Chick and duck embryos in the evaluation of pesticide toxicity

Chick and duck embryos in the evaluation of pesticide toxicity

TOXICOLOGY AND APPLIED Chick PHARMACOLOGY 13,1-15 (1968) and Duck Embryos in the Evaluation of Pesticide Toxicity K. S. KHERA AND D. A. LYON F...

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TOXICOLOGY

AND

APPLIED

Chick

PHARMACOLOGY

13,1-15

(1968)

and Duck Embryos in the Evaluation of Pesticide Toxicity K. S. KHERA AND D. A. LYON

Food and Drug

Directorate,

Department Ottawa, Ontario,

Receiced

December

of National Canada

Health

and Welfare,

II,1967

Chick and Duck Embryos in the Evaluation of Pesticide Toxicity. KHERA, K. S. and LYON, D. A. (1968). Toxicol. Appl. f%armacol. 13, l-15. Severalembryonalagesof chick and duck species wereevaluatedfor toxicity quantitation by injectinginto the yolk sacdosesof 1mg or lessof the following pesticides:Phosdrin, DDVP, Ruelene,dimefox, diazinon, parathion, malathion, Trithion, dimethoate,ethion, carbaryl, Bayer 37344,and Bayer 39007.Chick eggsinjected on incubation days 0, 4, and 7 and duck eggs injectedon days0,4,7, and 10appearedto beunsuitablefor toxicity studies. This wasdueto largevariation amongreplicationsor lack of doseresponse relationship,or low sensitivityto the specificlethal effect. At mid-incubation of both the species(10 days in chick and 13 days in duck embryos),there was a marked mortality responseoccurring primarily within 24 hours of injection; in many instancesanother period of mortality among the survivors was manifest near the hatching. The two avian speciesinjected at mid-incubationagearecapableof providing usefuldata for theassessment of pesticidetoxicity. There was a suggestionthat duck embryos provide lessvariable resultsthan chick embryos.

The chick embryo has been used for toxicity studies of a large number of chemicals. No standard technique has been recognized and methods used have varied, particularly the stage of embryonal development when the test chemical is injected. Injection on day 0 (McLaughlin et al., 1963), days 4 and 8 (Karnofsky et al., 1950; Ridgway and Karnofsky, 1952; Franciscis and Landauer, 1959), or less commonly, day 11 (Smith and Chapman, 1963; Milner and Finkelstein, 1966) day 13 (Halverson et al., 1965), and day 14 (Hadani and Egyed, 1967), have all been employed. The length of the post injection observation period also varied greatly in the above cited studies. Data from studies on the duck embryo are more limited (Limborgh and Meenen, 1963 ; Khera et a/., 1965). The most commonly used technique is to inject the chick egg prior to incubation, the toxicity being assessed on the basis of numbers of birds hatched (McLaughlin et al., 1964; Dunachie and Fletcher, 1966). The reliability of this technique has been questioned (Clegg, 1964; Walker, 1967). The present paper describes an investigation of the suitability for pesticide toxicity assessment of different embryonal ages of the chick and duck species. It involved almost 20,000 eggs. The conventional LD50 values were not sought in this study. The approach was based on comparison of percent mortalities accruing from the same @ 1968 by Academic

Press Inc.

1

2

K. S. KHERA

AND

D. A. LYON

three doses of every pesticide. The lowest of the three doses produced mortalities almost indistinguishable from the control mortalities; the highest dose produced mortalities from 0 to 100 ‘A depending upon the pesticide and embryonal age. MATERIALS

White Leghorn chick and Pekin duck eggs obtained from a commercial source,’ were carefully screened for defective eggs prior to use. The chick and duck eggs had average weights of approximately 55.4 and 82.5 g, respectively. Eggs, order of pesticide injection, and location within the incubator were almost completely randomized. Except for day 0 (preincubation injection), only viable eggs were injected. A Jamesway single-stage incubator-hatcher Model 252B was used and the temperature-humidity schedule recommended by the manufacturer was followed. The incubator was washed and disinfected after every hatch according to routine hatchery techniques. Chick eggs were injected on days 0,4,7, or 10, and duck eggs on days 0,4,7,10, or 13. Doses of 0, 10 pg, 100 pg and 1 mg/egg, dissolved in 0.1 ml of freshly prepared sterile propylene glycol, were injected into the yolk sac. Eggs were first candled on the day after injection (except for O-day injected eggs, which were candled on day 5 or 6 of incubation), and every l-4 days thereafter, until 2 days prior to anticipated hatching (day 21 for chick and day 28 for duck), when they were transferred to hatching trays. Two days after the anticipated hatching date, the numbers of hatched birds and of unhatched live embryos were counted and the sum of the two numbers was called “survival.” The experiments were then terminated. Pesticides

