-------
in
t/i
O
CD
2.5
2.0
1.5
LO
0.5
2.5 r
2.0
1.5
IX)
0.5
Control
Low
Medium
High
1.0
0.8
0.6
0.4
0.2
Control Low Medium High
TREATMENTS
Mar May Jun Jul Aug Sep Mar May Jun Jul Aug Sep
DATES in 1976
Figure 15.6.
Biomass dynamics of three collembolan families on Site I in
1976. A. Season mean biomass estimates of Poduridae. B.
Time traces of biomass estimates of Entomobryidae - Iso-
tomidae. C. Time traces of biomass estimates of Sminthuridae,
significant treatment-by-date interaction occurred for Sminthuridae (Figure
15.10) where the High treatment was significantly reduced from the Control
on the first sample date and the Low treatment on the third date. The most
notable aspect of the interaction is the consistently low densities on the
High treatment throughout the season. The significant interaction for the
family Laelapidae was erratic and not considered to reflect any trends
related to SC>2 fumigation.
Many of the microarthropod groups included in the ANOVA's had significant
population changes across time. Most of the groups had seasonal trends like
those depicted in Figures 15.5B, 15.6B, and 15.9A and B where the populations
were high in the first half of the season and sharply declined during the
second half. Because of the sharp population declines late in the season,
treatment effects were not observable among many groups. The S02 treatments
appeared not to cause any major changes in the timing of the late season
population declines.
DISCUSSION
Soil microarthropod populations were quite similar in size and structure
on the two field sites in 1976 when season mean populations are comparable.
Although not tested statistically, there was a substantial difference between
the 1975 and 1976 seasons on Site I (Table 15.1).
151
-------
o
c
LJ
O
125
100
75
50
?5
500
400
300
200
IUU
-
-
d
•
*
I — |
"5
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<§
3
2
.c
o>
i
400
300
200
100
5000
4000
3000
2000
1000
~|
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o
H-
6
-
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— 1
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"o
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J
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i
| — |
—
«
o
600
500
400
300
200
100
^000
3000
2000
KX)O
-
-
•
f
Control
1 -
r
Illl
TREATMENTS
Figure 15.7.
Density estimates of five prostigmatid mite families on Site
I on 10 August 77 and 8 July 78. A. Pediculochelidae, 1977;
B and C. The undescribed endeostigmatid family, 1977 and
1978 respectively; D. Eupodidae, 1978; E. Rhagidiidae,
1978; and F. Tetranychidae, 1978.
152
-------
150
125
i
E 100
in
o
c
"7R
> 75
1-
co
z
Q 50
25
-
-
Control
O
•o
£
-C
en
X
300
200
100
i-
-
o
'c
o
O
-1
•a
a>
x:
Ol
X
6000
5000
*T\J \)\J
3000
2000
1000
-
Control
3
o
T3
o
6
Figure 15.8.
TREATMENTS
Density estimates of three acarine families on Site II. A.
Season mean densities for Trombidiidae, 1976; B. Season
mean densities for Scutoverticidae, 1976; and C. Densities
on 10 August 77 for Nanorchestidae.
2000
,_ a
Control
I5,000r
10,000-
5000 -
Mar May Jun Jul Aug Sep Mar May Jun Jul Amg Sep
DATES in 1976
Figure 15.9. Time traces of density estimates for two prostigmatid mite
families on Site II in 1976. A. Eupodidae and B. Scuta-
caridae.
153
-------
4420
3870
3315
Control
Low
Medium
High
fT
'£
2760
in
O
LJ
Q
2210
1660
25 MAR
26 MAY 25 JUN 26 JUL 19 AUG
DATES in 1976
19 SEP
Figure 15.10. Time traces of density estimates for Sminthuridae (Collembola)
on ZAPS II in 1976.
Three of the four family categories of Collembola had significantly re-
duced populations on Site I in part or all of the 1976 season. The fourth
family, Onychiuridae, was not significantly affected by the S02 treatments
possibly because that group is more subsurface in hafritv- The three affected
families, Poduridae, Entomobryidae (including Isotomidae) and Sminthuridae,
are all generally surface or near surface dwelling (Leetham, unpublished
data from a shortgrass prairie). Since 1975 was the first year of treatment
(beginning in June), 862 treatment may not have had sufficient time to
affect the collembolan populations. This argument seems to hold true for
Site II in 1976 where only the Sminthuridae were affected by S02 enough to
154
-------
show population reductions. In 1977 and 1978, the single samplings may have
been made after the critical moist part of the season when it appears the
S02 may have its greatest impact. The 1976 date by treatment interactions
for the Entomobryiidae and Sminthuridae support this suggestion. Seasonal
dynamics of most all the microarthropod groups follow closely the seasonal
soil water dynamics - i.e, , wet in spring and early summer, drying out in
late summer and fall prior to rewetting by fall and winter storms (Dodd et
al., 1979).
Although trends of population declines among the soil acarines were
numerous, most were not statistically significant because of high sample
variability. Where acarine population declines were significant, most
occurred during the first half of the season much as among the Collembola.
The inconsistencies of apparent population changes among the acarine groups
across years and sites adds difficulty in drawing conclusions about the
effects of S02 fumigation. However, because there are so many trends, we
are concluding that S02 did have deleterious effects on the soil micro-
arthropods.
The results of this study are not conclusive enough to support a statement
of differential sensitivity of the three major trophic classifications used
in this study - herbivores, fungivores, and predators. Significant population
reductions occurred in representatives of all three. Population changes
observed in this study are likely due to direct toxicity of S02 or its
derivaties on the microarthropods themselves, or a reduction in their food
resources through toxicity to prey organisms. Reductions in fungivores is
probably due to reductions in food resources since there is ample evidence
that S02 and its derivatives have significant effects on soil microbial
activity (Babich and Stotzky, 1974) which probably reflects reduced microbial
populations.
The ecological implications of microarthropod population reductions
from S02 or other anthropogenic contaminants are largely speculative primarily
because the functional importance of these organisms in ecosystem processes
is poorly understood. In terms of energy flow as a function of density,
biomass, and respiration, the microarthropods are greatly outranked in the
soil by microbes and nematodes. However, the importance of the microarthropods
may be more along the lines of how they influence the functioning of other
groups. For example, the cryptostigmatid mites, which are largely fungivores,
can greatly enhance the activities of bacteria and fungi by distributing
inoculum (spores, etc.) among organic debris (Wallwork, 1970). Parkinson et
at. (1979) suggest that fungal grazing by Collembola not only can spread
fungal spores, but may very likely alter competitive relationships of fungal
species complexes in litter and/or soil. This same concept can be projected
to nematode-feeding microarthropods.
If direct toxicity of S02 is the reason for a decline in the population
of one or more microarthropod species, then those same organisms may function
in the future as sensitive indicators of changes in the soil-litter system
as a result of exposure to anthropogenic S02. The full impact of any changes
in the litter-soil system as a result of S02 exposure can only be evaluated
with time so that the long-term effects of small changes such as observed in
155
-------
this study can be related to long-term alterations in ecosystem structure
and/or function. Such was not the scope of this study.
CONCLUSIONS
Long-term, low-level S02 exposure resulted in significant population re-
ductions among a few of the soil microarthropod groups on the two field
sites, although their reductions were not large enough to affect the total
microarthropod population estimates or estimates of the dominant group, Acarina.
The acarines accounted for over 90 percent of the microarthropods density and 70
percent of the biomass. The most notable treatment effects occurred in
the apterygote insect order Collembola where significant population reductions
occurred in S02 treated plots for season mean collembolan density and the
season mean density and biomass of three family groups Poduridae, Entomo-
bryidae (including Isotomidae) and Sminthuridae. These reductions occurred
in 1976 on Site I, the second season of S02 fumigation. No significant
population changes were observed in the first year of fumigation on either
Site I or II with the exception of Sminthuridae on some sample dates on Site
II in 1976.
