Arch. Environm. Contam. Toxicol. 6, 435-446 (1977)
Archives of
Environmental
Contamination and Toxicology © Springer-Verlag New York Inc. 1977
The Effects of Low Dietary Levels of DDT on Breeding Performance in Hybrid Mice.1 T. A. Ledoux 2, J. R. Lodge, R. W. Touchberry3, and B. Magnus Francis Department of Dairy Science, University of Illinois, Urbana-Champaign
Abstract. A combination of breeding experiments was used to study the effects of various levels of dietary DDT on the reproductive efficiency in mammals. The results of feeding B6D2F1 hybrid mice 5, 10, or 20 ppm of DDT in the first breeding experiment showed an increase in litter size and number weaned over controls. Ten ppm of DDT caused increases over controls in the number born and alive on day 1 (P < 0.05). DDT caused decreases in the weight of pups at 5 ppm on days 15 and 30 (P < 0.05 and 0.01, respectively), at 10 ppm on day 15 (P < 0.005), and at 20 ppm on day 30 (P < 0.05). A second breeding experiment using 30, 60, or 120 ppm of DDT showed a general detrimental effect on reproduction. Thirty ppm DDT-fed animals produced fewer pups than controls (P < 0.01) at days 1, 15, and 30, and the pups weighed less than controls at 30 days of age (P < 0.05). DDT at 120 ppm resulted in fewer mice at birth and at 30 days of age (P < 0.01) than control diet. The third, fourth, and fifth breeding experiments involved diets with 0, 5, 10, and 20 ppm of DDT and with the addition of a 40-ppm level in the third and fourth studies. The data showed larger litter sizes on days 1, 15, and 30 for DDT-fed animals. This investigation indicates that 5, 10, 20, or 40 ppm of DDT in the diet often results in larger litter sizes and more pups weaned than controls. Animals fed levels of 30, 60, or 120 ppm of DDT generally produced fewer offspring than did control animals. DDT-fed mice often weighed less before weaning than control pups. Although variable responses were found, it appears from these studies that environmental levels of DDT are not detrimental to the reproductive efficiency of mice and may under certain circumstances be beneficial.
1 This work was supported in part by the Illinois Agricultural Experiment Station. 2 Present address: White Mountain Research Station, University of California, 3000 East Line Street, Bishop, California 93514. 3 Present address: Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55101.
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Introduction
The insecticide DDT has, in a mere thirty years of use, been hailed as a panacea and reviled as a xenobiotic whose continued use may destroy the entire biosphere. The very qualities responsible for its remarkable success are responsible for its dangers: Being highly lipid soluble and only marginally soluble in water, it is as persistent in insects and their predators as in fields or on walls. Small quantities of DDT absorbed from water or food are concentrated in fatty tissues, and as the trophic level increases from phytoplankton to fish to piscivores, repeated concentrations can lead to increasing levels of DDT which may affect physiological processes. Despite numerous articles on the hazards of DDT, few data are available for the effects of DDT on reproduction in mammals (Deichmann et al. 1971, Pimentel 1971, Ware 1975), and much of the published literature is controversial. The purpose of this investigation was to examine the effects o f plausible levels of DDT on the reproductive performance of mammals. To minimize the effects of extraneous factors, genetically uniform hybrid mice were used as the experimental species, and the DDT was added to the food at levels which might be found under less artificial conditions; for example, in a treated field or barn. A preliminary report of this work has been presented (Ledoux et al. 1971),
Materials and Methods B6D2F1/J hybrid mice (progeny of C57B1/6J X DBA/2J), from The Jackson Laboratory, Bar Harbor, Maine, and/or their descendants were used in these experiments. They were placed 12 per cage when approximately four weeks of age and allowed to adjust for three weeks to the local laboratory conditions. At the time of mating they were caged one male with one female (unless otherwise noted) in plastic cages having hardware cloth lids with San-i-cel litter. Mouse weights were determined on a Shadowgraph balance. Full-sib and parent-offspring matings were avoided at all times. Food and water were supplied ad libitum. Control and test groups were run concurrently. Room temperature during the winter months was thermostatically controlled between 20° and 22° C. Summer room temperatures were not controllable and at times exceeded 22° C. Ventilation was supplied by a large variable speed and cycle exhaust fan. Lights were regulated 6 A.M.--6 P.M. on; 6 P.M.-6 A.M, off. Technical DDT (control number 1807, Nutritional Biochemical Co., Cleveland, Ohio) was used in this investigation. Gas/liquid chromatography revealed the following analysis for 98.5% of the material: p,p'-DDT, 70.0%; o,p'-DDT, 21.0%; p,p'-DDD, 4.0%; and p,p'-DDE, 3.5% (analysis courtesy of Dr. Willis Bruce, Illinois State Natural History Survey, University of l~inois, UrbanaChampaign). Due to the quantities of feed required, stock mixes were prepared and used for making the final feeding diets. The DDT stock diet was prepared by sprinkling 2.0 g of technical grade DDT dissolved in 50.0 ml of analytical grade acetone over 2.0 kg of ground laboratory meal (either Rockland Mouse/ Rat diet or Purina Laboratory Chow) and thoroughly m~ing it. After allowing acetone evaporation in a well-ventilated room, the diet was transferred to a 1 cuft drum and thoroughly mixed. The resultant 1000 ppm DDT stock diet was stored in airtight containers in a cool, dark place until needed. Control stock diet was prepared in the same manner, omitting the DDT. The diluted feeding diets were prepared in 50-1b lots by adding the appropriate quantity of stock mix to untreated meat in a large Hobart mixer. The weight of control acetone-treated stock diet was the same as the amount of DDT stock diet used to make the mean DDT treatment level diet within a given experiment. After 15 man of mixing, 10 to 12% water by weight was added and mixing was
Effects of DDT on Breeding Performance in Mice
437
continued 15 additional min. The diets were then pelleted in a laboratory pellet mill, and dried for 48 to 72 hr. The dry pellets were stored in plastic containers in a cool place.
First Breeding Experiment. Diets of 5, 10, and 20 ppm of DDT were fed to separate groups, each consisting of 20 pairs of B6D2F] J mice. Control diet was fed to 40 pairs. The experiment started December 1, 1969, and continued for 86 days. Breeding parameters examined included i) time of first vaginal plug 2) date of littering, 3) number of live and dead pups on day of birth and on days t5 and 30, and 4) weight of live mice in litters on days 1, 15, and 30.
Second Breeding Experiment. Diets of control, 30, 60, and 120 ppm of DDT were fed to separate groups of Fz mice (derived from the first breeding experiment's large control group) in an effort to determine the effect of increased feeding levels. Each group consisted of 20 pairs, except the control group which again contained 40 pairs. The experiment started February 16, 1970, and continued for 130 days. The data recorded were the same as in the first breeding experiment.
Third Breeding Experiment. This experiment was designed as a replicate of the first breeding experiment using newly obtained B6D2FI/J hybrid mice. It started June 30, 1971, and ended September 20, 1971. Treatment consisted of control, 5, 10, 20, and 40 ppm of DDT diets. Each level used 20 breeding cages, each housing one female and one male, except in the 0 and 10 ppm levels where two females were caged with one male (one of the females was randomly assigned to another series of experiments not reported here). The data recorded were the same as in the first breeding experiment.
Fourth Breeding Experiment. The preceding experiment was repeated with the following modifications. Mice were chosen at random (except that no full-sib matings were allowed) from the first litters of the third breeding experiment and were maintained on their respective levels of control, 5, 10, 20, and 40 ppm of DDT. The experiment s t ~ e d November 24, 1971, and continued 50 days, except that pairs with first litters born on or before January 14, 1972 were kept to weaning. The breeding parameters examined were the number born live and dead on day of birth, and the number live and their weights on days 15 and 30.