The following pesticides (analytical grade) were injected in this study: (1) Phosdrin, methyl 3-hydroxy-a-crotonate, dimethyl phosphate; (2) DDVP, 2,2-dichlorovinyl dimethyl phosphate; (3) Ruelene, O-4-tert-butyl-2-chlorophenyl O-methyl methylphosphoramidate; (4) dimefox, N,N,N’,N’-tetramethyl phosphorodiamidic fluoride; (5) diazinon, O,O-diethyl 0-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate; (6) parathion, O,O-diethyl 0-p-nitrophenyl phosphorothioate; (7) malathion, diethyl mercaptosuccinate, S-ester with O,O-dimethyl phosphorodithioate; (8) Trithion, ,S-[(p-chlorophenylthio)methyl] O,O-diethyl phosphorodithioate; (9) dimethoate, O,O-dimethyl S-(methylcarbamoylmethyl) phosphorodithioate; (10) ethion, O,O,O’, O’-tetraethyl S,S’-methylene biphosphorodithioate; (11) carbaryl, 1-naphthyl methylcarbamate; (12) Bayer 37344, 4-(methylthio) 3,5-xylyl methylcarbamate; (13) Bayer 39007, o-isopropoxyphenyl methylcarbamate. Terminology and Notation Injection age: Embryonal age in days at the time of injection. Mid-incubation injection ages are the lo- and 13-day injection ages in chick and duck, respectively. Periods: The postinjection incubation life was divided into three periods for analysis

purposes: PI-the initial postinjection period of 5-6 days for injection age 0 and of one postinjection day for the other injection ages; Pz-the interval succeeding PI and l Chick eggs from Pioneer Hybrid Corn Co., Chatham, Ontario, and duck eggs from Brome Lake Limited, Knowlton, Quebec.

CHICK

AND

DUCK

EMBRYOS

3

extending until the transference of eggs to the hatching trays i.e., to day 19 for chick eggs and day 26 for duck eggs; P,-the hatching period from 19 to 23 days for chick embryos and 26 to 30 days for duck embryos. These periods varied slightly in a few replications, as shown in Fig. 1. The sum of these three periods, Pi + P, -t Ps is denoted as P,. Group: All eggs that were injected with one of the three doses of one of the 13 pesticides or with propylene glycol, at one injection age, in one trial. A pesticide-injected group consisted of 18-30 eggs; control groups (sizes are shown in Fig. 1) were sometimes larger. Replication or trial: A single incubation experiment, involving one species of egg, usually involving all three doses of pesticide and controls, and involving some or all of the pesticides and injection ages. SubrepZication: In the last trial of the mid-incubation injections for both species, some subdivision of eggs and methods was made in order to study the effects of local variations such as might arise from technique, spatial arrangement in the incubator, etc. For chicks, this last trial consisted of three simultaneously incubated lots of eggs, each lot having been injected with separately prepared pesticide solutions and consisting of 10 eggs per group. For ducks, this subreplication consisted merely of a stratification into two sections within the incubator, each section involving lo-egg groups. Szmioal (p): The proportion of the total number of injected embryos that remained alive after a period under investigation. Adjusted surciral: The test-group survival divided by the control survival. Survivals and adjusted survivals may be multiplied by 100 for percentage reference, and “mortality” is the complement of survival (l-p, etc.). Statistical