Among the Acarina, 11 families were observed to have significantly re-
duced populations on at least one site during the study. Although the popu-
lation reductions were statistically significant on only one site-year
and/or time-date within a year, trends for these same families and others
were observed at other times or dates in the study. High sample variability
was the principal reason for failure of many groups to show statistically
significant treatment effects.
Sulfur dioxide effects were not restricted to one functional or trophic
group. Among those families which had population reductions were representa-
tives of three important trophic groups, •i.e., herbivores, predators, and
fungivores. Most population reductions observed in this study occurred in
the first half of the growing season when soil water conditions were highest.
This suggests that the effects of S02 on microarthropods are magnified by
increased soil moisture, although the mechanism by which S02 caused the
population changes was not determined. It is possible that both direct
toxicity and toxicity to food resources could have been involved in the
population changes observed.
REFERENCES
Abrahamsen, G., J. Hovland, and S. Hagvar. 1978. Effects of Artificial
Acid Rain and Liming on Soil Organisms and the Decomposition of Organic
Matter. SNSF-contribution FA 28/78. 23 pp.
Baith, E., B. Berg, U. Lohm, B. Lundgren, H. Lundkvist, T. Rosswall, B.
SOderstrom, and A. Wiren. 1980. Effects of Experimental Acidification
and Liming on Soil Organisms and Decomposition in a Scots Pine Forest.
Pedobiologia. (In Press)
Babich, H. and G. Stotzky. 1974. Air Pollution and Microbial Ecology.
Grit. Rev. Environ. Control, 4:353-421.
156
-------
Dodd, J. L., J. W. Leetham, T. J. McNary, W. K. Lauenroth, and G. L. Thor.
1979. Baseline Characteristics of Producer and Invertebrate Populations
and Certain Abiotic Parameters in the Colstrip Vicinity, pp. 53-106.
In: E. M. Preston and T. L. Gullett (eds.) The Bioenvironmental Impact
of a Coal-fired Power Plant, Fourth Interim Report. EPA-600/3-79-044.
U. S. Environmental Protection Agency. Corvallis, Oregon.
Lebrun, P., G. Wauthy, C. Leblanc, and M. Goossens. 1977. Ecologic Test
of the Tolerance to SC>2 Toxicity in the Oribatid Mite EwneTobates
Tostrolcmel'latus (Grandjean, 1936) (Acari: Oribatei). Annales Soc. R.,
Zool., Belg., 106:193.
Lebrun, P., J. M. Jacques, M. Goossens and G. Wauthy. 1978. The Effect of
Interaction Between the Concentration of S02 and the Relative Humidity
of Air on the Survival of the Bark-living Bioindicator Mite Ewnevobates
rostrolamellatus. Water, Air, and Soil Pollut., 10:269-275.
Leetham, J. W., T. J. McNary, J. L. Dodd, and W. K. Lauenroth. 1979.
Response of Field Populations of Tardigrada to Various Levels of Chronic,
Low-level Sulfur Dioxide Exposure. Proceedings of VII International
Colloquim of Soil Zoology.
Merchant, V. A. and D. A. Crossley. 1970. An Inexpensive High Efficiency
Tullgren Extractor for Soil Microarthropods. J. Ga. Entomol. Soc.,
5:83-87.
Parkinson, D., S. Visser, and J. B. Whittaker. 1979. Effects of Collembolan
Grazing on Fungal Colonization of Leaf Litter. Soil Biol. Biochem.,
11:529-535.
Wallwork, J. A. 1970. Ecology of soil animals. McGraw-Hill Publishing
Company Limited, Maidenhead, Berkshire, England. 283 pp.
157
-------
SECTION 16
ARTHROPOD POPULATION RESPONSES TO THREE LEVELS OF CHRONIC
SULFUR DIOXIDE EXPOSURE IN A NORTHERN MIXED-GRASS ECOSYSTEM
II ABOVEGROUND ARTHROPODS
J. W. Leetham, J. L. Dodd, R. D. Deblinger, and W. K. Lauenroth
ABSTRACT
The effects of continuous, season-long exposure
of aboveground arthropods to various levels of S02
were investigated under field conditions in 1975 and
1976 in southeastern Montana. Three field plots
(0.52 ha each) on each of two grassland community
sites were fumigated with S02 on the ZAPS sites
(Section 1). Similar sized Control plots were in-
cluded in each site. Periodic samplings were made
throughout the 1975 and 1976 growing seasons. Above-
ground arthropods were sampled by dropping a 0.5 m
circular cage over predetermined (but randomly
chosen) sample locations and retrieving the arthro-
pods by vacuum. Berlese funnel extraction and
hand sorting were used to separate arthropods
from debris. Biomass and/or density reductions
occurred in a number of insect and acarine groups
on one or both sites. The groups included Acarina,
Diplura, Collembola (Poduridae), Hemiptera
(Pentatomidae), Homoptera (Cicadellidae), Thysanoptera
(Thripidae), Coleoptera (total) Staphylinidae and
Curculionidae, Diptera (Muscidae and Ceratopogonidae).
These taxonomic groups were considered to represent two
possible classifications of organisms showing popu-
lation reductions. One group included arthropods which
were relatively immobile and strongly associated with
the soil surface litter. A second group included
more mobile, flying insects. It is suggested that
reductions in populations in the former group most
likely involve toxic effects of S0£ or its derivatives
directly to the organisms or their food resources.
The mobility of the latter group introduces the
possibility of behavioral avoidance of the relatively
small experimental plots. Evidence to support these
hypotheses is given.
158
-------
INTRODUCTION
The origin and importance of this study as a portion of a large, interdis-
ciplinary research project concerned with coal-fired power plant emissions on
northern mixed-grass prairie were discussed in Leetham e~k at. (submitted b).
This paper will concern responses of aboveground arthropods to long-term, low-
level sulfur dioxide exposure under field conditions.
Three aspects of this study make it unique among studies of air pollutants
and arthropods: 1) a system level orientation to responses of arthropods in
a native prairie to exposure to a major component of coal-fired plant emissions;
2) the study area was considered pristine (i.e., no previous history of air
pollution exposure) prior to experimental fumigation with S02; and 3) the
study was conducted under field conditions. Of the few published studies
concerning insects and air pollutants, most are either a postenoi"i, in that
they were conducted sometime after the experimental areas were exposed to
pollution, or the studies were conducted under highly controlled laboratory
conditions. For example, Freitag et al. (1973) studied carabid beetle
populations near a Kraft mill in Thunder Bay, Ontario, some years after the
mill had been operating, and found lower beetle populations near the mill as
compared to farther downwind. Hillman and Benton (1972) concluded that S02
exposure from a coal-fired power plant in central Pennsylvania was responsi-
ble for reduced populations of social bees and parasitic wasps, and the
reduced wasp population was, in turn, responsible for increased aphid popula-
tions. They followed-up the field study by fumigating honey bee colonies
with S02 at various controlled levels between 0 and 500 pphm and found an
inverse relationship of brood-rearing and pollen collection with S02 con-
centration. Ginevan and Lane (1978), using Drosoph-ila melanogaster and
controlled laboratory conditions found long-term, low-level S02 exposure (70
and 40 pphm) caused significant increases in developmental time and decreases
in survival.
MATERIALS AND METHODS
The study area in southeastern Montana and design of the field experi-
mental plots were described in Leetham et al. (submitted b). Aboveground ar-
thropods were sampled on Site I 6 times in each 1975 and 1976 growing season
while Site II was sampled 6 times in the 1976 season only. Samplings for a
given year were spaced at approximately 3 week intervals throughout the
growing season (April - September).