Fifth Breeding Experiment. Since previous experimental results suggested that factors other than DDT treatment were causing inconsistent results, e.g., season, a fifth experiment was undertaken. Nineteen breeding pairs were placed on diets of control, 5, 10, or 20 ppm of DDT. Seventeen pairs at each level were B6D2F1 hybrids, raised in our laboratory, while two pairs on each treatment level were Fz females with F1 males. Animals were on their respective DDT diets from December 6, 1971, when the experiment started, until February 4, 1972, when the 5 ppm matings were shifted to 10 ppm and the 20 ppm raised to 40 ppm of DDT. On February 7, 1972, all mice receiving DDT were placed on 40 ppm until the end of the experiment, February 19, 1972. Mice on the control diet were maintained throughout. The data recorded were the same as in the fourth breeding experiment.
T . A . Ledoux et al.
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Biometrical Considerations. Least-squares procedures were utilized as outlined by Harvey (1960) for unequal subclass frequencies. The first and second breeding experiments were analyzed using the linear models: Y~k = u + Ti + Pj/Ti + Lk/pi/Ti Yi~k = u + T~ + P/Ti + bxD + Lk/PJTi
(Model A) (Model B)
where: YiJg = u = Ti = P]TI =
observation of the k th litter within the j ~hpair in the i th level. least-squares mean. deviation from the mean due to treatment. deviation from the mean due to treatment differences between individual pairs within a treatment. Lk/P]Ti = deviation from the mean due to differences between individual litters within pairs within treatment (random error). b1 = regression of the number of mice born in each litter on the average weight in the litter. D = number of mice born in each litter. In the analysis of experimental results, if the F ratio for treatment was significant at the 0.05 level or less, a two-sided Dunnett's test was used to compare the treatment means vs. the control mean for the particular dependent variable being evaluated (Steel and Torrie 1960). Whenever the F test in the preceding models was not significant for the pairs within treatment category, the pairs within treatment and the litters within pairs within treatment categories were pooled and the following models were used: YiJ = u + Ti + % Ya = u + Ti + b~D + ei~
(Model C) (Model D)
where: Yu = observation of the j th litter on the i t~ level. u = least-square mean. Ti = deviation from the mean due to treatment. e~ = random error. b l = regression of the number of mice born in each litter on the average weight in the litter. D = number of mice born in each litter. The dependent variables examined using models A and C in the first and second breeding experiments were; the number born, number live on days 1, 15, and 30, and the average live weight on days 1, 15, and 30. Average live weights adjusted for the number born on day one were analyzed using models B and D. Analysis of results from the first two breeding studies established the size of difference needed between treatment means and the magnitude of their standard errors necessary for statistical significance. If treatment means and standard errors were similar in magnitude or smaller than those required to previously achieve statistical significance, the data did not warrant further statistical analysis. Other experiments in which the results indicated a statistical analysis might be of value were analyzed using a one-way Classification.
Results and Discussion First B r e e d i n g E x p e r i m e n t . M o s t f e m a l e m i c e e x h i b i t e d vaginal plugs d u r i n g the first f o u r d a y s o f b e i n g paired with males. The highest incidence of mating occurred on the third day w i t h 38, 45, 60, a n d 4 5 % o f t h e f e m a l e s s h o w i n g p l u g s in t h e c o n t r o l , 5, 10, a n d 20
Effects of DDT on Breeding Performance in Mice
439
p p m DDT-fed groups, respectively. It has previously been reported that female mice caged in groups without a male have irregular estrous cycles and that pairing these females with males tends to synchronize their cycles so that on the third night the percentage of matings increases (Whitten 1966). This p h e n o m e n o n was frequently seen in our experiments. The number of females which produced first through fourth litters is shown in Table 1. Many of the females not producing successive litters were pregnant during the course of the experiment, but either did not deliver pups or killed and ate them during delivery. E a c h group of DDT-fed mice produced proportionately more litters than did the controls. This increased production of litters associated with D D T feeding is similar to that reported by Ottoboni (1969). These data were statistically analyzed using statistical Models A, B, C, and D and the resulting appropriate least-square treatment means with their standard errors are presented in Table 2. The results show a generalized increase in litter size for DDT-treated mice o v e r that of control mice. Mice consuming 10 p p m of D D T produced significant increases in number born and n u m b e r alive on day one compared to control values (P < 0.05). D D T at 5 and 10 p p m significantly decreased adjusted live weights on day 15 (P < 0.05) and 5 and 20 p p m significantly decreased adjusted live weights on day 30 (P < 0.01 and < 0.05, respectively). The regression for the n u m b e r born on day one (live weight) was highly significant (P < 0.001). These data showed that the reproductive capacity o f these mice was increased by the presence of D D T in the diet since the average litter size, which paralleled the results of Wrenn et al. (1970) in rats, and the number weaned were larger for all DDT-fed mice than the controls.
Second Breeding Experiment. The levels of DDT were increased to 30, 60, and 120 p p m in this experiment. The majority of females at all treatment levels had vaginal plugs by day four after pairing. The results on the number of females which produced first through fifth
Table 1. Number of litters produced by F~ mice fed DDT in the first breeding experiment ppm DDT fed 0
5 10 Pairs of mice set
20 Total
Litter no.
40
20
20
20
t00
First Second Third Fourth Total
40 36 t4 1 91
20 19 13 0 52
20 20 11 0 51
20 20 12 1 53
100 95 50 2 247
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Table 2. S u m m a r y of least-squares treatment m e a n s and their standard errors for F1 mice fed D D T in the first breeding experiment p p m D D T fed 0
5
10
20
20
20
20
6.19 + 0.29 (52) 5.92 ± 0.32 (48) 5.08 ± 0.49 (40) 5.00 ± 0.38 (39)
6.84 ± 0.30 ~ (51) 6.49 ± 0.32 a (49) 5.87 ± 0.48 (43) 5.65 ± 0.38 (43)
No. pairs set Day
40
1
No. born
1
No. live
15
No. five
30
No. live
5.65 -+ 0.24 O1) 5.21 ± 0.27 (84) 5.27 ± 0.41 (70) 4.66-+ 0.32 (70)
Day
6.55 ± 0.29 (53) 6.32 ± 0.32 (51) 5.87 +- 0.48 (46) 5.49 ± 0.38 (45)
L i v e w e i g h t ( g ) a ~ u s t e d f o r no, born
1 15 30
1.14 ± 0.03 6.23 ± 0.25 12.57 ± 0,51
1.17 ± 0.04 5.01 ± 0.29 a 9.98 ± 0.59 b
1.12 ± 0.04 5.22 ± 0.29" 10.72 ± 0,59
1 . 0 9 ± 0;04 5.48 ± 0.29 10.41 ± 0.58 a
a-~Significantly different from the control at the 0.05 and 0.01 levels o f probability, respectively, using Dunnett's test after first obtaining a significant F test using A N O V A on the variable ( ) = N u m b e r of fitters
litters (Table 3) show that proportionately only the second litters at 30 ppm and second and third litters at 60 ppm DDT exceeded the number of litters produced by the controls. As the experiment progressed, the percentage of DDT-treated mice which produced litters generally decreased faster than the control mice. The appropriate least-squares treatment means and standard errors are in Table 4. The results show a marked decrease in the parameters measured for the Table 3. N u m b e r of fitters produced by F 2 m i c e f e d D D T in the second breeding experiment ppmDDTfed 0
30 60 Pairs of mice set
120 Tot~
Litter no.