A4ethods

The analysis consisted entirely of determining which groups of eggs manifested pesticide toxicity. The index chosen for toxicity was embryo mortality. Three criteria were used for evaluating specific toxic response: (1) the comparison of survivals in pesticide-injected and control eggs, (2) the reproducibility of the results, and (3) the existence of a monotonic dose-response relationship. Although all data tabulated in this paper are in the form of “adjusted survivals,” all analyses were carried out using the raw survivals (p) form. The log (p) and arcsin (p) (Freeman and Tukey, 1950) transformations were also studied to some degree, but modified the results very little. Analyses of variance were carried out, employing techniques suggested by Cochran (1943) and Gabriel (1963). Pesticide x replication interactions were used as error terms. The IO-pg dose effects were usually so small that augmenting the control data by this lowest-dose data provided more powerful statistical tests of the 1-mg dose effects than was provided by using the control data alone. Reproducibility was evaluated by comparing the magnitudes of the replication interaction terms and the expected binomial sampling variations. It was also studied to a degree by means of Spearman rank correlation tests (Siegel, 1956). A nonparametric method, involving the notion of counting how often the two or three dose-survivals formed monotonic decreasing sequences, was developed in order to evaluate the reality of dose-response relationships. Details of the method will be published elsewhere.

4

K. S. KHERA AND D. A. LYON

RESULTS Survival in Control Groups

Survivals of control groups after PI, PI + Pz, and P, are graphed in Fig. 1. The ordering of the replications is the same for the controls in Fig. 1 and for the pesticides in Tables 1 and 2. Figure 1 also shows the total number of propylene glycol-injected control eggs in each trial. 90 80

22 73 48

23, 50 ,

NO. OF EGGS ,NJEC TED

I!?[-

20

CHICK

NJOT

DONE

I

;1

I

I

I 7

32 2044 2

IO 32 14 40

I 23 23 20 189 423 23

w

:

DUCK

2Ob-J

0

-

4 EMBRYONAL

-

I 7

AGES

AT

I IO INJECTION

1 tb”nYS,

FIG. 1. Survival of chick and duck control eggs after PI (0), PI + P2 (x), and PT (0). Deviations from the period definitions (given in the text) are indicated on the left of the period symbols.

The O-day injected chick controls showed an average of 48 % survival during PI. The 52% mortality included infertility estimated from other data as 10% for the source of supply. For the later injection ages, the control survival was higher during PI, averaging 95, 75, and 95 % for embryos injected on day 4, 7, and 10, respectively. The drop in PI survival for injection age 7, relative to that for injection ages 4 and 10 appeared real. Mortality during Pz + P3 was between 10 and 20 % of the original number of eggs, for all injection ages, excluding the first trial of day 4 injection which, in view of later experiments not reported here, appears to have been anomalous. The PI survivals were generally higher for duck eggs than for chick eggs. The O-day injected duck eggs showed an 81% survival during PI, and the 19 % mortality included an estimated infertility loss of 11 ‘A. The Pr survival of day 4-injected duck eggs was 87 %, of eggs injected on days 7,10, and 13, it was 95 % or more. However, the P2 and

CHICK

AND

DUCK

EMBRYOS

5

more particularly, the P3 losses were higher in duck than in chick embryos. For the mid-incubation injected eggs, the P, survivals in the two species (86 % for chicks and 75 e/, for ducks) were not significantly different in the statistical sense. Adjusted Suwical