For this study, "aboveground arthropods" were defined as those arthropods
occurring in or above the soil litter and aerial vegetation. The arthropods
were sampled by dropping a circular cage over predetermined, but randomly
chosen, sample locations and vacuuming out the contents, including litter and
vegetation. The cage covered 0.5 m2 and was dropped from a cart-mounted 18
foot (5.5 m) boom. Cage contents were vacuumed in two stages: 1) a light
vacuuming of cage walls and aerial vegetation for active arthropods, and 2)
clipping and bagging of all vegetation followed by hand vacuuming to retrieve
remaining litter and plant refuse. The first stage material was frozen then
hand sorted for arthropods while the second stage was subjected to Berlese
funnel extraction to retrieve the arthropods. The two stage process was
159
-------
found to have better efficiency of retrieval than other techniques such as
hand sorting and mechanical flotation. All the arthropods were preserved in
70 percent ethanol and later counted and identified. Five samples per repli-
cate (10 per treatment) were taken on all dates, the sample locations being
chosen by use of a random numbers table. Specific details of the collecting
and extracting equipment are given by Leetham (1975).
The arthropods were identified as far as possible which often was only
to family. When possible, voucher specimens were sent to recognized special-
ists for verification. Representatives of all taxa were dried at 65°C for
24 hours and weighed for dry weight biomass. A split plot analysis of variance
was performed on the data to test for treatment and date-within-season effects.
Because Site II was not fumigated until 1976, we concluded it would be illogi-
cal to include "sites" and "years" in the ANOVA design. The two sites were
not comparable in 1976 because of differences in fumigation history, and
first-year fumigation on Site I was not comparable to first-year fumigation
on Site II because of differences in growing seasons. Because of high sample
variability, the ANOVA was run at the order and family level on selected
groups based on whether or not there appeared to be population changes in the
treated plots (judged on season mean density and/or biomass summaries) .
Individual analyses were performed on both density and biomass data of each
group because either or both parameters can be used to measure population
changes in a given family or order. It should be stated at this point that
although the traditional significance level of P= 0.05 was used as a guideline
in judging population changes as significant, probability levels of 0.05 < p <
0.10 were accepted for one or more site-years for a given group if that group
showed similar trends (whether significant or not) in other site-years.
Tukey's Q procedure was used to calculate least significant ranges which were
used to compare treatments or dates when the ANOVA results indicated signifi-
cant population changes. In cases where both a main effect of treatment or
date and a date-by-treatment interaction were significant, the interaction
was given presidence over the main effect. In most cases where this situation
occurred, it was because the particular group was abundant only for a portion
of the season and significant treatment differences occurred at that time.
During the remainder of the season, the group did not occur or did so in such
low densities that treatment differences were not measurable.
RESULTS
The list of identified arthropods collected during this portion of the
study includes 15 orders and 60 families of insects, five families of spiders
(Araneida), five families of mites (Acarina), and centipedes (Geophilomorpha).
A general overview of the aboveground arthropod community structure on Sites
I and II is provided in Tables 16.1 and 16.2. Presented are density and
biomass estimates for the major orders and families. The major order on both
sites was Coleoptera with Hymenoptera and Homoptera following in that order
(based on biomass). The Curculionidae (weevils) was the major coleopteran
family, Formicidae (ants) the major hymenopteran family and the Cicadellidae
(leaf hoppers) the major homopteran family. A complete list of all families,
genera and species is far too voluminous to include here.
160
-------
TABLE 16.1.
GENERAL ABOVEGROUND ARTHROPOD COMMUNITY STRUCTURE OF SITE I
IN 1976*
Density (Nos • m~2)
Order
Araneida
n
Coleoptera
n
M
n
n
n
n
Collembola
n
Hemiptera
n
"
"
"
"
Homoptera
n
n
n
Hymenoptera
M
Lepidoptera
n
Orthoptera
11
Thysanoptera
n
Family
Lycosidae
Carabidae
Curculionidae
Elateridae
Staphylinidae
Tenebrionidae
Chrysomelidae
Poduridae
Cydnidae
Lygaeidae
Miridae
Nabidae
Scutelleridae
Cercopidae
Cicadellidae
Pseudococcidae
Formicidae
Noctuidae
Acrididae
Thripidae
Total Arthropods
Cont,
2.
<0.
55.
5.
10.
<0.
6.
0.
10.
173.
173.
11.
0.
8.
1.
0.
0.
50.
0.
12.
14.
22.
20.
1.
0.
0.
0.
83.
83.
420.
5
1
8
2
1
1
9
3
2
6
6
9
4
7
3
6
1
2
1
6
8
5
6
1
1
4
4
4
4
4
Low
3.3
0
44.3
4.0
10.6
0.2
4.5
0.7
7.9
6.1
6.1
14.3
0.2
10.9
1.3
0.9
0.1
21.2
0.2
14.0
4.4
27.7
25.4
0.5
0
0.4
0.3
27.8
27.8
164.1
Med.
2.9
0.2
42.3
3.5
8.2
<0.1
4.6
0.7
7.6
17.1
17.1
12.7
<0.1
9.9
1.8
0.6
<0.1
19.9
0.1
8.9
7.3
34.3
33.0
0.9
0
0.6
0.6
22.5
22.5
166.3
High
3.6
0.3
41.8
3.8
10.8
0.2
5.0
0.3
6.0
91.1
91.1
18.3
1.0
13.8
2.6
0.4
0.1
26.6
0.3
6.7
11.9
26.7
24.2
0.7
0.1
0.6
0.5
35.6
35.6
255.3
Biomass (nig
Cont.
1
0
64
7
28
<0
3
1
12
3
3
9
1
3
1
1
1
13
0
9
1
15
14
5
3
2
2
3
3
121
.6
.2
.4
.7
.5
.1
.8
.1
.0
.5
.5
.7
.6
.2
.2
.3
.2
.8
.7
.5
.3
.1
.4
.7
.4
.2
.2
.3
.3
.1
Low
1.3
0
56.8
5.5
30.0
1.5
2.3
1.7
8.8
0.1
0.1
8.8
0.8
3.2
1.3
1.9
0.7
9.9
0.8
8.1
0.4
14.0
12.9
1.3
0
3.8
1.5
1.0
1.0
98.4
. m-2;
Med.
6.3
5.0
53.6
7.5
23.9
0.1
1.7
2.3
9.6
0.3
0.3
6.5
0.1
2.8
1.5
1.3
0.3
7.7
0.5
5.7
0.7
18.3
17.4
2.0
0
8.2
7.4
0.9
0.9
105.6
)
High
5.0
3.5
62.6
7.3
29.5
2.7
2.9
1.3
11.0
1.8
1.8
12.1
3.9'
3.9
2.1
0.8
0.9
9.6
1.3
6.3
1.1
15.1
12.9
5.7
4.6
11.4
11.4
1.4
1.4
125.8
Data are season means. The list includes groups where 1.0 mg • m~2 occurred on
one or more treatments.
The three site-year combinations present difficulties in analyzing and
interpreting the resulting data. As mentioned previously, first year exposure
on Sites I and II are not directly comparable because they occurred in
separate seasons and the only second-year data collected were from Site I in
1976. Because of these problems, potential inconsistencies between site-
years in measured arthropod responses to S02 fumigation could very well be
expected and, in fact, did occur. Tables 16.3 and 16.4 list those orders
and families of arthropods which had trends of population density and/or
biomass changes with S0£ exposure, based on season mean density and biomass
estimates. Individual ANOVA's were performed for each of the groups listed.
The results will be discussed here by site-year.
1975 - Site I
Total coleopteran density was reduced for part of the 1975 season
resulting in a significant treatment-by-date interaction (Figure 16.1A).
This is quite probably due to a similar trend for the density and biomass of
161
-------
TABLE 16.2. GENERAL ABOVEGROUND ARTHROPOD COMMUNITY STRUCTURE OF SITE II
IN 1976*
Density (Nos • m~2)
Order
Araneida
it
Coleoptera
11
it
ti
ti
it
"
ii
Hemiptera
11
"
Homop ter a
ii
it
Hymenoptera
it
Lepidoptera
it
Orthoptera
ii
Thysanoptera
Family
Lycosidae
Carabidae
Chrysomelidae
Curculionidae
Elateridae
Orthoperidae
Staphylinidae
Tenebrionidae
Lygaeidae
Cydnidae
Cicadellidae
Pseudococcidae
Formic idae
Noctuidae
Acrididae
Thripidae
Cont.