40
20
20
20
100
First Second Third Fourth Fifth Total
40 36 34 29 11 150
20 19 14 7 1 61
20 20 17 12 3 72
20 17 14 10 4 65
100 92 79 58 t9 348
Effects of DDT on Breeding Performance in Mice
441
Table 4. Summary of least-squares treatment means and their standard errors for Fz mice fed DDT in the second breeding experiment ppm DDT fed
Day 1
No. born
1
No. live
15
No. live
30
No. live
0
30
40
20
7.55 ± 0.22 (150) 6.76 ± 0.43 (145) 4,91 ± 0,28 (113) 4.00 ± 0.28 (94)
Day 1 15 30
60 No. pairs set
4.56 ± 0.29 b (61) 4.03 ± 0.48 b (54) 2.85 _+ 0.37 ~ (38) 1.74 ± 0.37 b (26)
20
120
20 6,83 ± 0.27 (72) 6.37 ± 0.46 (69) 4.09 + 0,35 (51) 3,49 ± 0.35 (51)
6.29 ± 0,28 b (65) 6.25 _+ 1.08 (60) 4,12 ± 0.36 (45) 2.66 +_ 0.36 b (32)
Live weight (g) adjusted for no. born 1.32 ± 0.12 4.92 ± 0.28 8.64 ± 0.58
1.03 ± 0.13 4.44 _+ 0.36 5.95 ± 0,77 a
1.03 ± 0,12 4.62 ± 0.33 7.24 ± 0.70
1.24 ± 0.29 4.48 -+ 0.34 6.30 ± 0.72
a,b Significantly different from the control at the 0.05 and 0,01 levels of probability, respectively, using Dunnett's test after first obtaining a significant F test using ANOVA on the variable ( ) = N u m b e r of litters
30 ppm DDT-fed mice compared with controls. The results for mice on 60 and 120 ppm of DDT showed a greater reproductive capacity than those for 30 ppm but were still below those for the controls. The percentage postnatal mortality to day 15 or 30 was similar for all treatment groups except at day 30 where the percentage tended to be higher for the 30 and 120 ppm groups. Statistical analysis results showed that mice receiving 30 ppm of DDT had significantly fewer pups born; fewer alive on days 1, 15, and 30 (P < 0.01); and significantly lower pup weights on day 30 when adjusted for number born (P < 0.05). At 120 ppm there were fewer pups born and alive on day 30 (P < 0.01). The regression for number born on day one for live weight was not significant. The results, in contrast to those from the first experiment, suggest that these higher levels of DDT were detrimental to reproductive response. However, the lack of a dose response with the effect occurring with 30 and 120 ppm and not with 60 ppm suggest that factors other than DDT may have been involved.
Third Breeding Experiment. This experiment was designed to see if the results from the first experiment were repeatable. The results for littering response (Table 5) and breeding capacity (Table 6) showed that DDT levels of 5, 10, 20 or 40 ppm had no significant effect on the parameters measured.
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Table 5. Number of litters produced by F1 mice fed DDT in the third breeding experiment ppmDDTfed 0
5
10 Pairs of mice set
20
40 'l'ot~
Litter no.
20
20
20
20
20
100
First Second Third TotN
20 20 16 56
20 20 13 53
20 19 14 53
20 20 17 57
20 20 15 55
100 99 75 274
During this experiment there was a much higher female mortality rate than w a s s e e n i n t h e p r e v i o u s e x p e r i m e n t s , b u t t h e d e a t h s w e r e d i s t r i b u t e d a c r o s s all treatments and thus not treatment-related. All of the females were pregnant and nursing a previous litter at the time of death. The Veterinary Diagnostic Laboratory identified no specific cause of death. Replacement foster females were chosen from a preliminary experiment from the same or next lower level of DDT. The combined stress of pregnancy, lactation, and heat of summer may have c o n t r i b u t e d t o t h e h i g h e r m o r t a l i t y a m o n g t h e f e m a l e s . L e s (1967) r e p o r t e d t h a t 95 ° F, b u t n o t 86 ° F , e x e r t e d a d e t r i m e n t a l e f f e c t o n g r o w t h a n d r e p r o d u c t i o n o f m a n y i n b r e d s t r a i n s o f m i c e . H e n o t e d n o e f f e c t s o f r e l a t i v e h u m i d i t y i n t h e 30 t o 60% range.
Table 6. Treatment means and their standard errors for F~ mice fed DDT in the third breeding experiment ppm DDT fed 0
Day 1
No. born
1
No. live
15
No. live
30
No. live
5
10 No. pairs set
20
40
20
20
20
20
20
8.86 ± 0.21 (56) 8.77 .+_ 0.22 (56) 8.27 ± 0.38 (45) 8.74 ± 0.39 (35)
8.60 +- 0.30 (53) 8.67 ± 0.28 (52) 8.98 ± 0.26 (41) 8.79 ±_ 0.25 (38)
8.22 ± 0.29 (53) 8.30 ± 0.28 (52) 8.t5 ± 0.32 (40) 7.76 ± 0.38 (33)
8.54 ± 0.27 (57) 8.53 ± 0.27 (57) 8.34 -+ 0.30 (45) 8.01--+ 0.28 (39)
8.71 _+ 0.29 (55) 8.89 ± 0.24 (53) 8.82 ± 0.32 (38) 8.62 ± 0.40 (34)
1.33 _+ 0.02 7.25 ± 0.14 14.92 ± 0.22
1.33 ± 0.02 6.88 ± 0.17 14.44 ± 0.34
Live weight (g) 1 15 30
1.29 ± 0.02 6.96 ± 0.19 14.20 ± 0.38
( ) = Number of litters
1.32 ± 0.02 6.93 _+ 0.11 13.82 ± 0.33
1.29 ± 0.02 6.95 ± 0.17 15.01 ± 0.28
Effects of DDT on Breeding Performance in Mice
443
Table 7. Number of litters produced by Fz mice fed DDT continuously in the fourth breeding experiment ppm DDTfed 0
5
10 20 Pairs of mice set
40 Total
Litter no,
20
18
20
19
20
97
First Second TotN
20 13 33
18 14 32
20 12 32
19 ll 30
20 9 29
97 59 155
Fourth Breeding Experiment. T h e m i c e u s e d in t h i s e x p e r i m e n t w e r e r a n d o m l y s e l e c t e d f r o m t h e first l i t t e r s o f the third experiment and thus were subjected continuously to their respective t r e a t m e n t f r o m the t i m e o f c o n c e p t i o n . T h e d a t a p r e s e n t e d in T a b l e s 7 a n d 8 s h o w n o s i g n i f i c a n t d i f f e r e n c e s in t h e p a r a m e t e r s m e a s u r e d b e t w e e n t h e c o n t r o l s and any of the treatment levels.
Table 8. Treatment means and their standmd errors for F2 mice fed DDT continuously in the fourth breeding experiment ppm DDT ted 0
5
20
18
8.33 _+ 0.38 (33) 8.21 +_ 0.40 (33) 7.63 ± 0.64 (19) 7.33 + 0.62 (18)
8.69 ± 0.44 (32) 8,87 + 0.39 (31) 8,47 ± 0.54 (17) 8,13 _+ 0.58 (16)
Day 1
No. born
1
No. live
15
No. live
30
No. live
I0 No. pairs set
20
40
20
19
20
8.09 :#: 0.45 (32) 8.19 ~:: 0.41 (31) 8.25 ± 0.54 (20) 8.00 ± 0.51 (20)
8.37 ± 0.44 (30) 8.37 _+ 0.44 (30) 8.26 ± 0.51 (19) 7.47 ± 0.58 (15)
8,76 ± 0.49 (29) 8.76 _+ 0.49 (29) 8.35 _+ 0.51 (20) 8.05 ± 0.57 (18)
6.48 ± 0.28 14.18 ± 0.40
6.I8 ± 0.32 13.12 _+ 0,51
Live weight (g) 15 30
6.20 + 0.25 13.57 ± 0.49
( ) = Number of litters
6.50 ± 0,29 14.34 ± 0,59
6.39 ± 0.30 14.22 + 0.59
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Table 9. N u m b e r of fitters produced by F1 and F~ mice fed D D T in the fifth breeding experiment
ppm DDT fed 0
5 10 Pairs of mice set
20 Total
Litter no.