in Pesticide-Groups

All adjusted survivals after the total period of observation (Pr) are given in Table 1 (chicks) and Table 2 (ducks). Values greater than 100%, although probably impossible, have been shown as calculated in order not to obscure the data variation. Rapid perusal of these data indicates that toxic effects were produced in both speciesand that theseeffects depended upon the pesticide, its dose, and the age of the embryo at injection. The results were reasonably similar in the two species.Somequite large variations are noted among these observations, particularly for the 0- and 4-day injected chick embryos. It appears plain, from Tables 1 and 2, that a dose-responserelationship obtained, at least for eggsinjected at the mid-incubation age. The results of statistical tests for monotonicity of this relationship, using P, data, are shown under test I in Table 3. There was evidence for the existence of a monotonic relationship in the O-day injected chicks, as well as in the mid-incubation injected chick and duck embryos. One cannot rank the magnitude of the effect in accordance with the significance levels which appear in Table 3. In fact, no significant difference was found between the mid-incubation results for the two species, but the IO-day chick result was significantly stronger (P -- 0.05) than the O-day chick one. For the mid-incubation data, the survivals after P, showed almost asclear a dose-responserelationship as did the survivals after P,. Data Variation and Reproducibility Standard error estimates (sr) for single-group (30 eggs)p estimates based on P, survivals of 1-mg doses,are given in Table 4A. If one denotes the expected binomial or sampling error estimate by sn, then the ratio s&a must be at least asgreat asunity, on the average, and study of this ratio provides somenotion of the extent of uncontrolled variation in the data. No significant effect of injection age upon this variation was found (Table 4A). Average values of these ratios were 1.64 for chicks and 0.99 for ducks, which suggeststhat duck eggs produce significantly less variable results. The 0.99 value for the duck data signifies no variation whatsoever, beyond that due to sampling; while this isdoubtlessan exaggerated interpretation, the result still constitutes good evidence for reproducibility of the duck results. Table 4B provides some variation estimatesfor the subreplication data from within the last mid-incubation trials which were carried out for each species.Again there was a suggestion of more variation in chick data than in duck data. In both species,the variation tended to increase with increasing dose of the pesticidesand with increasing time after injection, but not with increasing injection age. The significancesof the rank correlations of the pesticide mortalities from replication to replication are summarized under test II in Table 3. Again, the O-day chick data and the mid-incubation injected chick and duck data appeared significant (P < 0.05). However, no significant differentiation could be made between the three setsof rank correlations, probably becauseof the weaknessof the test. These correlations, where they occurred, constituted evidence for both reproducibility and toxicity.

Parathion

Diazinon

Dimefox

Ruelene

DDVP

Phosdrin

Pesticide

1: III IV

I II III IV I II III IV I II III IV I II III IV I II III IV

Replication No.

53 91

69 59

53 124

66 46

108 85

14 85 170

13 33

0 0

53 65

53 85

66 117

57 65 145

0 0

0 0

0 0

79 91

53 78

66 59 194

29 108

0 67

14

57 103

43 31

14 98

86

57 93

43

82

92 43

100

186

lop&!

Embryonal

29

143 57

14

82

29

1 mg

83

100

102

64

80

109 148

lop!3

96

80

102

83

128

89 122

100 tG3

(days)

26

91

87

84

113

96 109

1 mg

(P.,) INTHECHICKEMBRYOS

age at injection

TABLE 1 VERENTIREINCUBATIONF%RIOD

10 PEC 100kc3 1 mg

0

ADJUSTED SURVIVALS(%)•

79 80 85 86 71 91 85 111 95 103 95 99 104 91 106 123 105 103 85 111 99 80 95 111

21 57 64 86 56 80 95 99 100 68 85 111 89 80 85 99 105 91 64 99 32 68 64 49

lob

0 11 11 62 0 57 11 74 16 34 32 37 16 68 64 99 0 11 11 12 0 23 0 37

1 mg

I II III IV I II III IV I II III IV I II III IV I II III IV I II III IV I II III IV

30 78

15 72

65 170

104 72

192 72

40 33

40 13 73

104 111

8.5

118 85 236

30 72

185 85

106 20

13 39 73

74 104

44 39

44 59 121

30 72

133 39

53 13

64 20 109

100

57

0 98 100

0

43

-

129

14

14 113 76

29

14

10

88

71

121

93 117

117

117

121

115 102

(described under Materials).

29

43

0 72 59

14

0

82

105

88

82

71

1-continued

57

n First replication at 7 days is PI + P2 data. DLast three rows of IO-day data dewe trom the three subreplications ’ Doses were given at levels of 10 pg, 100 p*g, and 1 mg per egg.

Bayer 39007

Bayer 31344

Carbaryl

Ethion

Dimethoate

Trithion

Malathion

TABLE

96

115 78

95

77

120

77 141

133

102 78

112

113

128

121 117

74 57 95 86 103 114 106 111 103 91 106 99 126 114 106 111 115 91 106 111 107 80 106 123 73 103 95 99

89 103 95 111 109 114 106 86 92 91 106 123 113 114 95 99 75 114 85 111 112 91 74 99 114 68 74 123

84 80 95 86 109 101 85 49 69 103 106 99 107 103 74 86 84 46 95 74 51 34 64 49 86 80 74 99

-

I II IV

I II III IV

I III IV

I II IV

I II III

I IV

I IV

Phosdrin

DDVP

Ruelene

Dimefox

Diazinon

Parathion

Malathion

Pesticide

Replication No.