2
0
49
4
8
15
0
4
5
0
10
7
0
19
4
11
20
19
1
<0
0
0
31
.5
.7
.4
.3
.5
.6
.9
.6
.9
.7
.3
.7
.9
.5
.2
.9
.8
.5
.1
.1
.5
.5
.4
Low
1.
0.
53.
6.
4.
17.
1.
5.
5.
3.
14.
8.
0.
24.
4.
11.
36.
35.
1.
<0.
0.
0.
22.
4
1
6
1
6
8
8
5
0
2
0
8
2
4
2
6
6
7
5
1
7
7
2
Med.
1.7
0.1
41.3
4.2
5.3
14.8
0.5
2.9
3.0
4.0
9.7
7.8
0.1
14.9
2.3
11.0
32.8
32.4
1.2
0
0.8
0.8
23.2
High
1.9
0.2
24.4
2.4
3.2
9.8
0
2.1
3.1
1.0
9.0
5.9
<0.1
13.4
3.6
9.1
29.7
29.2
1.0
<0.1
0.9
0.8
10.3
Biomass (mg • m~2)
Cont.
3.0
1.6
80.3
7.6
14.8
47.5
2.2
1.0
3.1
2.6
10.3
2.8
3.6
5.5
3.6
1.1
9.9
9.5
2.8
1.1
1.9
1.9
1.1
Low
5.1
4.3
90.3
6.9
7.2
53.2
6.0
1.2
2.5
11.5
9.3
2.8
0.7
7.6
4.7
1.5
16.6
16.1
2.7
1.1
3.6
3.6
0.9
Med.
4.4
3.1
77 .1
7.4
7.7
42.0
1.9
0.6
1.4
14.6
6.4
2.7
0.4
5.2
3.0
1.0
14.6
14.5
1.0
0
22.3
22.3
0.9
High
6.6
5.3
40.9
2.2
4.4
28.5
0
0.5
0.9
3.2
7.2
1.7
0.1
6.1
4.4
0.8
13.2
12.8
4.0
2.3
19.8
17.4
0.4
Total Arthropods 157.9 170.7 141.0 100.5 117.2 138.9 133.7 99.8
"* ~
Data are season means. The list includes groups where 1.0 mg • m~z occurred on
one or more treatments.
the major coleopteran family Curculionidae (Figure 16.IB). Nine species of
curculionid beetles were collected on Site I in 1975 and 1976, two of which
account for over 90 percent of the total density and biomass for the family
Hyperodes grypidioides Dietz and E. vittieollis (Kirby). The E. grypidioides
was collected only in the spring and fall (probably overwintering as adults)
and E. vi-ttioollis was collected only during the mid part of the growing
season.
Significant treatment effects occurred for both density and biomass for
the order Homoptera and the family Pseudococcidae. However, the significant
reductions occurred in the Low and Medium treatments while the Control and
High plots were not significantly different from each other. The date by
treatment interactions reveal high pseudococcid (and total Homoptera) popula-
tions on the Control in spring and similarly high populations on the High
treatment in the fall (data are not presented). Because of these erratic
trends, the population differences among the treatments are not considered
as the result of S02 fumigation but chance variation among the field plots.
Although both density and biomass of the acarine family Parasitidae
were reduced in all treated plots, none were significant because of high
sample variability.
162
-------
TABLE 16.3. ANOVA RESULTS FOR ARTHROPOD BIOMASS ON THREE SITE-YEARS. NUMBERS
ARE THE SIGNIFICANCE LEVELS FOR EACH TEST.
Site I - 1975 Site I - 1976 Site II - 1976
Group Treat* TxDt Treat TxD Treat TxD
Geophilomorpha - - - .060
Araneida Dictinidae .008 .000
Acarina (total)
" Tydeidae .030
" Oribatulidae - .032
" Parasitidae
Collembola (total) - .007
" Poduridae - .007
" Sminthuridae
Diplura
Hemiptera Pentatomldae .009
Homoptera (total) .012 - .003 .000
" Cicadellidae .055
Pseudococcidae .013 .000 .030 .001
" Psyllidae - .000
" Aphididae
Thysanoptera Thripldae .038 .007 .013 .071
Coleoptera (total) - - .011 .029
" Anthicldae
" Curcullonldae - .075
" Leptodiridae
" Staphyllnidae .047
Diptera (total) .089 .002
" Ceratopogonidae .007 .000
" Chloropidae
" Muscidae .049 .058
11 Sphaeroceridae
Hymenoptera Dlapriidae - .060
* Treatment main effect.
t Treatment by date interaction.
t Indicates the group showed a trend of season mean population reduction with
S02 exposure, but reductions were not significant at P < 0.10. Blank spaces
indicate no trends and hence not tested statistically.
163
-------
TABLE 16.-4. ANOVA RESULTS FOR ARTHROPOD DENSITIES ON THREE SITE-YEARS. NUMBERS
ARE THE SIGNIFICANCE LEVELS FOR EACH TEST.
Site I - 1975 Site I - 1976 Site II - 1976
Group Treat* TxDt Treat TxD Treat TxD
Geophilomorpha - ~ ~ ~
Araneida Dictinidae .008 .000
Acarina (total) .084
Tydeidae .030
Oribatulidae - .032
" Parasitidae -t
Collembola (total) - .007
" Poduridae - .007
" Sminthuridae
Diplura -048
Hemiptera Pentatomidae
Homoptera (total) .005 .000 .020 .000
" Cicadellidae
" Pseudococcidae .012 .000 .032 .001
Psyllidae .045 .000
" Aphididae
Thysanoptera Thripidae .033 .004 .025 .026
Coleoptera (total) - .020 - .006 .075 .043
" Anthicidae
" Curculionidae - .008
" Leptodiridae
" Staphylinidae
Diptera (total)
" Ceratopogonidae .007 .000
" Chloropidae - -
" Muscidae .048 .056
" Sphaeroceridae
Hymenoptera Diapriidae - .003
* Treatment main effect.
t Treatment by date interaction.
t Indicates the group showed a trend of season mean population reduction with
S02 exposure, but reductions were not significant at P ^ 0.10. Blank spaces
indicate no trends and hence not tested statistically.
164
-------
80-|
(M
'E
L 60-
JD
40-
co
z
UJ
Q
20-
o CONTROL
x LOW
A MEDIUM
a HIGH
•-x
CM
•E
lOOn
80-
E
*- 60-1
V)
o
CD
40-
20-
O-
1-X
%
Figure 16.1.
DATES in 1975
Time traces of two insect groups on Site I in 1975. (A) Coleoptera (Total)
density. (B) Curculionidae biomass.
-------
1976 - Site I
As expected, there was an increase in the number of groups showing
apparent responses to second year SC>2 fumigation on Site I. Fifteen groups
had reductions in season mean density and/or biomass in one or more fumigated
plots (Tables 16.3 and 16.4). However, high sample variability again was
responsible for some of the reductions not being statistically significant,
including Geophilomorpha and two dipteran families Chloropidae and
Sphaeroceridae. Many of the significant reductions were confused by signifi-
cant treatment-by-date interactions. Each of these cases will be discussed
separately.
The collembolan family Poduridae which was represented in these data by
only one species, Hyogastriwa avmata (Nicolet), had reduced densities and
biomass on all treated plots but none were significant. However, the treatment-
by-date interactions were significant and a plot of the biomass data
(Figure 16.2A) shows H. aimata was collected only on the first two sample
dates at which time there were significantly smaller populations on the ow
and Medium plots. The High treatment was reduced but not significantly.
Certainly E. armata is not the only collembolan occurring on the field
plots, but it is the major species occurring on the soil surface in late
winter and early spring. It often is found in large numbers on or near
water puddles.