19
19
19
19
76
First Second Third Total
19 19 10 48
19 17 7 43
19 17 7 43
19 17 5 4t
76 70 29 175
Fifth Breeding Experiment. The results of this experiment are shown in Tables 9 and 10. There was a small, nonsignificant increase in litter size in favor of the DDT-treated mice. The small weight differences at various times were also insignificant. The combined experiments involved over 470 breeding pairs of mice which produced 1190 litters and 8800 pups for a total of over 10,500 observable units. In addition to the rigorously analyzed parameters, certain general conclusions can be drawn about the reproductive state of the colony. No macroscopic evidence of tumors was observed in any of the mice, Table 10. Treatment m e a n s and their standard errors for F~ and F2 mice fed D D T in the fifth breeding experiment
p p m D D T fed 0
5
I0
20
19
19
No. pairs set 19
Day 1
No. born
1
No. live
15~
No. live
30
No. live
8.40 _ 0.31 (48) 8.47 ± 0.29 (47) 8.05 ± 0.26 (19) 7.75 ± 0.53 (28)
t9 8.51 ± 0.28 (43) 8.66 ± 0.25 (41) 7.50 -+ 0.24 (18) 7.61 ± 0.26 (23)
8.63 _+ 0.30 (43) 8.46 ± 0.34 (43) 8.00 _+ 0.24 (19) 8.11 ___ 0.28 (28)
8.85 --- 0.24 (41) 8.86 ± 0.24 (41) 7.67 --- 0.38 (18) 7.54 ± 0.30 (22)
6.31 +_ 0.25 13.64 ± 0.33
6.09 --- 0.29 13.81 _+ 0.46
Live weight (g) t5 a 30 a First litter data only ( ) = N u m b e r of fitters
6.40 + 0.30 14.10 --- 0.39
7.23 + 0.26 14.36 _+ 0.21
Effects of DDT on Breeding Performance in Mice
445
suggesting that DDT treatment was not carcinogenic. The duration of treatment was not, however, long enough for rigorous tests. The few gross abnormalities seen in the offspring were distributed equally across treatment groups. They consisted of hydrocephalus, characteristic of the C57BL/6J parental strain, (experiments 1 and 2); runts (experiments 1, 2, 4, and 5); and transient hair loss at weaning (experiments 1 and 2). Hydrocephalus and runting were rare occurrences while the hair loss sometimes affected entire fitters. Mean gestation length in the colony was 19 days, with no differences among treatments. A few females were infertile or aborted or destroyed fitters before births were recorded. Such losses were not treatment related and were rare in all experiments. The combined results of the third through fifth breeding experiments show that DDT-fed mice tended to produce larger litters than controls. The short interval between the beginning of treatment and mating argues against an increased ovulation rate, and consequently for a decreased embryonic mortality. The data therefore support the conclusion that, under these experimental conditions, DDT was neither teratogenic nor highly carcinogenic, and improved the reproductive performance of treated pairs. Inconsistencies between experiments must be ascribed to unidentified variables other than noise, pheromones, temperature, and crowding. The mice were housed in an infrequently used area, and no inconsistent noises were generated within the room. All treatment groups were equally crowded, and treated and experimental mice were housed in the same parts of the room. The room was not temperature controlled in summer, but variations should have affected all treatment groups equally. The most feasible explanation lies in an interaction of DDT with other stress factors, as was elegantly demonstrated for toxaphene in fish (Mehrle and Mayer 1975a and 1975b). Inconsistent data are familiar in studies on DDT, and the present results are similar to those reported by Ware and Good (1967) who also fed low dietary levels of DDT to mice. The present results also partially support the results reported for rats by Fitzhugh (1948), Ottoboni (1969), Treon and Cleveland (1955), Read et al. (1965) and Phillips et al. (1971), but are in disagreement with the results of Fahim et al. (1970) in rats, Hart et al. (1971) in rabbits, and Deichmann et al. (1971) in beagles. It is obvious from the results of this study and others that levels of DDT which might be encountered in the environment are not detrimental to the reproductive efficiency of mice and may under certain circumstances be beneficial. There are still some questions which need to be answered regarding the cause(s) of the variable responses found which may only be due to biological variability. The results presented here with the variable response help also to explain the variability and confusion among reports in the literature on the effect of DDT in the reproduction of animals.
References Deichmann, W. B., W. E. MacDonald, A. G. Beasley, and D. A. Cubit: Subnormal reproduction in beagle dogs induced by DDT and aldrin. Ind. Med. Surg. 40, 10 (1971).
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Fahim, M. S., R. Bennett, and D. Goodsell: Effect of DDT on the nursing neonate. Nature 228, 1222 (1970). Fitzhu~a, O. G.: Use of DDT insecticides on food products. Industr. Eng. Chem. 4, 704 (1948). Hart, M. M., S. Fabro, and R. H. Adamson: Prematurity and intrauterine growth retardation induced by DDT in the rabbit. Arch. Int. Pharmacodyn. Thera. 192, 286 (1971). Harvey, W. R.: Least-squares analysis of data with unequal subclass numbers. U.S.D.A. Agr. Res. Ser. Publ. ARS 20-8 (1%0). Ledoux, T. A., J. R. Lodge, and R. W. Touchberry: Reproductive performance of B6D2F~/J mice fed DDT. Program of the Fourth Annual Meeting of the Society for the Study of" Reproduction, Boston. Biol. Reprod., 5, 95 (1971). Les, E. P.: Effects of temperature and humidity on mice. In: The Jackson Laboratory 38th Annual Report 1%6-1967, p. 102. Bar Harbor, Maine: The Jackson Laboratory (I%7). Mehrle, P. M. and F. L. Mayer, Jr.: Toxaphene effects on growth and bone composition of fathead minnows, Pimephah, s promelas. J. Fish. Res. Board Can. 32, 593 (1975a). Mehrle, P. M. and F. L. Mayer, Jr.: Toxaphene effects on growth and development of brook trout (Salvelinus fontinalis). J. Fish. Res. Board Can. 32, 609 (1975b). Ottoboni, A.: Effect of DDT on reproduction in the rat. Toxicol. Appl. Pharmacol. 14, 74 (1969). Phillips, W. E. J., G. Hatina, D. C. Villeneuve, and D. L. Grant: Multi-generation studies on the effect of dietary DDT on the vitamin A status of the weanling rat. Can. J. Physiol. Pharmacol. 49, 382 (1971). l~mentel, D.: Ecological effects of"pesticides on non-target species. U. S. Government Printing Office #4106~0029 (1971). Read, S. I., T. K. Murray, and W. P. McKinley: The effect of DDT on the liver carboxylesterase and vitamin A utilization of mother rats and their young. Can. J. Biochem. 43, 317 (1965). Steel, R. G. D., and J. H. Torrie: Principles and procedures of statistics. New York: McGraw-Hill Book Company, Inc. (I960). Treon, J. F., and F. P. Cleveland: Toxicity of certain chlorinated hydrocarbon insecticides for laboratory animals, with special reference to aldrin and dieldrin. J. Agr. Food Chem. 3, 402 (1955). Ware, G. W.: The effects of DDT on reproduction in higher mammals. Residue Reviews 59, 119 (1975). Ware, G. W., and E. E. Good: Effects of insecticides on reproduction in the laboratory mouse: II. Mirex, Telodrin, and DDT. Toxicol. Appl. Pharmacol. 10, 54 (1967). Whitten, W. K.: Pheromones and mammalian reproduction. Adv. Reprod. Physiol. 1, 155 (t966). Wrenn, T. R., J. R. Wood, G. F. Fries, and J. Bitman: Tests of Estrogenicity in rats fed low levels of o,p'-DDT. Bull. Environ. Contamin. Toxicol. 5, 61 (1970).
