w. intoxication
3w.
Fig. 8. The number of lymphocytes and monocytes in the control (C) and after Ekatin intoxication.
Analysis of the results obtained justify the statement that the direction and intensity of haematological changes in quails intoxicated with Ekatin exhibit some degree of relation to the sex of the birds. In males changes in erythrocytes, that is, a considerable reduction in the number of erythrocytes, haemoglobin level and haematocrit, agree with the results of studies by a large number of authors using both the above and other organophosphorus compounds (Giermaziak, 1973; Patyra et al., 1974a;
46
K. GROMYSZ-KAXKOWSKA
Chaitov et al., 1975; Giermaziak et al., 1975; Giermaziak and Andrzejewski, 1976; Smigielska, 1977; Ali and Shakon, 1981; Gromysz-Kalkowska et al., 1981; Szubartowska, 1983). A decrease in the number of erythrocytes and the haemoglobin level, by only a small percentage, was observed in females, whereas the haematocrit value showed a distinct increase both 3 hr and 1 week after intoxication. Similar results were obtained by Kalinowska et al. (1972). These authors found a decrease in the number of erythrocytes in hens intoxicated with Dermafos, while the haemoglobin level and haematocrit value exhibited a slight tendency to increase. Sex differences in the reaction of erythrocytes have been found not only in the early period of intoxication, but also 3 weeks after intoxication. While the values of the parameters of this system were close to the control in the case of males, a distinct decrease in the number of erythrocytes was observed in females. Both erythroblastosis and reticulocytosis were observed in both sexes. It must be emphasized that a greater number of erythroblasts was observed in males during the initial period of intoxication, while a greater number of reticulocytes was found in females. The increase in the number of young elements in the blood was reflected in the mean corpuscular volume, which increased in males 3 hr and 1 week after intoxication, then returned to the control value. In females the greatest mean erythrocyte volume was observed 3 weeks after intoxication, that is, when the number of erythrocytes had been greatly reduced. The reduction in the number of erythrocytes in Ekatin intoxication may take place as a result of the occurrence of haemorrhages and clotting. As demonstrated by Ziolo et al. (1973) extensive haemorrhaging to the pulmonary parenchyma and also in the mucous membrane of the stomach occur in Ekatin intoxication and veiling clots form in the arterioles. Erythropaenia may also take place as a result of disintegration of blood cells due to the destructive action of this compound on their zona. This is suggested by the results of studies by Truchlinski and JabIonski (1976), who found cytolysis of human fibroblasts after adding Intration to the incubation fluid. Gromysz-Kalkowska et al. (198 1) have drawn attention to the haemolytic action of organophosphorus pesticides. The appearance in the peripheral blood of young forms of erythrocytes observed in the present studies, the increase in mean erythrocyte volume and also the increased osmotic resistance of blood cells suggest an intensified compensating activity of the haematopoietic system in response to the haemolytic action of Ekatin. Similar results were obtained by Smigielska (1977) in studies on rabbits intoxicated by foschlor and by Gromysz-Kalkowska et al. (1981, 1984) on quails intoxicated by trichlorfon and chlorfenvinfos. The effect of Ekatin on leucocytes in quails was a neutrophil leucocytosis with lymphopaenia and eosinopaenia. A similar reaction of this system has been observed in other species of animals intoxicated with different pesticides (Bieljajev, 1969; Vartic et al., 1972; Giermaziak, 1973; Patyra et al., 1974b; Chaitov
and E.