One hemipteran family, Pentatomidae, represented largely by Neoti-glossa
sulc'Lfvons Stal. late in the growing season, had significantly reduced
season mean biomass on the Medium and High treatment plots (Figure 16.3A).
The order Homoptera had significant treatment effects for both density and
biomass, however, in both cases there also were significant interactions. A
plot of the density data (Figure 16.2B), which is similar to that for biomass,
shows a significant late season increase in the populations on the Control
plot while similar increases did not occur in any treated plot. The families
Cicadellidae and Pseudococcidae made up a majority of the Homoptera. The
cicadellids had significantly reduced biomass on the Medium and High treatments
(Figure 16.3B). Numerous genera and species of cicadellids were collected
thoughout the season with various seasonal abundance patterns. The population
reductions appeared not to be the result of one major species being affected
but a general reduction of most of the dominant species. Only one species
of pseudococcid was collected (Distiohli-ooaous sp.) and it occurred throughout
the season. It had significantly reduced densities and biomass on the low
and Medium plots on the first sample date as determined from the interaction.
Trends were quite erratic and possibly not a reflection of S02 fumigation.
The family Psyllidae had reduced biomass and densities on all treated plots
late in the season, but the only significant treatment differences occurred
on the fifth sample date (Figure 16.2C). No representatives of the only
genus collected, Craspedolepta, were collected in any treated plot after the
third sampling.
Both density and biomass of Thripidae (Thysanoptera) were reduced late
in the season. Significant treatment differences occurred only on the fifth
date (Figure 16.2D), where all treatments were significantly lower than the
Control , however a trend of reduced populations in all treatments occurred
across the last four sample dates. At least eight species of thrips, all of
166
-------
2.5-1
160-1
DATES
1976
Figure 16.2.
Time traces of five insect groups on Site I in 1976. (A)
Poduridae (Collembola), (B) Homoptera (Total), (C)
Psyllidae (Homoptera), (D) Thripidae (Thysanoptera), (E)
Diptera (Total)-
167
-------
Jl
0.15-
c
0.10-
0.05-
tNI M n o.oo-
CID CONTROL
ESLOW
IZ3 MEDIUM
C3HIGH
d
TREATMENTS
Figure 16.3.
Season mean biomass of four insect groups on the four treat-
ment plots of Site I in 1976. (A) Pentatomidae (Hemiptera),
(B) Cicadellidae (Homoptera), (C) Ceratopogonidae (Diptera),
and (D) Muscldae (Diptera).
the family Thripidae, were collected, only two of which were identified to
genus - Chirothrips sp. and FTarikl'in'iella sp. Neither of these two species
were of major importance among those collected. Four other species comprised
the majority of the thrip biomass and they were generally most abundant in
the last half of the season.
Significant treatment-by-date interactions for total Coleoptera density
and biomass resulted when the Control differed from the Medium treatment on
the third date and the Eigh treatment on the fifth date (data not presented).
No other significant treatment differences occurred which suggests the
differences were chance happenings.
The significant treatment-by-date interaction for total dipteran biomass
resulted when the High treatment was significantly lower than the Control on
the first sample date and all treated plots were lower than the Control on
the fourth date (Figure 16.2E). As with the total Coleoptera, there are no
consistent trends across all dates, so it becomes questionable if the two
significant points represent treatment differences. Both dipteran families
with significant population reductions, Muscidae and Ceratopogonidae, were
collected in the larval stages from the soil surface litter. Fannla sp.
(Muscidae) was collected on only two sample dates (17 May and 12 July) and
the ceratopogonid (species unidentified) were collected from all treatments
on 22 March 1976 but only from the Control plot on 17 May and was not collected
168
-------
from any of the plots during the remainder of the season (Figures 16.3C and
16.3D). The significant interactions were the result of the temporary
occurrence on the plots and not from erratic seasonal trends.
1976 - Site II
First year fumigation on Site II resulted in more groups showing popu-
lation changes than occurred during the first year on Site I. However,
again because of high sample variability some of the changes were not signi-
ficant including Sminthuridae (Collembola), Homoptera (total), and Leptodiridae
(Coleoptera). Each of these groups showed trends of density and/or biomass
reductions in the treated plots, based on season means.
A plot of the treatment-by-date interaction for centipedes (Geophilo-
morpha) shows erratic collections in the first half of the season with no
collections thereafter (Figure 16.4A). The most consistent characteristic
is that no representatives were taken in the High treatment plot and very
few in the edium treatment. However, considering the reductions as being
the result of S02 fumigation is tenuous. One family of spiders (Dictynidae),
represented by Di,ctyna oonsutta Gertch and Ivie and D, terrestr-i-is Emerton
had significantly reduced density and biomass in the Medium and High treat-
ment plots on- the third sample date and in all treated plots on the fifth
sample date (Figure 16.4B). Total acarine density was reduced on all treated
plots although the significance level was only P = 0.084 (Figure 16.5A). The
density and biomass of the acarine family Tydeidae was significantly reduced
on all treated plots (Figure 16.5B). A plot of the date by treatment inter-
action for Oribatulidae biomass shows a significant reduction on the fifth
sampling when populations were at their highest (Figure 16.4C)„ The density
of Diplura was reduced on all treated plots, however, the reduced biomass
was not significant (Figure 16.5C).
Both density and biomass of Thripidae (Thysanoptera) were significantly
reduced on the High treatment plot (Figure 16.5D) although the significant
interaction shows the reduction occurred late in the growing season much the
same as on Site I. Total beetle (Coleoptera) biomass was significantly
reduced on the High treatment plot (Figure 16.5E) while a significant inter-
action occurred for density. A plot of the interaction (Figure 16.4D) shows
the High treatment to be consistently lower than the Control, Low, and
Medium plots, but that significant differences occurred on the third, fifth,
and sixth dates. Only one coleopteran family, Staphylinidae, had a signifi-
cant biomass reduction, where both the Medium and High treatments were
reduced from the Control (Figure 16.5F). Three species made up the majority
of the Staphylinidae - Philonthus sp., Aleochara sp., and Taohyporus sp.
The Philonthus was collected throughout the season while the Aleookara sp.
and Tachyporus were collected almost exclusively in the first three samplings.
At least six other species were collected but only rarely. The reduction in
Staphylinidae was due to reductions in all three of the principal species.
One other coleopteran family, Curculionidae, showed substantially reduced
biomass on the High treatment, but the data were untested.
One parasitic hymenopteran family, Diapriidae, showed significant
interactions for both density and biomass, particularly density (Figure 16 .4E).
169
-------
0.8-1
o CONTROL
X LOW
A MEDIUM
D HIGH
0.8
\ '\\\\**<
>
>
0.4-,
DATES in 1976
Figure 16.4.
Time traces of five arthropod groups on four treatment plots
of Site II in 1976. (A) Geophilomorpha, (B) Dictynidae
(Araneida), (C) Oribatulidae (Acarina), (D) Coleoptera
(Total), (E) Diapriidae (Hymenoptera).
170
-------
6-
4-
2-
0.6-
—
'£ 0.4-
~ 200-
co
co
5
0
i — i — /-.
d
r\
\
\
\
\
\
\
\
\
\
\
\
\
\
^
/
/
/
/
X
/
/
/
/
/
/
/
/
e
\
\
\
\
\
\
\
\
\
\
\
\
|-
UJ ^
20-
c
10-
. — |
m._ 0:
f
^1
\
\
\
\
\
\
\
\
\
\
'/
/
/
/
/
f
—
Figure 16.5.
TREATMENTS
Season mean density or biomass of six arthropod groups on four
treatment plots of Site II in 1976. (A) Acarina, (B)
Tydeidae (Acarina), (C) Diplura, (D) Thripidae (Thysanoptera),
(E) Coleoptera (Total), (F) Staphylinidae (Coleoptera).