Manuscript received December 4, 1976; accepted May 16, 1977
Archives of
Environmental
Contamination and Toxicology © Springer-Verlag New York Inc. 1977
The Effects of Low Dietary Levels of DDT on Breeding Performance in Hybrid Mice.1 T. A. Ledoux 2, J. R. Lodge, R. W. Touchberry3, and B. Magnus Francis Department of Dairy Science, University of Illinois, Urbana-Champaign
Abstract. A combination of breeding experiments was used to study the effects of various levels of dietary DDT on the reproductive efficiency in mammals. The results of feeding B6D2F1 hybrid mice 5, 10, or 20 ppm of DDT in the first breeding experiment showed an increase in litter size and number weaned over controls. Ten ppm of DDT caused increases over controls in the number born and alive on day 1 (P < 0.05). DDT caused decreases in the weight of pups at 5 ppm on days 15 and 30 (P < 0.05 and 0.01, respectively), at 10 ppm on day 15 (P < 0.005), and at 20 ppm on day 30 (P < 0.05). A second breeding experiment using 30, 60, or 120 ppm of DDT showed a general detrimental effect on reproduction. Thirty ppm DDT-fed animals produced fewer pups than controls (P < 0.01) at days 1, 15, and 30, and the pups weighed less than controls at 30 days of age (P < 0.05). DDT at 120 ppm resulted in fewer mice at birth and at 30 days of age (P < 0.01) than control diet. The third, fourth, and fifth breeding experiments involved diets with 0, 5, 10, and 20 ppm of DDT and with the addition of a 40-ppm level in the third and fourth studies. The data showed larger litter sizes on days 1, 15, and 30 for DDT-fed animals. This investigation indicates that 5, 10, 20, or 40 ppm of DDT in the diet often results in larger litter sizes and more pups weaned than controls. Animals fed levels of 30, 60, or 120 ppm of DDT generally produced fewer offspring than did control animals. DDT-fed mice often weighed less before weaning than control pups. Although variable responses were found, it appears from these studies that environmental levels of DDT are not detrimental to the reproductive efficiency of mice and may under certain circumstances be beneficial.
1 This work was supported in part by the Illinois Agricultural Experiment Station. 2 Present address: White Mountain Research Station, University of California, 3000 East Line Street, Bishop, California 93514. 3 Present address: Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55101.
436
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Introduction
The insecticide DDT has, in a mere thirty years of use, been hailed as a panacea and reviled as a xenobiotic whose continued use may destroy the entire biosphere. The very qualities responsible for its remarkable success are responsible for its dangers: Being highly lipid soluble and only marginally soluble in water, it is as persistent in insects and their predators as in fields or on walls. Small quantities of DDT absorbed from water or food are concentrated in fatty tissues, and as the trophic level increases from phytoplankton to fish to piscivores, repeated concentrations can lead to increasing levels of DDT which may affect physiological processes. Despite numerous articles on the hazards of DDT, few data are available for the effects of DDT on reproduction in mammals (Deichmann et al. 1971, Pimentel 1971, Ware 1975), and much of the published literature is controversial. The purpose of this investigation was to examine the effects o f plausible levels of DDT on the reproductive performance of mammals. To minimize the effects of extraneous factors, genetically uniform hybrid mice were used as the experimental species, and the DDT was added to the food at levels which might be found under less artificial conditions; for example, in a treated field or barn. A preliminary report of this work has been presented (Ledoux et al. 1971),
Materials and Methods B6D2F1/J hybrid mice (progeny of C57B1/6J X DBA/2J), from The Jackson Laboratory, Bar Harbor, Maine, and/or their descendants were used in these experiments. They were placed 12 per cage when approximately four weeks of age and allowed to adjust for three weeks to the local laboratory conditions. At the time of mating they were caged one male with one female (unless otherwise noted) in plastic cages having hardware cloth lids with San-i-cel litter. Mouse weights were determined on a Shadowgraph balance. Full-sib and parent-offspring matings were avoided at all times. Food and water were supplied ad libitum. Control and test groups were run concurrently. Room temperature during the winter months was thermostatically controlled between 20° and 22° C. Summer room temperatures were not controllable and at times exceeded 22° C. Ventilation was supplied by a large variable speed and cycle exhaust fan. Lights were regulated 6 A.M.--6 P.M. on; 6 P.M.-6 A.M, off. Technical DDT (control number 1807, Nutritional Biochemical Co., Cleveland, Ohio) was used in this investigation. Gas/liquid chromatography revealed the following analysis for 98.5% of the material: p,p'-DDT, 70.0%; o,p'-DDT, 21.0%; p,p'-DDD, 4.0%; and p,p'-DDE, 3.5% (analysis courtesy of Dr. Willis Bruce, Illinois State Natural History Survey, University of l~inois, UrbanaChampaign). Due to the quantities of feed required, stock mixes were prepared and used for making the final feeding diets. The DDT stock diet was prepared by sprinkling 2.0 g of technical grade DDT dissolved in 50.0 ml of analytical grade acetone over 2.0 kg of ground laboratory meal (either Rockland Mouse/ Rat diet or Purina Laboratory Chow) and thoroughly m~ing it. After allowing acetone evaporation in a well-ventilated room, the diet was transferred to a 1 cuft drum and thoroughly mixed. The resultant 1000 ppm DDT stock diet was stored in airtight containers in a cool, dark place until needed. Control stock diet was prepared in the same manner, omitting the DDT. The diluted feeding diets were prepared in 50-1b lots by adding the appropriate quantity of stock mix to untreated meat in a large Hobart mixer. The weight of control acetone-treated stock diet was the same as the amount of DDT stock diet used to make the mean DDT treatment level diet within a given experiment. After 15 man of mixing, 10 to 12% water by weight was added and mixing was
Effects of DDT on Breeding Performance in Mice
437
continued 15 additional min. The diets were then pelleted in a laboratory pellet mill, and dried for 48 to 72 hr. The dry pellets were stored in plastic containers in a cool place.
First Breeding Experiment. Diets of 5, 10, and 20 ppm of DDT were fed to separate groups, each consisting of 20 pairs of B6D2F] J mice. Control diet was fed to 40 pairs. The experiment started December 1, 1969, and continued for 86 days. Breeding parameters examined included i) time of first vaginal plug 2) date of littering, 3) number of live and dead pups on day of birth and on days t5 and 30, and 4) weight of live mice in litters on days 1, 15, and 30.
Second Breeding Experiment. Diets of control, 30, 60, and 120 ppm of DDT were fed to separate groups of Fz mice (derived from the first breeding experiment's large control group) in an effort to determine the effect of increased feeding levels. Each group consisted of 20 pairs, except the control group which again contained 40 pairs. The experiment started February 16, 1970, and continued for 130 days. The data recorded were the same as in the first breeding experiment.
Third Breeding Experiment. This experiment was designed as a replicate of the first breeding experiment using newly obtained B6D2FI/J hybrid mice. It started June 30, 1971, and ended September 20, 1971. Treatment consisted of control, 5, 10, 20, and 40 ppm of DDT diets. Each level used 20 breeding cages, each housing one female and one male, except in the 0 and 10 ppm levels where two females were caged with one male (one of the females was randomly assigned to another series of experiments not reported here). The data recorded were the same as in the first breeding experiment.
Fourth Breeding Experiment. The preceding experiment was repeated with the following modifications. Mice were chosen at random (except that no full-sib matings were allowed) from the first litters of the third breeding experiment and were maintained on their respective levels of control, 5, 10, 20, and 40 ppm of DDT. The experiment s t ~ e d November 24, 1971, and continued 50 days, except that pairs with first litters born on or before January 14, 1972 were kept to weaning. The breeding parameters examined were the number born live and dead on day of birth, and the number live and their weights on days 15 and 30.
