Pestic. Sci. 1975, 6, 337-345
'I
Glenn L. Crouch and Mohamed A. Radwan
Pacific Northwest Forest and Range Experiment Station, Olympia, Washington, USA
(Manuscript received 31 December 1974 and accepted 24 March 1975)
Chemically treated and untreated Douglas fir seed was evaluated in the laboratory and at four locations on National Forests in Oregon and Washington. Seed was treated by coating with endrin or endrin-thiram and by impregnation with endrin.
In the laboratory, germination was adversely affected by impregnation and by field exposure. Also, bioassays of treated seed showed no differences in consump­ tion by deer mice among endrin treatments, but more seed. was eaten after exposure in the field than before. Deer mice were the most common seed eaters found on all field study areas, and their numbers exceeded the level prescribed as a minimum requiring protection prior to seeding. Observations during and at the end of two growing seasons indicated that seedling production and stocking were much greater from endrin-treated than untreated seed, but there were no signi­ ficant differences among the three endrin treatments. In addition, performance of treated and untreated seed differed by area, with best results at the two locations known for favourable climatic conditions.
1. Introduction
Endrina has been extensively used in a coating treatment to protect field-sown seed from rodents since the treatment was recommended by the Bureau of Sport Fisheries and Wildlife.! Despite recent declines, direct seeding of conifers was still employed to regenerate more than 34 425 ha in the Pacific Northwest in 1972.2 Seed of Douglas fir
(Pseudotsuga menziesii) was applied to most of these hectares at 1.12 kg or less per hectare, and virtually all the seed was coated with endrin.
Although efficacy of endrin as a seed protectant on Douglas fir has previously been demonstrated in the field,5,6 many failures have occurred where seed-eating rodents were believed responsible. Accordingly, various modifications of the original treatment were tried, including elimination of thiram
tetramethylthiuram disulphide
and increasing the amount of endrin. Unfortunately, these changes were largely empirical and benefits, if any, were not documented.
Recently, Douglas fir seed was impregnated with endrin without impairing laboratory germination.7 Bioassays of impregnated seed with caged deer mice a
1,2,3,4,10,10-Hexachloro-6,7-epoxy 1,4,4a,5,6,7,8,8a-octahydro-exo-l,4-exo-5,8-dimethanonaph­ thalene. Mention of chemical compounds and chemical companies does not represent endorsement by the US Forest Service or the Department of Agriculture. Endrin is registered for forest tree seed protection in the United States. However, at this writing the compound is not used on lands owned by the Federal Government.
337
1

338 G. L. Crouch and M. A. Radwan
(Peromyscus maniculatus) suggested that impregnation would provide better field protection from these common seed eaters and with less endrin than coating with endrin alone or endrin in combination with thiram. Moreover, it appeared that impregnation would provide longer lasting protection than coating since the chemical would be less susceptible to losses from weathering.7 Subsequent bioassays also showed that addition of thiram to ¢ndrin did not improve seed protection. 3
Results of these laboratory tests and the urgency to reduce amounts of chemicals applied to forest lands prompted a test of different seed treatments under field con­ ditions. The test had three objectives: (a) to evaluate impregnation with endrin as an alternative to coating ; (b) to determine if thiram adds to the protective value of endrin, and (c) to reassess the need for chemical protection of field-sown Douglas fir seed.
Treated and untreated seed was used and the test was replicated at four widely sepa­ rated locations representative of Douglas fir sites on National Forests in western
Oregon and Washington.
2. Study areas
One study site was located in the Oregon Coast Ranges (Alsea), another in the
Washington Cascade Range (Randle), a third in the northern Cascades of Oregon
(Clackamas), and the fourth in the southern Oregon Cascades (Tiller). All sites were recent clearcuts, with slash burned in the year of seeding. All had supported Douglas fir, although the three Cascade Range sites also had been stocked with other conifer species, including true firs (Abies spp.) and western hemlock (Tsuga heterophylla).
All areas were located on moderate north- or east-facing slopes-the Cascade sites at about 915 m in elevation and the Coast Ranges site at 183 m.
3. Materials and methods
3.1. Seed
Four 2 . 27 kg samples of seed from lots appropriate to the four study areas were obtained from storage at the US Forest Service Nursery" Wind River, Washing­ ton.