The reduction occurred early in the season when they were most abundant. No
species identifications were made.
DISCUSSION
All arthropod population changes noted here as associated with S02
fumigation have been reductions in density and/or biomass. No significant
171
-------
population increases were observed for any group in any season although
increases certainly could have been expected as a result of upsetting predator-
prey balances or changes in plant host susceptability. Both concepts have
been argued as causes of increased insect damage to plants in areas of high
air pollutant exposure (Hillman and Benton, 1972; Heagle, 1973) .
Although numerous groups showed significant population reductions
associated with the S02 fumigation in the three site-years, we are concerned
over the lack of consistency between sites and years. Only three groups had
significant population reductions in more than one site year. Total homopteran
density and biomass were significantly affected in 1975 and 1976 on Site I;
the family Thripidae was significantly affected on both sites in 1976; and
total coleopteran density and biomass were variously reduced in all three
site-years. All other groups that showed population changes did so in only
one site-year. As stated previously, these inconsistencies were not completely
unexpected because of the way the field study was designed and implemented,
*i.e. , first year fumigation on the two sites being in different seasons and
only one set of second season data. Despite these inconsistencies, there
are enough significant population reductions and trends of such to lead to
the conclusion that S02 fumigation did have detrimental effects on many
aboveground arthropod groups and that the reductions were not chance occur-
rences among a large number of groups.
The treatment responses of arthropods to long-term low-level S02
exposure observed here fall into two general categories - 1) those arthropods
(or stages) which are associated with the soil surface litter and relatively
non-mobile, and 2) those arthropods associated with aerial vegetation and
generally quite mobile. The former group includes the Geophilomorpha,
Araneida, Diplura, Acarina, Poduridae, Pseudococcidae, Muscidae (larvae) and
Ceratopogonidae (larvae). The latter group includes the Cicadellidae,
Thripidae, Staphylinidae, and Curculionidae. Direct toxicity or toxicity to
food resources would conveniently explain the population reductions in the
first group simply because those organisms cannot leave the plots for mere
avoidance of the S02. A possible exception here would be the two dipteran
families where the population reductions could be the result of avoidance of
the treated plots by the adults during oviposition. Since all of the members
of the first group were found to occur in the soil surface litter it is
quite possible to link their population reductions to other determined
effects of S02 on the litter, i,e., reduction of decomposition rates of
plant litter as a consequence of reduced microbial activity (Leetham et al.,
submitted a; Dodd and Lauenroth, 1980). Decreased microbial activity may re-
flect decreased available microbial food reserves for arthropod groups listed,
most of which are considered to be utilizing these resources wholly or
partially. Certainly direct toxicity is a real possibility for explaining
population decreases in all the arthropods since other studies have shown
S02 can be toxic in relatively small concentrations (Lebrun et at., 1977;
Ginevan and Lane, 1978).
The fact that many of the population reductions in litter inhabiting
groups occurred in the early part of the growing season was not unexpected
since it is well known that S02 is highly attracted to moist surfaces (Saunders,
1966; Hocking and Hocking, 1977) and S02 toxicity to arthropods can be
172
-------
increased with relative humidity (Lebrun et al., 1978). The soil moisture
conditions are wettest in the spring and early summer (Dodd et al., 1978).
Another important possible explanation for the reduction in populations
of the more mobile arthropod groups (listed in group two previously) on the
fumigated plots may involve behavioral avoidance. Since ea-.h treated plot
was only 0.52 ha in size, groups such as Staphylinidae, Curculionidae, and
Thripidae, which are active fliers, could easily move out of the plots or at
least not move into them in their various random movements. Under control
conditions, the population of a given species of active arthropod in a given
small plot of ground may be maintained by a rough balance of emigration and
immigration. This balance could be upset if individuals are repulsed by the
presence of S02 and hence avoid the plots in their random flight movements.
Bromenshenk and Gordon (1978) using the same field sites as this study have
shown that the dung beetle Canthon sp. (Scarabaeidae) could not be attracted
to carrion baits in the S02 treated plots in the densities attracted to
similar baits on the Control plots (both sites). The differences were
significant and suggest that the beetles' behavior was influenced by the
S02. The critical factor in this explanation is the size of the field
plots. The effect of S02 on the active arthropod groups may be quite differ-
ent if a large enough region were exposed so as to rule out behavioral
avoidance of the polluted atmosphere. Normal behavioral patterns may or may
not be affected to the extent of significantly changing the population size
and/or dynamics of a given arthropod species. Hillman and Benton (1972)
found reduced foraging activities of honey bees when exposed to S02 in low
concentrations of 100 to 600 pphm. The resolution of this question remains
to be made.
CONCLUSIONS
Significant population reductions in density and/or biomass were observed
on S02 treated plots during the two-season study although the reductions
were not large enough or involve enough of the dominant groups to cause a
significant change in tha overall total aboveground arthropod population
estimated of either site. The arthropod groups which showed significant
density and/or biomass changes are listed in Tables 16.3 and 16.4. Inconsis-
tencies between site-years are cause for concern about the extent of the
effects of S02 fumigation. However, the numerous significant population
reductions are accepted as evidence that S02 did have a deleterious effect
on the above ground arthropod community.
The arthropods listed in Tables 16.3 and 16.4 can be generally categorized
into two groups. One group would include the relatively immobile types that
are strongly associated with the soil surface litter (Acarina, Diplura,
Collembola, and Diptera larvae). The other group would include the relatively
mobile, flying insects which may only partially be associated with the
surface litter or not at all. The former group members possibly are reduced
by direct toxicity of S02 or toxic effects on food resources or a change in
soil acidity. The second group, in addition to being affected by toxicity
(direct or indirect), may also be reduced by behavioral changes due to S02
which may result in a change in emigration/immigration ratios, i.e,, avoidance
of the relatively small field plots. There is published evidence to support
173
-------
both concepts, but our data are not definitive enough to conclude if either
or both mechanisms were involved.
REFERENCES
Bromenshenk, J. J. and C. C. Gordon. 1978. Terrestrial Insects Sense Air
Pollutants. In: Conference Proceeding, Forest Joint Conference on the
Sensing of Environmental Pollutants, 1977. Am. Chem. Soc., Wash., D.C.
pp. 66-70.
Dodd, J. L., J. W. Leetham, T. J. McNary, W. K. Lauenroth, and G. L. Thor.
1979. Baseline Characteristics of Producer and Invertebrate Populations
and Certain Abiotic Parameters in the Colstrip Vicinity. In: E. M.
Preston and T. L. Gullett (eds.) The Bioenvironmental Impact of a Coal-
Fired Power Plant, Fourth Interim Report. EPA-600/3-80-044. U. S.
Environmental Protection Agency, Corvallis, Oregon, pp. 53-106.
Dodd, J. L., and W. K. Lauenroth. 1980. Effects of Low Level S02 Fumigation
on Decomposition of Western Wheatgrass Litter in a Mixed-grass Prairie.
In: E. M. Preston and D. W. O'Guinn (eds.) The Bioenvironmental Impact
of a Coal-Fired Power Plant, Fifth Interim Report. EPA 600/3-80-052.
U.S. Environmental Protection Agency, Corvallis, Oregon, pp. 212-215.
Freitag, R., L. Hastings, W. R. Mercer, and A. Smith. 1973. Ground Beetle
Populations Near a Kraft Mill. Can. Entomol., 105:299-310.
Ginevan, M. E. and D. D. Lane. 1978. Effects of Sulfur Dioxide in Air on
the Fruit Fly, Drosophila melanogaster. Environ. Sci. Technol., 12:821-
831.
Heagle, A. S. 1973. Interactions Between Air Pollutants and Plant Parasites.
Ann. Rev. Phytopathol., 11:365-388.
Hillman, R. C. and A. W. Benton. 1972. Biological Effects of Air Pollution
on Insects, Emphasizing the Reactions of the Honey Bee (Apis metl-ifeva
L.) to Sulfur Dioxide. J. Elisa Mitchell Sci. Soc., 88:195.