Fifth Breeding Experiment. Since previous experimental results suggested that factors other than DDT treatment were causing inconsistent results, e.g., season, a fifth experiment was undertaken. Nineteen breeding pairs were placed on diets of control, 5, 10, or 20 ppm of DDT. Seventeen pairs at each level were B6D2F1 hybrids, raised in our laboratory, while two pairs on each treatment level were Fz females with F1 males. Animals were on their respective DDT diets from December 6, 1971, when the experiment started, until February 4, 1972, when the 5 ppm matings were shifted to 10 ppm and the 20 ppm raised to 40 ppm of DDT. On February 7, 1972, all mice receiving DDT were placed on 40 ppm until the end of the experiment, February 19, 1972. Mice on the control diet were maintained throughout. The data recorded were the same as in the fourth breeding experiment.
T . A . Ledoux et al.
438
Biometrical Considerations. Least-squares procedures were utilized as outlined by Harvey (1960) for unequal subclass frequencies. The first and second breeding experiments were analyzed using the linear models: Y~k = u + Ti + Pj/Ti + Lk/pi/Ti Yi~k = u + T~ + P/Ti + bxD + Lk/PJTi
(Model A) (Model B)
where: YiJg = u = Ti = P]TI =
observation of the k th litter within the j ~hpair in the i th level. least-squares mean. deviation from the mean due to treatment. deviation from the mean due to treatment differences between individual pairs within a treatment. Lk/P]Ti = deviation from the mean due to differences between individual litters within pairs within treatment (random error). b1 = regression of the number of mice born in each litter on the average weight in the litter. D = number of mice born in each litter. In the analysis of experimental results, if the F ratio for treatment was significant at the 0.05 level or less, a two-sided Dunnett's test was used to compare the treatment means vs. the control mean for the particular dependent variable being evaluated (Steel and Torrie 1960). Whenever the F test in the preceding models was not significant for the pairs within treatment category, the pairs within treatment and the litters within pairs within treatment categories were pooled and the following models were used: YiJ = u + Ti + % Ya = u + Ti + b~D + ei~
(Model C) (Model D)
where: Yu = observation of the j th litter on the i t~ level. u = least-square mean. Ti = deviation from the mean due to treatment. e~ = random error. b l = regression of the number of mice born in each litter on the average weight in the litter. D = number of mice born in each litter. The dependent variables examined using models A and C in the first and second breeding experiments were; the number born, number live on days 1, 15, and 30, and the average live weight on days 1, 15, and 30. Average live weights adjusted for the number born on day one were analyzed using models B and D. Analysis of results from the first two breeding studies established the size of difference needed between treatment means and the magnitude of their standard errors necessary for statistical significance. If treatment means and standard errors were similar in magnitude or smaller than those required to previously achieve statistical significance, the data did not warrant further statistical analysis. Other experiments in which the results indicated a statistical analysis might be of value were analyzed using a one-way Classification.
Results and Discussion First B r e e d i n g E x p e r i m e n t . M o s t f e m a l e m i c e e x h i b i t e d vaginal plugs d u r i n g the first f o u r d a y s o f b e i n g paired with males. The highest incidence of mating occurred on the third day w i t h 38, 45, 60, a n d 4 5 % o f t h e f e m a l e s s h o w i n g p l u g s in t h e c o n t r o l , 5, 10, a n d 20
Effects of DDT on Breeding Performance in Mice
439
p p m DDT-fed groups, respectively. It has previously been reported that female mice caged in groups without a male have irregular estrous cycles and that pairing these females with males tends to synchronize their cycles so that on the third night the percentage of matings increases (Whitten 1966). This p h e n o m e n o n was frequently seen in our experiments. The number of females which produced first through fourth litters is shown in Table 1. Many of the females not producing successive litters were pregnant during the course of the experiment, but either did not deliver pups or killed and ate them during delivery. E a c h group of DDT-fed mice produced proportionately more litters than did the controls. This increased production of litters associated with D D T feeding is similar to that reported by Ottoboni (1969). These data were statistically analyzed using statistical Models A, B, C, and D and the resulting appropriate least-square treatment means with their standard errors are presented in Table 2. The results show a generalized increase in litter size for DDT-treated mice o v e r that of control mice. Mice consuming 10 p p m of D D T produced significant increases in number born and n u m b e r alive on day one compared to control values (P < 0.05). D D T at 5 and 10 p p m significantly decreased adjusted live weights on day 15 (P < 0.05) and 5 and 20 p p m significantly decreased adjusted live weights on day 30 (P < 0.01 and < 0.05, respectively). The regression for the n u m b e r born on day one (live weight) was highly significant (P < 0.001). These data showed that the reproductive capacity o f these mice was increased by the presence of D D T in the diet since the average litter size, which paralleled the results of Wrenn et al. (1970) in rats, and the number weaned were larger for all DDT-fed mice than the controls.
Second Breeding Experiment. The levels of DDT were increased to 30, 60, and 120 p p m in this experiment. The majority of females at all treatment levels had vaginal plugs by day four after pairing. The results on the number of females which produced first through fifth
Table 1. Number of litters produced by F~ mice fed DDT in the first breeding experiment ppm DDT fed 0
5 10 Pairs of mice set
20 Total
Litter no.
40
20
20
20
t00
First Second Third Fourth Total
40 36 t4 1 91
20 19 13 0 52
20 20 11 0 51
20 20 12 1 53
100 95 50 2 247
440
T.A.
L e d o u x et al.
Table 2. S u m m a r y of least-squares treatment m e a n s and their standard errors for F1 mice fed D D T in the first breeding experiment p p m D D T fed 0
5
10
20
20
20
20
6.19 + 0.29 (52) 5.92 ± 0.32 (48) 5.08 ± 0.49 (40) 5.00 ± 0.38 (39)
6.84 ± 0.30 ~ (51) 6.49 ± 0.32 a (49) 5.87 ± 0.48 (43) 5.65 ± 0.38 (43)
No. pairs set Day
40
1
No. born
1
No. live
15
No. five
30
No. live
5.65 -+ 0.24 O1) 5.21 ± 0.27 (84) 5.27 ± 0.41 (70) 4.66-+ 0.32 (70)
Day
6.55 ± 0.29 (53) 6.32 ± 0.32 (51) 5.87 +- 0.48 (46) 5.49 ± 0.38 (45)
L i v e w e i g h t ( g ) a ~ u s t e d f o r no, born
1 15 30
1.14 ± 0.03 6.23 ± 0.25 12.57 ± 0,51
1.17 ± 0.04 5.01 ± 0.29 a 9.98 ± 0.59 b
1.12 ± 0.04 5.22 ± 0.29" 10.72 ± 0,59
1 . 0 9 ± 0;04 5.48 ± 0.29 10.41 ± 0.58 a
a-~Significantly different from the control at the 0.05 and 0.01 levels o f probability, respectively, using Dunnett's test after first obtaining a significant F test using A N O V A on the variable ( ) = N u m b e r of fitters
litters (Table 3) show that proportionately only the second litters at 30 ppm and second and third litters at 60 ppm DDT exceeded the number of litters produced by the controls. As the experiment progressed, the percentage of DDT-treated mice which produced litters generally decreased faster than the control mice. The appropriate least-squares treatment means and standard errors are in Table 4. The results show a marked decrease in the parameters measured for the Table 3. N u m b e r of fitters produced by F 2 m i c e f e d D D T in the second breeding experiment ppmDDTfed 0
30 60 Pairs of mice set
120 Tot~
Litter no.