48

96

92 144

72

96 87

74

80

80

1Opg”

64

104

75

64

80

104

96

1OOpg

0

84

0 53

98

55 68

55

56

88 160

55 137

84

49

80

2

4

104

73

49

57

82

67

55

73

19

55 93

47

55 95

147

73

82

1mg

65

80

80 93

87

95 124

47

109

95

1opLg

Embryonal

65

87

95

80

95

65

109

100 PLg

7

58

0

80 87

65

95 124

70

102

102

1 mg

87

65

102 84

102

73 90

62

80

95

(days)

109

73

95

102

73

58

73

100 I-G

10

114

0

73 73

65

95 90

103

116

44

1mg

(PT) INTHEDUCKEMBRYOS

age at injection

VERENTIREINCUBATIONPERIOD

1otLg l~pg

100 117

61

72

72

1 mg

ADJUSTED SURVIVALS(%)•

TABLE

120 99

19 74

100 143

71 111

99

99

88

71 124

76

0

112

loo

83

65

98

0

41 36

71 99

43

56

83

12 25

1mg

CHICK

AND

DUCK

EMBRYOS

9

K.S.KHERAANDD.A.LYON

10

TABLE ONE-TAIL

Species Chicks Ducks

SIGNIFICANCE

3

LEVELS ATTAINED (PT DATA)

BY Two

STATISTICAL

Injection

age (days)

TESTS

Tests

0

4

7

10

13

I” IIb I” IIb

0.05 0.05 NS NS

NS’ NS NS NS

NS NAd NS NS

IO-11 0.01 NS NS

0.001 0.01

UDose response test. b Correlation of pesticide survival ranks test. c NS, not significant. d PT data not available; PI, Pz data not significant. TABLE

4A

EXPERIMENTALERROR(REPLICATION x PESTICIDEINTERACTION) (DOSE:~MG;PEFUOD: PT) Chick Injection age (days) 0 4 7 10 13 Weighted

Duck

__ ST

0.13 0.17 NDb 0.15 mean

STha

ST

1.59 1.83 ND 1.56 -

STbB

0.05

0.68 1.14 1.00 0.45 1.18

0.10

0.09 0.04 0.11

1.64

TABLE

0.99

4B

VARIATION BETWEEN SURREPLICATIONS (PERIOD: PT)

Dose 1 n-42

1oPg

Chick embryos injection age day 10

injection

ST

ST/SB

ST

ST/SB

0.12 0.06

1.30 1.03

0.13 0.06

1.13 0.78

Duck embryos age day

13

a sT = estimated total standard error; sg = estimated binomial sampling error, of proportion in a group. b Not done.