3.2. Chemicals
Endrin 50-WP (50 % wettable powder from Stauffer Chemical Company) and Arasan
42-S (42 % liquid thiram from the DuPont Company) were the active ingredients of the treatments. For seed coating, Dow Latex 512-R (from the Dow Chemical
Company) served as adhesive to bind the active ingredients to the seed, and aluminium powder was used to minimise seed clustering and to identify treated seed. For impreg­ nation, endrin solutions containing 2 % active endrin were prepared by dissolving ndrin 50-WP in 1,2-dichloroethane and filtering out the insoluble ingredients of the powder.7

Protecting Douglas fir seed with endrin 339
3.3. Seed treatments
Each 2.27 kg sample of seed was divided into four equal parts and treated as follows:
1. Untreated (control): Seed was not chemically treated.
2. 0.5 % endrin: Seed was coated with endrin at a concentration of O. 5 % by weight
(1. 0 % of Endrin 50WP) using 12 ml of diluted adhesive (1 adhesive: 9 water by volume) and 1 .2 g of aluminium powder per 100 g seed.
3. O. 5 % endrin + 8 .0 % thiram: Seed was coated with endrin at O. 5 % by weight and with a volume of liquid Arasan 42-S that provided solid thiram at 8 % by weight.
Undiluted adhesive and aluminium powder were used at a rate of 2.8 ml and 1.2 g, respectively, per 100 g seed.
In treatments 2 and 3, seed was wet with slurries of endrin or endrin-thiram in the adhesive, covered with aluminium powder, spread in a thin layer, and allowed to dry overnight under a fume hood.
4. Impregnation with endrin: Seed was dried in a forced-air oven at 40°C for 48 h.
After cooling, the seed was enclosed in cheesecloth, immersed in the endrin­ dichloroethane solution for 1 h, and dried ina forced-air oven at 400e for 24 h.
3.4. Field seeding
On each clearcut, five, square, 0.41 ha test blocks were established. Blocks were separated by at least 64. 05 m and placed 64.05 m or more from uncut timber. 0.45 kg of seed of each treatment was hand-broadcast with a cyclone seeder on one block at each location. In addition to the four treatments, an unseeded block was also included at each location to measure recruitment from natural seedfall. Block assignments were made at random, and seed was sown across slopes in November and December 1969.
Prior to seeding, 100 live traps were set within each clearcut for one night to esti­ mate abundance of deer mice and other seed-eating mammals. Animals caught were released alive after identification and tallying.
Tp evaluate seedling production, 25 circular (4.05 m2) plots were established in each treatment block. Plots were systematically spaced and centre-marked by wooden stakes. They were searched for seedlings in May, July and September 1970 and June and September 1971. Seedlings found at each inspection were marked with wire pins to permit their relocation and identification of new plants at subsequent examinations.
To provide seed for laboratory tests, eight 0.19 m
2 subplots were established at each field location. About 0.03 kg of seedof each treatment were distributed at seeding time on two of the subplots and covered with 0.85 cm mesh screening. At the Tiller and Alsea sites, one of the two subplots containing seed of each treatment was provided with a window screen bottom to prevent rodents from removing seed from below.
Seed from selected subplots was collected prior to germination for laboratory testing.
3.5. Germination
Shortly after treatment, four 100 seed replicates of seed of each treatment were germinated in the laboratory on perlite at 24 °e ± 1 °e after stratification for 21 days at 3 to 5 °e.· Similar replicates of seed, collected from the protected subplots at each field location after 4 to 5 months of exposure, were germinated as above, but without stratification. In all tests, germinants were counted at weekly intervals for 4 weeks.

340 G. L. Crouch and M. A. Radwan
3.6. Bioassay
Deer mice live-trapped near Tumwater, Washington, were used to evaluate the effects of field exposure on the protective capacity of the treatments. Seed tested consisted of unweathered samples held under refrigeration and seed weathered for 4 to 5 months at the Alsea and Tiller sites. For the bioassays, ten seeds of one treatment were fed daily to each of eight freshly caught mice for five consecutive days. Water and a commercial pelleted ration were offered ad libitum before and during each test.
4. Statistical analysis
All data were subjected to analysis of variance with arc-sine transformation when appropriate. Means 'Yere separated according to Tukey's test9 or appropriate ortho­ gonal comparisons were made at the 5 % probability level, as required.
5. Results
5.1. Rodent populations
Numbers and species of rodents caught prior to seeding are shown in Table 1. As expected, deer mice were the most common seed eaters which justified the use of this species in bioassay tests. It is likely that trapping beyond one night would have caught other species, but this was unnecessary to accomplish the purpose of trapping.
Table 1. Numbers and species of rodents live-trapped and released on four National Forest locations in the Pacific Northwest prior to seeding with endrin-treated and unt.t;eated Douglas fir seeda
Animal species Alsea
Location (number caughtb)
Randle Tiller Clackamas
Peromyscus maniculatus
(deer mouse)
Spermophilus sp.