40
20
20
20
100
First Second Third Fourth Fifth Total
40 36 34 29 11 150
20 19 14 7 1 61
20 20 17 12 3 72
20 17 14 10 4 65
100 92 79 58 t9 348
Effects of DDT on Breeding Performance in Mice
441
Table 4. Summary of least-squares treatment means and their standard errors for Fz mice fed DDT in the second breeding experiment ppm DDT fed
Day 1
No. born
1
No. live
15
No. live
30
No. live
0
30
40
20
7.55 ± 0.22 (150) 6.76 ± 0.43 (145) 4,91 ± 0,28 (113) 4.00 ± 0.28 (94)
Day 1 15 30
60 No. pairs set
4.56 ± 0.29 b (61) 4.03 ± 0.48 b (54) 2.85 _+ 0.37 ~ (38) 1.74 ± 0.37 b (26)
20
120
20 6,83 ± 0.27 (72) 6.37 ± 0.46 (69) 4.09 + 0,35 (51) 3,49 ± 0.35 (51)
6.29 ± 0,28 b (65) 6.25 _+ 1.08 (60) 4,12 ± 0.36 (45) 2.66 +_ 0.36 b (32)
Live weight (g) adjusted for no. born 1.32 ± 0.12 4.92 ± 0.28 8.64 ± 0.58
1.03 ± 0.13 4.44 _+ 0.36 5.95 ± 0,77 a
1.03 ± 0,12 4.62 ± 0.33 7.24 ± 0.70
1.24 ± 0.29 4.48 -+ 0.34 6.30 ± 0.72
a,b Significantly different from the control at the 0.05 and 0,01 levels of probability, respectively, using Dunnett's test after first obtaining a significant F test using ANOVA on the variable ( ) = N u m b e r of litters
30 ppm DDT-fed mice compared with controls. The results for mice on 60 and 120 ppm of DDT showed a greater reproductive capacity than those for 30 ppm but were still below those for the controls. The percentage postnatal mortality to day 15 or 30 was similar for all treatment groups except at day 30 where the percentage tended to be higher for the 30 and 120 ppm groups. Statistical analysis results showed that mice receiving 30 ppm of DDT had significantly fewer pups born; fewer alive on days 1, 15, and 30 (P < 0.01); and significantly lower pup weights on day 30 when adjusted for number born (P < 0.05). At 120 ppm there were fewer pups born and alive on day 30 (P < 0.01). The regression for number born on day one for live weight was not significant. The results, in contrast to those from the first experiment, suggest that these higher levels of DDT were detrimental to reproductive response. However, the lack of a dose response with the effect occurring with 30 and 120 ppm and not with 60 ppm suggest that factors other than DDT may have been involved.
Third Breeding Experiment. This experiment was designed to see if the results from the first experiment were repeatable. The results for littering response (Table 5) and breeding capacity (Table 6) showed that DDT levels of 5, 10, 20 or 40 ppm had no significant effect on the parameters measured.
442
T . A . Ledoux e t al.
Table 5. Number of litters produced by F1 mice fed DDT in the third breeding experiment ppmDDTfed 0
5
10 Pairs of mice set
20
40 'l'ot~
Litter no.
20
20
20
20
20
100
First Second Third TotN
20 20 16 56
20 20 13 53
20 19 14 53
20 20 17 57
20 20 15 55
100 99 75 274
During this experiment there was a much higher female mortality rate than w a s s e e n i n t h e p r e v i o u s e x p e r i m e n t s , b u t t h e d e a t h s w e r e d i s t r i b u t e d a c r o s s all treatments and thus not treatment-related. All of the females were pregnant and nursing a previous litter at the time of death. The Veterinary Diagnostic Laboratory identified no specific cause of death. Replacement foster females were chosen from a preliminary experiment from the same or next lower level of DDT. The combined stress of pregnancy, lactation, and heat of summer may have c o n t r i b u t e d t o t h e h i g h e r m o r t a l i t y a m o n g t h e f e m a l e s . L e s (1967) r e p o r t e d t h a t 95 ° F, b u t n o t 86 ° F , e x e r t e d a d e t r i m e n t a l e f f e c t o n g r o w t h a n d r e p r o d u c t i o n o f m a n y i n b r e d s t r a i n s o f m i c e . H e n o t e d n o e f f e c t s o f r e l a t i v e h u m i d i t y i n t h e 30 t o 60% range.
Table 6. Treatment means and their standard errors for F~ mice fed DDT in the third breeding experiment ppm DDT fed 0
Day 1
No. born
1
No. live
15
No. live
30
No. live
5
10 No. pairs set
20
40
20
20
20
20
20
8.86 ± 0.21 (56) 8.77 .+_ 0.22 (56) 8.27 ± 0.38 (45) 8.74 ± 0.39 (35)
8.60 +- 0.30 (53) 8.67 ± 0.28 (52) 8.98 ± 0.26 (41) 8.79 ±_ 0.25 (38)
8.22 ± 0.29 (53) 8.30 ± 0.28 (52) 8.t5 ± 0.32 (40) 7.76 ± 0.38 (33)
8.54 ± 0.27 (57) 8.53 ± 0.27 (57) 8.34 -+ 0.30 (45) 8.01--+ 0.28 (39)
8.71 _+ 0.29 (55) 8.89 ± 0.24 (53) 8.82 ± 0.32 (38) 8.62 ± 0.40 (34)
1.33 _+ 0.02 7.25 ± 0.14 14.92 ± 0.22
1.33 ± 0.02 6.88 ± 0.17 14.44 ± 0.34
Live weight (g) 1 15 30
1.29 ± 0.02 6.96 ± 0.19 14.20 ± 0.38
( ) = Number of litters
1.32 ± 0.02 6.93 _+ 0.11 13.82 ± 0.33
1.29 ± 0.02 6.95 ± 0.17 15.01 ± 0.28
Effects of DDT on Breeding Performance in Mice
443
Table 7. Number of litters produced by Fz mice fed DDT continuously in the fourth breeding experiment ppm DDTfed 0
5
10 20 Pairs of mice set
40 Total
Litter no,
20
18
20
19
20
97
First Second TotN
20 13 33
18 14 32
20 12 32
19 ll 30
20 9 29
97 59 155
Fourth Breeding Experiment. T h e m i c e u s e d in t h i s e x p e r i m e n t w e r e r a n d o m l y s e l e c t e d f r o m t h e first l i t t e r s o f the third experiment and thus were subjected continuously to their respective t r e a t m e n t f r o m the t i m e o f c o n c e p t i o n . T h e d a t a p r e s e n t e d in T a b l e s 7 a n d 8 s h o w n o s i g n i f i c a n t d i f f e r e n c e s in t h e p a r a m e t e r s m e a s u r e d b e t w e e n t h e c o n t r o l s and any of the treatment levels.
Table 8. Treatment means and their standmd errors for F2 mice fed DDT continuously in the fourth breeding experiment ppm DDT ted 0
5
20
18
8.33 _+ 0.38 (33) 8.21 +_ 0.40 (33) 7.63 ± 0.64 (19) 7.33 + 0.62 (18)
8.69 ± 0.44 (32) 8,87 + 0.39 (31) 8,47 ± 0.54 (17) 8,13 _+ 0.58 (16)
Day 1
No. born
1
No. live
15
No. live
30
No. live
I0 No. pairs set
20
40
20
19
20
8.09 :#: 0.45 (32) 8.19 ~:: 0.41 (31) 8.25 ± 0.54 (20) 8.00 ± 0.51 (20)
8.37 ± 0.44 (30) 8.37 _+ 0.44 (30) 8.26 ± 0.51 (19) 7.47 ± 0.58 (15)
8,76 ± 0.49 (29) 8.76 _+ 0.49 (29) 8.35 _+ 0.51 (20) 8.05 ± 0.57 (18)
6.48 ± 0.28 14.18 ± 0.40
6.I8 ± 0.32 13.12 _+ 0,51
Live weight (g) 15 30
6.20 + 0.25 13.57 ± 0.49
( ) = Number of litters
6.50 ± 0,29 14.34 ± 0,59
6.39 ± 0.30 14.22 + 0.59
444
T.A.
L e d o u x et al.
Table 9. N u m b e r of fitters produced by F1 and F~ mice fed D D T in the fifth breeding experiment
ppm DDT fed 0
5 10 Pairs of mice set
20 Total
Litter no.