SZUBARTOWSKA
et al., 1975; Giermaziak et al., 1975; KlukowskaSiczek, 1980; Gromysz-Kalkowska et al., 1981, 1984; Szubartowska, 1983). It would seem that both leucocytosis and also changes in the leucogram should be attributed to the effect of Ekatin as a chemical stressor, since there have been reports that organophosphorus compounds activate the pituitary-adrenal axis (Vartic et al., 1972; Brzezinski, 1973; Wojcik, 1975). In leucocytes the intensity of change was also to some extent dependent on the animals’ sex. In males a considerable increase in the number of neutrophils and a decrease in the number of lymphocytes was observed as early as 3 hr after intoxication. Changes in the number of monocytes are interesting. In males the number of monocytes decreased 3 hr after intoxication and this decrease continued in successive determinations. In females the number of monocytes increased during the first determination and did not decrease until successive measurements were made, but even then to a far lesser degree than in males. The decrease in the number of monocytes observed in males in the present study points to a deficiency in the functioning of the reticula-endothelial system, even during the initial period of intoxication, and is confirmed by studies made by Kossakowski (1970) and Norkowski (1972), who recorded the inhibiting effect of organophosphorus compounds on this system. The monocytopaenia found agrees with the data given by Ziolo et al. (1973). In the histopathological studies made by these authors they did not find any defensive reaction round the foci of necrosis after Ekatin intoxication. Our suggestions on the inhibiting effect of Ekatin on the reticula-endothelial system also agree with the reduced level of phosphatases found by Kadziolka et al. (1976) in hens intoxicated by this preparation. The slight increase in the number of monocytes found in females 3 hr after intoxication in the present studies, followed by their decrease, leads to the assumption that Ekatin mobilizes the reticuloendothelial system in females only during the early period of intoxication and only to a slight degree. This assumption can be confirmed by the results of Giermaziak’s studies (1973) in which he found an increase in phosphatases in rabbits several hours after Intration intoxication, which is a pesticide in which the active substance, as in Ekatin, is thiometon. These data indicate that in males changes appear in the blood far earlier than in females. This would appear to be due to the more rapid rate of metabolising of this pesticide in males than in females. It is known that the metabolic rate of poisons is connected with the activity of certain microsomal enzymes of the liver (Przeidziecki, 1976) which in turn exhibits a dependence on the kind and level of sex hormones. It has been shown in studies on rats that the metabolic rate of poisons increases in females after administering testosterone, while in males administration of estradiol reduces metabolic activity (Dutkiewicz, 1974). Ekatin, like other thiophosphoric and dithiophosphoric pesticides which contain a sulphur atom, is metabolized to the corresponding oxygen analogues. The resulting compounds exhibit far greater
Blood changes in quails after Ekatin
toxicity than the initial compounds (Przeidziecki, 1976). When considering the differences in the time of appearance of changes in the blood in males and females it is aIso necessary to take into account the fact that in females not only desulphuration-a process conditioning the occurrence of toxic metabolites of Ekatin-but also detoxication of metabolites may take place more slowly than in males. As Benke and Murphy (1975) demonstrated in their studies the oxygen analogue paraoxon, a metabolite of parathion, undergoes dearylation to a far lesser degree in females than in males, and as already mentioned, Ekatin is also metabolized to the corresponding oxygen analogues. Thus single estimations of changes in the blood during intoxication by pesticides within a short time after their administration may Iead to the conclusion that females are less susceptible to intoxication. Wong and Terriere (1965) make this statement with respect to females intoxicated by such organochloride insecticides as aldrine and heptachlor, which also undergo rapid transformation into more toxic epoxides. It would seem that long-term examination of changes and their intensity is essential in order to assess the susceptibility of males and females. The results obtained in the present study show that Ekatin is a compound with marked toxic effects on quails. The different reaction of females from males to intoxication by this pesticide may be due to the different LD,, values of this compound to the two sexes, since it has been found that LD,, values of organophosphorus pesticides exhibit considerable differences depending on the sex of animals (Gaines, 1969; Rusiecki, 1973). To sum up it may be said that although Ekatin intoxication leads to profound changes in the blood of quails, they are capable of compensating for these changes, since 3 weeks after intoxication the majority of the haematological parameters examined return to the values of the control group. REFERENCES
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K. GROMYSZ-KAZKOWSKA and E. SZUBARTOWSKA
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