(mantled ground squirrel)
Clethl'ionomys sp.
(red-backed mouse)
Eutamias sp.
(chipmunk)
Neotoma sp.
(woodrat)
Totals
20
0
0
0
0
10
0
0
0
0
15
0
0
0
1
21
1
1
1
0
20 10 16 24 a Trapping was conducted by F. H. Armstrong, US Forest Service, Pacific Northwest Region,
Portland, Oregon. b In 100 live traps exposed for one night on each area.
Numbers of deer mice from each study area exceeded the level prescribed as a minimum requiring protection from seed-eating rodents in operational artificial-seeding pro­ grammes on National Forests in the Pacific Northwest Region.10

Protecting Douglas fir seed with endrin 341
Additional traps set in uncut timber adjacent to each study area caught even more deer mice per trap-night than those exposed on the clearcuts.
5.2. Seedling production
Snow cover prevented inspection of the higher elevation areas until late April 1970.
At that time, germinants were found on all treatment plots and the small protected subplots. Considerable sound seed was found on most endrin-treatment blocks, but many seed hulls and fragments were also evident.
In late May, the first systematic examination for seedlings found many ungerminated seeds and many others which had begun to germinate but then apparently ceased activity. Such seeds were most prevalent on the Clackamas area. Numbers of germi­ nants and percentages of 4. 05 m
2 plots stocked with:one or more seedlings are shown in Figure 1 (a) and l(b). Throughout the 2 year examination period, only two seedlings were found on unseeded plots. During this time, endrin-treated seed on all the study sites produced about six times more seedlings than untreated seed, but orthogonal comparisons indicated no significant differences among the three endrin treatments.
D Cumulative seedlings
_ Net seedlings a.
V> c:
=0
Q)
C/)
10 000 r
7500 1-(1 )
5000
2500
0
(2) (3)
(4)
First season
I
I
I
I
I
I
I
I
I
I
I
(a)
1
I
I
(I) v
(2) (3)
(4) r;J
Second seasan
£...
. N
E l{)
0
<i
0 ti
V>
:§ a.
40
20
80
:-
60
'-(I) (2) (3)
0
First season
(4)
I
I
I
I
I
I
I
( b)
I
I
I
I (I)
1
I
I
I v
(2) (3)
II
Second season
Figure 1. Cumulative and net seedlings (a) and stocking (b) from endrin-treated and untreated
Douglas fir seed after two growing seasons on four National Forest locations in the Pacific Northwest.
(In (a) and (5), combined endrin treatments differ significantly [P=O.05] from the controls in each category and season. Differences among endrin treatments are not significant.) (1) Endrin coated;
(2) endrin impregnated; (3) endrin+thiram coated; (4) untreated (control).
Also, numbers of surviving seedlings after two growing seasons were more than four times greater from endrin-treated than untreated seed. Again, there were no significant differences among endrin treatments.
Percentages of 4. 05 m
2 plots stocked with one or more seedlings followed a similar pattern; but differences, although significant between endrin-treatment and control plots, were not as dramatic [Figure 1 (b)]. Records of individual 4. 05 m
2 plots revealed that after two growing seasons 60 % of the stocked endrin-treatment plots had two or more seedlings compared with less than 20 % of the control plots.

342 G. L. Crouch and M. A. Radwan
As expected, seedling production and survival were markedly different among study sites [Figure 2(a)]. For all treatments, cumulative numbers of seedlings produced at
Alsea and Randle were significantly higher and subsequent losses lower than those at
Tiller and Clackamas. After two growing seasons at Alsea and Randle, numbers of seedlings present on endrin-treatment plots, 4843/ha and 3361/ha, respectively, were also enough to stock these areas satisfactorily.
CJ
Cumulative seedlings
_ Net seedlings
10 000 (a) 80-
( b)
(/)
0'
,5
=0
Q)
(j)
7500-
5000 f-
2500
I-
0
'0
601--
-
Alsea Randle
-
I rJ
Tiller
Location
Cl ackamas
"'" o ti
(/)
-0
0..
N
E o
<i
401-
20-
-
Alsea Randle Tiller Clackamas
Locotion
Figure 2. Cumulative and net seedlings (a) and stocking (b) from endrin-treated and untreated
Douglas fir seed after two growing seasons on four National Forests in the Pacific Northwest. (a),
Alsea and Randle do not differ, but combined differ significantly (P
=
0.05) from Tiller and Clackamas.