19
19
19
19
76
First Second Third Total
19 19 10 48
19 17 7 43
19 17 7 43
19 17 5 4t
76 70 29 175
Fifth Breeding Experiment. The results of this experiment are shown in Tables 9 and 10. There was a small, nonsignificant increase in litter size in favor of the DDT-treated mice. The small weight differences at various times were also insignificant. The combined experiments involved over 470 breeding pairs of mice which produced 1190 litters and 8800 pups for a total of over 10,500 observable units. In addition to the rigorously analyzed parameters, certain general conclusions can be drawn about the reproductive state of the colony. No macroscopic evidence of tumors was observed in any of the mice, Table 10. Treatment m e a n s and their standard errors for F~ and F2 mice fed D D T in the fifth breeding experiment
p p m D D T fed 0
5
I0
20
19
19
No. pairs set 19
Day 1
No. born
1
No. live
15~
No. live
30
No. live
8.40 _ 0.31 (48) 8.47 ± 0.29 (47) 8.05 ± 0.26 (19) 7.75 ± 0.53 (28)
t9 8.51 ± 0.28 (43) 8.66 ± 0.25 (41) 7.50 -+ 0.24 (18) 7.61 ± 0.26 (23)
8.63 _+ 0.30 (43) 8.46 ± 0.34 (43) 8.00 _+ 0.24 (19) 8.11 ___ 0.28 (28)
8.85 --- 0.24 (41) 8.86 ± 0.24 (41) 7.67 --- 0.38 (18) 7.54 ± 0.30 (22)
6.31 +_ 0.25 13.64 ± 0.33
6.09 --- 0.29 13.81 _+ 0.46
Live weight (g) t5 a 30 a First litter data only ( ) = N u m b e r of fitters
6.40 + 0.30 14.10 --- 0.39
7.23 + 0.26 14.36 _+ 0.21
Effects of DDT on Breeding Performance in Mice
445
suggesting that DDT treatment was not carcinogenic. The duration of treatment was not, however, long enough for rigorous tests. The few gross abnormalities seen in the offspring were distributed equally across treatment groups. They consisted of hydrocephalus, characteristic of the C57BL/6J parental strain, (experiments 1 and 2); runts (experiments 1, 2, 4, and 5); and transient hair loss at weaning (experiments 1 and 2). Hydrocephalus and runting were rare occurrences while the hair loss sometimes affected entire fitters. Mean gestation length in the colony was 19 days, with no differences among treatments. A few females were infertile or aborted or destroyed fitters before births were recorded. Such losses were not treatment related and were rare in all experiments. The combined results of the third through fifth breeding experiments show that DDT-fed mice tended to produce larger litters than controls. The short interval between the beginning of treatment and mating argues against an increased ovulation rate, and consequently for a decreased embryonic mortality. The data therefore support the conclusion that, under these experimental conditions, DDT was neither teratogenic nor highly carcinogenic, and improved the reproductive performance of treated pairs. Inconsistencies between experiments must be ascribed to unidentified variables other than noise, pheromones, temperature, and crowding. The mice were housed in an infrequently used area, and no inconsistent noises were generated within the room. All treatment groups were equally crowded, and treated and experimental mice were housed in the same parts of the room. The room was not temperature controlled in summer, but variations should have affected all treatment groups equally. The most feasible explanation lies in an interaction of DDT with other stress factors, as was elegantly demonstrated for toxaphene in fish (Mehrle and Mayer 1975a and 1975b). Inconsistent data are familiar in studies on DDT, and the present results are similar to those reported by Ware and Good (1967) who also fed low dietary levels of DDT to mice. The present results also partially support the results reported for rats by Fitzhugh (1948), Ottoboni (1969), Treon and Cleveland (1955), Read et al. (1965) and Phillips et al. (1971), but are in disagreement with the results of Fahim et al. (1970) in rats, Hart et al. (1971) in rabbits, and Deichmann et al. (1971) in beagles. It is obvious from the results of this study and others that levels of DDT which might be encountered in the environment are not detrimental to the reproductive efficiency of mice and may under certain circumstances be beneficial. There are still some questions which need to be answered regarding the cause(s) of the variable responses found which may only be due to biological variability. The results presented here with the variable response help also to explain the variability and confusion among reports in the literature on the effect of DDT in the reproduction of animals.
References Deichmann, W. B., W. E. MacDonald, A. G. Beasley, and D. A. Cubit: Subnormal reproduction in beagle dogs induced by DDT and aldrin. Ind. Med. Surg. 40, 10 (1971).
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Fahim, M. S., R. Bennett, and D. Goodsell: Effect of DDT on the nursing neonate. Nature 228, 1222 (1970). Fitzhu~a, O. G.: Use of DDT insecticides on food products. Industr. Eng. Chem. 4, 704 (1948). Hart, M. M., S. Fabro, and R. H. Adamson: Prematurity and intrauterine growth retardation induced by DDT in the rabbit. Arch. Int. Pharmacodyn. Thera. 192, 286 (1971). Harvey, W. R.: Least-squares analysis of data with unequal subclass numbers. U.S.D.A. Agr. Res. Ser. Publ. ARS 20-8 (1%0). Ledoux, T. A., J. R. Lodge, and R. W. Touchberry: Reproductive performance of B6D2F~/J mice fed DDT. Program of the Fourth Annual Meeting of the Society for the Study of" Reproduction, Boston. Biol. Reprod., 5, 95 (1971). Les, E. P.: Effects of temperature and humidity on mice. In: The Jackson Laboratory 38th Annual Report 1%6-1967, p. 102. Bar Harbor, Maine: The Jackson Laboratory (I%7). Mehrle, P. M. and F. L. Mayer, Jr.: Toxaphene effects on growth and bone composition of fathead minnows, Pimephah, s promelas. J. Fish. Res. Board Can. 32, 593 (1975a). Mehrle, P. M. and F. L. Mayer, Jr.: Toxaphene effects on growth and development of brook trout (Salvelinus fontinalis). J. Fish. Res. Board Can. 32, 609 (1975b). Ottoboni, A.: Effect of DDT on reproduction in the rat. Toxicol. Appl. Pharmacol. 14, 74 (1969). Phillips, W. E. J., G. Hatina, D. C. Villeneuve, and D. L. Grant: Multi-generation studies on the effect of dietary DDT on the vitamin A status of the weanling rat. Can. J. Physiol. Pharmacol. 49, 382 (1971). l~mentel, D.: Ecological effects of"pesticides on non-target species. U. S. Government Printing Office #4106~0029 (1971). Read, S. I., T. K. Murray, and W. P. McKinley: The effect of DDT on the liver carboxylesterase and vitamin A utilization of mother rats and their young. Can. J. Biochem. 43, 317 (1965). Steel, R. G. D., and J. H. Torrie: Principles and procedures of statistics. New York: McGraw-Hill Book Company, Inc. (I960). Treon, J. F., and F. P. Cleveland: Toxicity of certain chlorinated hydrocarbon insecticides for laboratory animals, with special reference to aldrin and dieldrin. J. Agr. Food Chem. 3, 402 (1955). Ware, G. W.: The effects of DDT on reproduction in higher mammals. Residue Reviews 59, 119 (1975). Ware, G. W., and E. E. Good: Effects of insecticides on reproduction in the laboratory mouse: II. Mirex, Telodrin, and DDT. Toxicol. Appl. Pharmacol. 10, 54 (1967). Whitten, W. K.: Pheromones and mammalian reproduction. Adv. Reprod. Physiol. 1, 155 (t966). Wrenn, T. R., J. R. Wood, G. F. Fries, and J. Bitman: Tests of Estrogenicity in rats fed low levels of o,p'-DDT. Bull. Environ. Contamin. Toxicol. 5, 61 (1970).
Manuscript received December 4, 1976; accepted May 16, 1977