Hocking, D. and M. B. Hocking. 1977. Equilibrium Solubility of Trace
Atmospheric Sulfur Dioxide in Water and its Bearing on Air Pollution
Injury to Plants. Environ. Pollut., 13:57-64.
Lebrun, P ., G. Wauthy, C . Leblanc, and M. Goossens. 1977. Ecologic Test
of the Tolerance to S02 Toxicity in the Oribatid Mite EwneTobates
rostrolamellatus (Grandjean, 1936) (Acari:0ribatei) Annales Soc. r.,
Zool., Belg., 106:193.
Lebrun, p ., J. M. Jacques, M. Goossens, and G. Wauthy. 1978. The Effect
of Interaction Between the Concentration of S02 and the Relative Humidity
of Air on the Survival of the Bark-living Bioindicator Mite Himevobates
rostrolamellatus. Water, Air, and Soil Pollu., 10:269-275.
174
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Leetham, J. W. 1975. A Summary of Field Collecting and Laboratory Processing
Equipment and Procedures for Sampling Arthropods at Pawnee Site. U.S./
IBP Grassland Biome Tech. Rep. 284. Colorado State Univ., Ft. Collins.
49 pp.
Leetham, J. W., J. L. Dodd, and W. K. Lauenroth. Effects of Low-Level Sulfur
Dioxide Exposure on Decomposition of Western Wheatgrass (Agropyron
smith-ii.) Litter Under Laboratory Conditions. (Submitted a).
Leetham, J. W., J. L. Dodd, R. D. Deblinger, and W. K. Lauenroth. Arthropod
Population Responses in Three Levels of Chronic Sulfur Dioxide Exposure
in a Northern Mixed-grass Ecosystem. I. Microarthropods. (Submitted b).
Saunders, P. J. W. 1966. The Toxicity of Sulfur Dioxide to Diploaarpon
rosae Wolf Causing Blackspot of Roses. Ann. Appl. Biol., 58:103<-114.
175
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SECTION 17
RESPONSE OF MELANOPLUS SANGUINIPES TO LOW-LEVEL SULFUR DIOXIDE
EXPOSURE FROM EGG HATCH TO ADULT (ORTHOPTERA:ACRIDIDAE)
J. W. Leetham, J. L. Dodd, J. A. Logan, and W. K. Lauenroth
ABSTRACT
The effects of low-level S02 exposure on the
migratory grasshopper Melanoplus sanguini-pes (Fab.)
from egg hatch to egg-laying adult were investigated
by rearing the grasshoppers in controlled laboratory
environments. A nondiapausing strain of M. sangu-inipes
was reared from eggs in control and S02 environments,
the S02 concentration being 17 pphm (468 yg • m~3).
No significant difference in egg-hatching success,
mean developmental time for each nymphal instar, adult
dry weight biomass, and egg production per female per
day were found between control and S02-exposed indivi-
duals. There was, however, a significant reduction in
the variability around the mean developmental time for
instars three, four, and five in the S02~exposed nymphs.
At the same time, there was evidence of increased
mortality of S02~exposed nymphs. It is postulated that
the physiologically marginal individuals of a given
instar are more vulnerable to the S02 stress and hence
are eliminated rather than continue to develop slower
than the remaining members of the same instar. There
were more slower-developing third, fourth, and fifth
instar nymphs in the control group.
INTRODUCTION
Studies of the direct toxicity of S02 on arthropods are few and
generally do not address the question of the effects of realistic
atmospheric S02 concentrations on arthropod survival. For example, Weedon
et al, (1939) studied the deleterious effects of very high S02 concentra-
tions on various vertebrate and invertebrate animals. They calculated an
LD50 for the grasshopper Melanoplus differentialis (Thomas) at 10700 pphm
for 5 days. Sulfur dioxide has been shown to be effective in high concen-
trations as an insecticide for stored-grain insect pests (Kanaga, 1956).
176
-------
Only a few studies have been concerned with low-level S(>2 exposure on
arthropods. Hillman and Benton (1972) found reduced brood rearing and
pollen collection in honey bee (Apis mellifera L.) colonies fumigated with
SC>2 concentrations of 0 to 500 pphm over 9- and 14-week periods. Ginevan
and Lane (1978) found increased developmental times and decreased survivability
of fruit flies (DTOsophila metanogastev Meigen) exposed to 40 and 70 pphm
S02 during the larval stage. Lebrun et at. (1977) found Ewnerobates
rostrolamellatus (Grandjean), a bark-living oribatid mite, very sensitive to
S02. Exposure to 45 pphm for 2 days resulted in 50 percent mortality in experi-
mental populations. All of these aforementioned three studies used S02
concentrations much higher than generally encountered even in highly polluted
areas which makes the results questionably applicable to real life situations.
This study was undertaken in hopes of clarifying the results of a field
study of the effects of low-level, long-term S02 exposure on rangeland
grasshoppers in southeastern Montana (McNary et at., submitted). In that study
season-long exposure of field plots of native northern mixed-grass prairie
were exposed to controlled levels of SC>2 of less than 10 pphm (260 yg • m~3)
throughout the growing season (April-October). Significantly reduced
grasshopper populations were found, especially for the species M, sangui-n-
ipes (Fab.)- The mechanism by which the populations were reduced was
not resolved between direct toxicity and behavioral changes. The field
plots were relatively small (0.52 ha), which would have allowed the grass-
hoppers to leave the area, or at least not enter the area, because of
repulsion by the S02. This study was performed to test for direct toxicity
of S02 to a laboratory strain of M. sanguinipes reared under continuous S02
exposure from egg hatch to egg-laying adult.
The dynamics of nymphal development and nymphal mortality were of prime
interest in this study. Low-level, chronic toxicant exposure may affect
developmental rates in two ways. Median time to complete an instar may be
altered and/or the variation in developmental rates may be affected. Modifi-
cation of either the time required to complete a stadium or the variation in
developmental rates may have profound ramifications upon an organism's total
life system. Taylor (1980) discussed in detail the importance of timing
critical life history events in insect population dynamics. Stinner et at.
(1977) discussed the importance of accurately representing variation in life
history events. They further presented an example of variation in develop-
mental rates dramatically affecting individual fitness in a Hetiothi-s zea
(Boddie) population.
Also of interest in this study were adult dry weight biomass (newly
emerged) and egg laying success, since both parameters are indicators of the
fitness of the individuals within a population. We hypothesized that S02
effects on such parameters as food intake rate, digestion rate, and as-
similation efficiency would be reflected in the ultimate size and/or egg
production of adults. If either, or both, parameters are measurably affected
by S02 exposure, major population level change would be expected.
177
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MATERIALS AND METHODS
This study was conducted at the headquarters buildings of the field
research site of the Natural Resource Ecology Laboratory. Since the site is
approximately 35 miles northeast of Fort Collins, Colorado, in the Pawnee
National Grasslands, it afforded relatively clean ambient air that could be
used without scrubbing. Also, the ambient relative humidity is very low,
eliminating the need to artificially lower the humidity before circulation
through the exposure chambers. Low humidity is considered critical for
successful laboratory rearing of the grasshoppers used in this study.
The exposure chambers used were simple continuous-flow, single-pass
cabinets measuring 2 ft by 2 ft by 6 ft (61 cm x 61 cm x 183 cm). They were
constructed completely of V (0.64 cm) clear plexiglass with large side-
opening doors for easy access. Air movement through the chamber was at an
approximately linear velocity of 0.46 to 0.91 m • min , resulting in a com-
plete air turnover once every 1 to 2 minutes. The chamber inlet and exhaust
fans were set up to create a slight negative internal pressure to prevent
pollutant leakage. Source SC>2 was fed directly into the inlet air stream.