Tiller does not differ from Clackamas. (b), In cumulative stocking, Alsea and Randle do not differ, but combined are significantly different from Tiller and Clackamas. Tiller does not differ from
Clackamas. After two seasons, Alsea and Randle differ significantly from Tiller and Clackamas,
Alsea differs from Randle, and Tiller differs from Clackamas.
Similarly, 'during the 2 year study period, more 4.05 m
2 plots were stocked for all treatments on the Alsea and Randle areas than on the Tiller and Clackamas sites
[Figure 2(b)]. Cumulative stocking was also higher at Tiller than at Clackamas, and stocking percent after two growing seasons was highest at Alsea, followed by Randle,
Tiller and Clackamas. In addition, stocking on endrin-treatment plots was significantly higher than on the control plots at all locations on a cumulative and net basis after two growing seasons.
, 5.3. Germination
Results of laboratory germination tests are shown in Table 2. Overall, seed germination was significantly higher before than after field exposure, although the difference amounted to only 7 %. Germination was sjgnificantly higher in seed lots from Alsea and Randle compared with those from Clackamas and Tiller. This same pattern carried through into field seedling production, although Clackamas and Tiller were reversed in order in the field. Only the impregnation treatment caused a significant

Protecting Douglas fir seed with endrin 343 reduction in germination, and this result was caused by the poor performance of impregnated seed in the lot from Tiller.
Table 2. Laboratory germination of endrin-treated and untreated Douglas fir seed before and after exposure in the field on four National Forest locations in the Pacific Northwest
Seed treatmenta Alsea
Location (%b)
Randle Tiller Clackamas
Before exposure
Untreated (control)
0.5E
0.5E+8T
EDI
After 4 month exposure in the field
Untreated (control)
0.5E
0.5E+8T
EDI
87
85
78
76
90
92
88
88
89
87
85
72
92
69
75
81
78
71
72
26
68
65
59
25
80
86
84
72
60
39
68
68 a O. 5E=endrin-coated at 0.5%, 0 . 5E+8T=coated with 0.5%-endrin and 8% thiram, EDl=
,endrin-impregnated. b Germination percentages are means of four replications each. Grand means for main effects are:
Treatments: Control = 80a, O. 5E=74a, O. 5E+ 8T=76a, EDI= 64b; Location: Alsea = 86a, Randle
=81a, Tiller = 58b, Clackamas=70c; Exposure: Before=77a, after=70b.
Means within each category followed by the same letter do not differ significantly at the 5 % level.
In September 1970, seeds remaining in the protected subplots were collected at
Alsea and Clackamas. Examination under low-power magnification revealed that some seeds had germinated but not rooted or grown further. Others had apparently begun to germinate, but progressed no further than swelling and cracking of seedcoats, and a third group of seeds appeared intact, with no evidence of germination activity. Intact and cracked seeds were germinated, and results indicated that some viable seed was probably still present on the ground after the first growing season, especially on the
Alsea area. Subsequent field data confirmed this observation, since an average of more than 1730 new seedlings per hectare were counted on endrin-treatment plots at Alsea during the second year of the study.
5.4. Bioassays
Bioassays of endrin-treated seed from Alsea and Tiller before and after exposure in the field were conducted in October 1970. Results depicted in Figure 3 show no significant differences in feeding b
mice among endrin treatments, but that more seed was eaten after exposure than before. Analyses also indicate that significantly more seed of the
Alsea lot was eaten compared with the Tiller lot. The data further suggest that endrin­ coated seed from Alsea was less resistant to feeding after weathering than that from
Tiller.

344
100r
80f-
c
.9
Q.
E
::>
(J) c o u
"0
Q)
U)
60f­
40f­
201-
G. L. Crouch and M. A. Radwan
_
Before exposure c:::J After 4 month exposure
( 2) r-
(3 )
Alsea
Location
Figure 3. Consumption by deer mice of endrin-treated Douglas fir seed before and after exposure in the field at two National Forest locations in the Pacific Northwest. Each bar is the average percent­ age consumption by eight mice fed ten seeds per day for 5 days. Grand means for main effects are as follows. Treatment: (1)=63a, (2)= 60a, (3) = 55a. Location: Alsea=65a, Tiller = 54b. Exposure: before=51a, after = 68b. Means within each category followed by the same letter do not differ significantly at the 5 % level. (1) Endrin coated; (2) endrin
+ thiram coated; (3) endrin impregnated.
6. Discussion
Results show no significant differences in seedling production from field-sown Douglas fir seed coated with endrin, with or without thiram. Thiram, therefore, did not add to the protective value of endrin. Results of earlier studies in the laboratory3 and in the field
4 agree with this finding.