alive

CHICK

AND

DUCK

EMBRYOS

11

Toxicity at I-Mg Dose The most powerful statistical tests for toxicity were probably the comparisons of 1-mg pesticide survivals and the control survivals, over time periods whose initial point was the time of injection. The P, data were studied most extensively. If the survival over P, in the pesticide-treated group was found to be significantly lower than the control survival, then the survivals over the component periods were also analyzed in an attempt to localize when the mortality occurred. The results of this analysis for 1-mg doses are shown in Table 5. Rather than providing all the details, this table is restricted to showing those (adjusted) survivals which appeared to be significantly lower than 100 %. (The product of the adjusted survivalsin fractional form-over Pi, PZ, and P, is the adjusted survival over P,.) It should be plain that the determination of the toxic responses has been quite conservative throughout these analyses, but one should not be so severe as to hide salient parts of the pattern. For this reason, Table 5 displays both very significant mortalities (entries not in parentheses: P < 0.02 by one-tailed tests) and mortalities of more borderline significance (entries in parentheses: P < 0.10 by one-tailed tests). Cases where there was only one replication are indicated, and they were more cautiously evaluated by the use of probability levels that were half those stated above. Naturally, a few of the entries shown as significant in Table 5 are probably wrongly indicated (false positives): however, one suspects that there are more false negatives (blanks), because of the large amount of sampling and other kinds of variation.The consequences of data variation may be better appreciated by realizing that the following were the two extreme situations in the present analysis: in the worst cases (chick data, only one replication), there was about a 50: 50 chance of detecting a specific mortality of 50?, at borderline level and, in the best cases (duck data, three replications), there was about the same chance of detecting a specific mortality of 15 II!. Despite the probability of some false entries in Table 5, it is felt that the pattern of mortality incidence shown is substantially correct. Some support for this is provided by the reasonable similarity between the patterns for the two species. Among the various embryonal ages investigated, the maximum number of pesticides was found to be toxic in IO-day chick and 13-day duck embryos. Evidence of toxicity of Ruelene in duck and Phosdrin, DDVP, carbaryl, and Ruelene in chick were not observed at any other injection ages. Conversely, the toxicity of malathion and Trithion in chick and of dimefox, Trithion, and carbaryl in duck demonstrated at earlier injection ages, could not be detected after mid-incubation injection. Malathion produced variable results and may have a nonmonotonic response to dose. Results of carbaryl in the duck were generally ambiguous and the evidence for toxicity in the 4-day injected embryo rested on a single trial. Response to dimefox at all early duck stages, though consistent, was of borderline significance; the average adjusted survival of 897, for dimefox at the 13-day age, while not statistically significant, was also statistically indistinguishable from the survival values at the earlier injection ages. Only Trithion produced definite evidence of specific lethality after early injection but not after mid-incubation injection, and this was observed in both species. With progressive embryonal age at injection, the observations suggested that (1) the interval between in.jection and onset of lethality became shorter, (2) the interval between

(80)

6

P,

P3

PT

(56)” (4 (50)

(6

(50)

(61)

(42)

(63)

(65)

< 2c (4 t2 <2 t2 (72) (48) (49) 26

P2

0

12 ~8

P2

(75)

61 31b 35b 33b

63 34 (56) 36

(47Y

lob

<2 (2

P,

19

11

-

P3

SURVIVAL

(71) 26

(76) (75)

PI

4

ADJUSTED

TABLE

5

P2

~.

7

ND

ND ND

ND ND ND

(72)

16 (23

(68)

Duck embryos

ND

24

NDd ND ND ND ND

P3

SHOWING

Chick embryos

PI

IN CASES

(59)

~4~

(65) b

b

PT

SIGNIFICANT

75 53

13 24 37 66 28 < 14

PI

50

(78)

PZ

Tox1c1~~”

PJ

(76) < 7

(59)

67 63 ~25 X47

i68

10

-__

(73) i 4b

(66)b

(44)b

74 50

t9 < 13 26 42 ~7 -C4

PT

(83) 33

(77) 30

16 71 62

_-.--

(41)