Seedling production from seed impregnated with endrin indicates that as much impregnated as coated seed would be required to produce a given number of seedlings.
Since impregnation and coating at 0.5 % require about the same amoupts of endrin per seed,7 there appears to be no justification for employing the laborious and costly impregnation technique in place of the relatively simple coating procedure. With
Douglas fir seed, impregnation with endrin allows little of the chemical to enter the seed and leaves most of the compound in and on the seedcoat.7 It is believed that impregnating with chemicals that are not as strongly adsorbed to seedcoats as endrin would permit protection with less amounts than with coating. Under these conditions, impregnation would have the advantage of introducing smaller amounts of chemicals into the environment.
The bioassay data of endrin-treated seed before and after exposure in the field most probably reflect effects of weathering on endrin losses from the seed.8 Such losses, especially from endrin-coated seed, were evidently higher at Alsea where seed was subjected to much greater weathering effects than at Tiller. Winter weather at the
183 m elevation Alsea site is mainly one of constant overcast and rainfall, about
127 cm between sowing and germination, broken only by occasional clear periods,

Protecting Douglas fir seed with endrin 345 and short-term snowfall. On the other hand, the Tiller site at 915 m was covered by snow soon after the seed was sown in November 1969 and remained snow-covered into
March 1970. Thus, endrin was subject to almost constant leaching at Alsea, but presumably much less at Tiller.
At all locations, performance of untreated seed was poor. Lacking protection by endrin, most of the seed was apparently consumed by seed-eating animals. Results, therefore, demonstrate that protecting Douglas fir seed from seed eaters is a major requirement for success of artificial regeneration of the species by direct seeding in the
Pacific Northwest. In earlier studies, other investigators5,6 came to a similar conclusion.
Although this study was not designed to evaluate artificial seeding as a means of reforestation, the results from Alsea and Randle reaffirm the utility of seeding if employed with judicious selection of areas, adequate provision for seed protection, and favourable weather conditions. Adequate stocking at these two locations occurred despite the absence of natural seedfall during the first year, and the relatively small amount of treated and untreated seed (1 . 82 kg/12 . 15 ha of clem'cut) available to seed eaters.
The apparent failure of the sown seed to restock the Tiller and Clackamas plots was attributed to unfavourable environmental conditions for Douglas fir seedling pro­ duction at these sites, a fact well known to local foresters. Also, the lower germination of the Tiller seed lot in the laboratory, especially after exposure and with impregnation, must have been a factor at that site.
Finally, there is little doubt that artificial seeding is a useful means of reforestation under suitable conditions. However, the probability of success is variable and dependent upon a multitude of uncontrollable physical and biological interactions.
Regardless of other variables, the writers reaffirm their belief that Douglas fir seed sown at 1.12 kg or less per hectare must be protected with endrin or another equally effective chemical if seeding effort is to have any reasonable chance for success.
References
1'. Anonymous. 1956
2. Anonymous.
Department of Agriculture, Forest Service, Washington, DC, 1972, 13 pp.
3. formulation for treatment 0/ coniferous tree seed. US Fish and Wildlife Service,
Wildlife Research Laboratory special release, Denver, Colorado, 1956, 2 pp.
Report 0/ forest and windbarrier planting and seeding in the United States. US
Crouch, G. L.; Radwan, M. A. US Department of Agriculture, Forest Service Research Paper
PNW-136, Pacific Northwest Forest and Range Experiment Station, Portland, Oregon, 1972,
7 pp.
4. Dick, J. ; Finnis, J. M.; Hunt, L. 0. ; Kverno, N. B. J. Forestry 1958, 56, 660.
5. Dimock, E. J. US Department of Agriculture, Forest Service Research Paper 20, Pacific North­ west Forest and Range Experiment Station, Portland, Oregon, 1957, 17 pp.
6. Hooven, E. F. Oregon Forest Lands Research Center Research Note 28, 1957, 8 pp.
7. Radwan, M. A.; Crouch, G. L. ; Ellis, W. D. US Department of Agriculture, Forest Service
Research Paper PNW-94, Pacific Northwest Forest and Range Experiment Station, 1970, 17 pp.
8. Radwan, M. A.; Ellis, W. D. Northwest Science 1971, 45,188.
9. Snedecor, G. W. Statistical methods applied to experiments in agriculture and biology. Iowa
State University Press, Ames, Iowa, 1961, 534 pp.
10. US Department of Agriculture. Animal damage control handbook.
Forest Service, Washington, DC. 1968, Section 32 . 12-5.
US Department of Agriculture,