p3

(81) 54

(56) 53 < 23

~~~~~~~ PI Pz

13

66 17

38 ~2

Fz

19

PT

’ Blanks indicate values not significantly different from controls, values not in parentheses are highly significant; values in parentheses are of borderline significance. * Only one replication. ’ -Cindicates zero survivals in one or more replications.

Phosdrin DDVP Ruelene Dimefox Diazinon Parathion Malathion Trithion Dimethoate Ethion Carbaryl Bayer 37344 Bayer 39007

Phosdrin DDVP Ruelene Dimefox Diazinon Parathion Malathion Trithion Dimethoate Ethion Carbaryl Bayer 37344 Bayer 39007

Postinjection periods (PT = P, + P2 + P3):

Embryonal age at injection:

-

AVERAGE

CHICK AND DUCK EMBRYOS

13

onset and end of most of the mortalities was shorter, and (3) the total amount of mortality was greater. While none of the 13 pesticides completely displayed all three of these features, most of the toxic ones displayed some of them. An example is the adjusted survival history shown in Fig. 2 for Phosdrin in the duck embryos. 0

4

7

IO

I. 0 1 -__ --g--w... 1 ‘\ ‘-t ‘\

0.9

‘\ L.”

0.8

13 . ..' .*7

.. v. \ \: /. L., t

. . . . ...*.

-.

l . . . . . . . . . . e..’

-.--+

----, \ \

*.\ .-.

--\

*.-*-.-.--C'\-'

\\

44 \ LO

y'?‘ \ \ \ \ \

i IO

0.2 c 0 0 2

6

IO 14 INCUBATION

18 22 DAY

26

30

FIG. 2. Adjusted survivals in duck eggs injected on incubation days 0,4,7, 10, and 13 (arrows) with 1 mg of Phosdrin.

The pesticide-induced mortalities were usually detected in only one of the three postinjection periods. At mid-incubation of the two species, specific mortality was always sharply defined at the time of first observation (PI : usually 24 hours, occasionally 48 hours). This mortality was frequently followed by a second period of mortality during P3 (Table 5). DISCUSSION

The unsuitability of O-day chick eggs for toxicity evaluation is evident from the data presented herein and confirms earlier findings (Clegg, 1964; Walker, 1967). The low survival in the background control was in contrast to that reported by Verrett et al. (1964). The discrepancy could be due to a biological difference in the strain of eggs. The cause of unsuitability of the O-day chick egg for toxicity evaluation was not determined. High background mortality, however, points out that the chick egg responded to the act of injection or the propylene glycol, with a high rate of lethality. The extreme

14

K. S. KHERA

AND

D. A. LYON

sensitivity of the preincubated chick egg appeared to be one of the factors in the lack of appropriate specific toxic response. However, in the O-day duck egg, there was less interference of background mortality (P1 + P2 data in Fig. 1) and other types of variation; therefore, the failure to observe a dose-response relationship may be due to causes inherent in the egg. In the 7-day chick embryo the lethal response and number of pesticides identified as toxic at 1-mg dose, was minimal (P1 + P2 data). A low sensitivity of the g-day chick embryo has been noted previously (Ridgway and Karnofsky, 1952; Franciscis and Landauer, 1959). The 4-day chick and 4-, 7-, and IO-day duck embryos were unsatisfactory because of low sensitivity or lack of a dose-response relationship. The mid-incubation age in both species satisfied the necessary criteria for toxicity quantitation. The mortality did include deaths due to nonspecific causes. These were, however, not of a magnitude to obviate the value of the injection age. The embryocidal effect of pesticides of diverse chemical compositions such as thiophosphates (P=S), phosphates (P=O), or carbamates was manifest generally within 24 hours. The rapidity of the response might have obvious advantages in certain applications for screening, etc. The middle of incubation may be particularly suitable because the absorptive surface and vascularization of the yolk sac are quite extensive at this time (Bellairs, 1963) and thus probably favourable for rapid uptake and transport across the yolk sac endoderm. Within 24 hours of yolk injection in the 11-day-old chick embryo, deposits of tetracycline were demonstrable in the skeleton (Smith and Chapman, 1963). By mid-incubation age it is probable that the metabolizing enzymes and the pesticide target enzyme(s) have acquired specific functions to such a degree that good assessment of pesticide toxicity is possible. The 1Cday chick embryo has been described as a faithful model for studying the cholinesterase-inhibiting agents (Hadani and Egyed, 1967). A precise comparison of the toxic response in chick and duck embryos was not feasible since at the chosen injection ages, the embryonal stages did not correspond. Further, equal doses of pesticides were administered despite the different egg weights. With few exceptions, the pesticides appeared equally toxic in both species. Duck embryos, however, appeared to provide more reproducible results than the chick embryos. The data cited by Kenaga (1966) concerning oral toxicity in rats and the present study, permit some interesting comparisons of toxicity of pesticides in the mammalian and avian species. LDSO’s were calculated from the present data in the usual way for those pesticides showing more than 50% mortality at the 1-mg dose level. It appears that, at mid-incubation, Phosdrin, DDVP, and parathion have similar LDSO’s in the rodent and avian hosts, dimefox has a higher LD50 and ruelene, diazinon, and the two Bayer preparations have lower ones in avian embryos than in rodents. ACKNOWLEDGMENTS

The authors are indebtedto Dr. H. C. Grice for assistancein all phasesof the study and to Drs. W. P. McKinley, D. F. Bray, and D. G. Clegg for their valued suggestionsand discussion. REFERENCES BELLAIRS, R. (1963).Differentiation of the yolk sacof the chick studiedby electron microscope. J. Embryol. Exptl. Morphol. 11,201-225.

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D. J. (1964). The hen egg in toxicity and teratogenicity studies. Food Cosmet. Toxicol. 2,717-727. COCHRAN, W. G. (1943). Analysis of variance for percentagesbasedon unequal numbers. J. Am. Statist. Assoc. 38, 287-301. DUNACHIE, J. F., and FLETCHER, W. W. (1966). Effect of someinsecticideson the hatching rate of hens’eggs.Nature 212,1062-1063. FRANCISCIS, P., and LANDAUER, W. (1959). Combined effects of cortisone and insulin on developingchicken embryos.Nature 184, 101-103. FREEMAN, M. F., and TUKEY, J. W. (1950). Transformationsrelated to the angular and the squareroot. Ann. Math. Statist. 21, 607-611. GABRIEL, K. R. (1963).Analysis of variance of proportions with unequalfrequencies.J. Am. Statist. Assoc. 58, 1133-l 157. HADANI, A., and EGYED, M. N. (1967). Use of the chick embryo for testing the toxicity of cholinesterase-inhibitingcompounds.Toxicol. Appl. Pharmacol. 10, 313-321. HALVERSON, A. W., JERDE, L. G., and HILLS, C. L. (1965). Toxicity of inorganic selenium saltsto chick embryos. Toxicol. Appl. Pharmacol. 7.675-679. KARNOFSKY, D. A., RIDGWAY, L. P., and PATTERSON, P. A. (1950).Production of achondroplasiain the chick embryo with thallium. Proc. Sot. Exptl. Biol. Med. 73,255-259. KENAGA, E. E. (1966). Commercial and experimental organic insecticides.Bull. Entomol. Sot. Am. 12,161-217. KHERA, K. S., LAHAM, Q. N., and GRICE, II. C. (1965).Toxic effects in ducklings hatched from embryosinoculatedwith EPN or Systox. Food Cosmet. Toxicol. 3,581-586. LIMBORGH, J. V., and MEENEN, F. V. (1963).Effect of reserpineon rate of mortality and growth of duck embryos.Nature 197,615-616. MCLAUGHLIN, J., JR., MARLIAC, J. P.,VERRETT, M. J., MUTCHLER, M. K., andFITZHUGH, 0. G. (1963).The injection of chemicalsinto the yolk sacof fertile eggsprior to incubation asa toxicity test. Toxicol. Appl. Pharmacol. 5,760-771. MCLAUGHLIN, J., JR., MARLIAC, J. P., VERRETT, M. J., MUTCHLER, M. K., andFITZHUGH, 0. G. (1964).Toxicity of fourteen volatile chemicalsas measuredby the chick embryo method. CLEGG,

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K. C., and FINKELSTEIN, R. A. (1966).Bioassayof endotoxin: Correlation between pyrogenicity for rabbits and lethality for chick embryos.J. Infectious Diseases 116,529-536. RIDGWAY, L. P., and KARNOFSKY, D. A. (1952).The effects of metalson the chick embryo: Toxicity andproduction of abnormalitiesin development.Ann. N. Y. Acad. Sci. 55,203-215. SIEGEL, S. (1956).Nonparametric Statistics for the Behavioral Sciences. pp. 203-213.McGrawHill, New York. SMITH, H., and CHAPMAN, I. V. (1963).Useof living chick embryo asa biological indicator of the effectivenessof chelating agents.Nature 198, 32-33. VERRETT, M. J., MARLIAC, J. P., and MCLAUGHLIN, J., JR. (1964).Useof the chickenembryo in the assayof aflatoxin toxicity. J. Assoc. Ofic. Agr. Chem. 47, 1003-1006. WALKER, N. E. (1967).Distribution of chemicalsinjected into fertile eggsand its effect upon apparenttoxicity. Toxicol. Appl. Pharmacol. 10, 290-299.