Details of the chamber construction and operation are given by Leetham et-
a.1. (submitted). Sulfur dioxide concentration was maintained at 17 pphm through-
out the study. Measurement of the SC>2 was by two methods. A flame photometric
sulfur gas analyzer (Meloy Laboratories, Inc., Model SA-160) was used to get
accurate point measurements at the outset and conclusion of the study and
occasionally during the interim. For continuous monitoring, a technique of
chemical absorption (Pararosaniline method) was used to give average concen-
trations over time (CFR, 1975).
Rearing and adult maintenance was in clear acetate tubes of two sizes,
capped with aluminum screen-covered lids. Large tubes (9 cm diameter x 51 cm
long) were used for egg hatching and nymphal development, while smaller
tubes (5 cm dia. x 20 cm long) were used to pair adults for egg production.
The large tubes were held horizontally on an aluminum rack, while the small
tubes were held vertically in moist sand within a styrofoam cup.
Two exposure chambers, control and S02, were housed in a temperature-
controlled room maintained at 35°C. Relative humidity was uncontrolled but
averaged 5-15 percent throughout the study except when passing storm fronts
temporarily elevated the humidity.
A nondiapausing strain of M. sanguln-ipes was used for this study. Eggs
were obtained from the Range Insect Laboratory, Montana State University,
Bozeman, where a disease-free culture is maintained. The strain was originally
developed at the Saskatoon Research Station of the Canada Department of
Agriculture (Pickford and Randall, 1969). Eggs were maintained in moist
vermiculite until hatch. All life stages were fed a diet of head lettuce
and a dry-mix medium composed of 50 g alfalfa meal, 50 g wheat midlings, 25
g soybean meal, 5 g brewer's yeast, and 11.5 ml corn oil.
The study was initiated by placing eight groups of eggs numbering 100
to 200 eggs per group in each chamber 7 days prior to hatching to test
hatching success and to ensure that the nymphs received the desired exposure
178
-------
from the moment of hatching. All newly hatched nymphs were counted at 24-hr
intervals, at which time groups of 20 were placed in the larger acetate
tubes for rearing to the adult stage. This ensured that the nymphs were all
nearly the same age in any given group of 20, making determination of develop-
ment stage easier. A total of 16 groups of 20 nymphs were placed in each
chamber. At 48-hr intervals, each group of 20 nymphs was evaluated for
instar structure and mortality until all survivors reached adulthood. From
the surviving adults, male-female pairs were set up in the small acetate
tubes for egg production. A reserve of males was maintained to replace
those that died before the female died. A total of 48 excess male and 48
excess female adults from each chamber were killed by freezing, dried at
65°C for 24 hr and weighed. Egg pods were retrieved and counted at 48-hr
intervals. Moist sand from an ephemeral stream bed was used for the oviposi-
tion medium. All eggs laid were counted but not maintained for second-
generation studies. Egg-laying was continued for 41 days, with 32 pairs in
each chamber at the outset.
A preliminary trial study was performed with just eight groups of 20 nymphs
in each chamber. Only nymphal mortality and adult egg-laying success data
are included here.
Simple t-tests were used to compare the precent egg hatch in each
chamber and, at the end of the study, to compare the mean number of eggs per
female per day in each chamber. A paired t-test was used to compare mortality
rates while a two-way factorial ANOVA (treatment, sex) was used to compare
dry weight biomass data. For the developmental data a probit analysis
(Finney, 1971) provided a means for testing the effect of the low-level S02
atmosphere on both median and variance in developmental rate (rate of
development = I/time to complete a life stage). A probit transformation was
made for the cumulative percent of individuals completing each instar.
Because experimental replicate (tubes) were treated identically within
treatment and replication was entirely a function of experimental convenience
(.i.e. > the grasshoppers were confined in plastic tubes to facilitate counting),
the replicates were pooled and probits were computed for the total treatment
population. Sample size varied from 250 to 320- The transformed data were
then regressed on the elapsed time (from eclosion) to completion of an
instar. Median (M) time to completion of an instar was then computed as
m = £ (5 - I) (1)
where b is the slope from the regression line and I is the y-intercept. The
standard deviation in time to completion of an instar was estimated from
(2)
p
RESULTS AND DISCUSSION
The percent hatch was similar for the Control and S02—71.8 percent (S.D. =
18.0 percent) and 67.4 percent (S.D. = 12.6 percent), respectively. The slight-
ly reduced rate in the S02 chamber was not significant (P = 0.05). These per-
centages compare favorably with those (55-80 percent) of Pfadt et al. (1979),
who used the same strain. They also compare favorably with the 53-77 percent
179
-------
found by Pickford (1960), who used a wild strain of the same species. The fact
that the eggs were not maintained under treatment conditions throughout their
embryological development confounds the apparent lack of SC^ effect.
From the probit analysis confidence intervals about median development
times indicated no statistically significant difference between control and
S02 nymphs, and no observable trends existed in median developmental times.
An analysis of covariance for homogeneity of regression coefficients,
however, indicated that the difference between control and treatment variance
in developmental rates was highly significant (P < 0.05) for the third,
fourth, and fifth instars. Data are summarized in Table 17.1.
The variation in developmental rates is significantly and consistently
reduced in the S02-treatment group (Table 17.1). A possible biological
interpretation of the reduction in variation of developmental rates is that
physiologically marginal individuals are adversely affected by the S02
atmosphere. This interpretation may be logical if the assumption that
physiologically less-fit individuals exhibit a retarded developmental rate
under normal circumstances is valid. If so, it follows that these physio-
logically less-fit individuals would be less likely to withstand the addi-
tional stress of S02 intoxication. This interpretation is consistent with
the observed trend of reduced survival in the S02-treatment group (Figure
17.1). Further circumstantial evidence to support the conjecture that
reduced variation is the result of increased mortality of slow-developing
individuals was gained by performing an analysis for the fifth instar,
ignoring the end point (100 percent-completed life stage) for the Control group.
When the end point (i.e., those individuals with the most retarded developmental
rates) was ignored, no significant difference was found between the Control and
treatment groups.
The trend of increased mortality in the S02 nymphs appears to support
the significant reduction in the variation around the mean developmental
time for the latter instars. Mortality data for both trials were tested
with a paired t-test. In both trials mortality rates were significantly
greater in the S02-exposed grasshoppers (P = 0.001 in both trials). Total
mortality was a bit greater in the first trial (36.9 and 41.2 percent for
Control and S02, respectively) than in the second (30 percent in both Control
and S02, respectivlly) than in the second (30 percent in both control and S02).
These figures compare favorably with the 35-39 percent mortality found by Pfadt
et al. (1979) using the same strain of grasshopper and nearly the same rearing
conditions. The rearing conditions other than S02 appear not to have been
stressful on the nymphal instars such that greater than normal mortality oc-
curred.
Pfadt et al. (1979) concluded that rearing temperature had a substantial
effect on the number of nymphal instars that M. sanguinipes may have. Six
instars usually result when rearing temperatures are below 30°C, and five is
common when the temperatures are over 30°C. In their study, 97% of the
females and 51% of the males had six instars when reared at 30°C. In this
study we found that all grasshoppers had five instars. Our rearing tempera-
ture was 35°C.
180
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TABLE 17.1. PROBIT ANALYSIS OF DEVELOPMENTAL DATA
00
Ins tar
3
4
5
5
Treatment
Control
S02
Control
S02
Control
S02
Control**
S02
n
6
6
8
10
6
8
5
8
I
-0.760
-6.279
-1.746
-6.144
-5.557
-10.816
-8.388
-10.816
b*
0.4441
0.8811
0.4092
0.6094
0.4111
0.5924
0.5160
0.5924
r2
0.90
0.99
0.95
0.98
0.95
0.96
0.99
0.96
m
12.97
12.80
16.49
16.84
25.67
26.70
25.95
26.70
s
2.25
1.13
2.44
1.64
2.43
1.69
1.94
1.69
P
0.0066
0.0013
0.0270
0.2642
n = number
P = probab:
of days.
Llity of obtaii
ling
the observed
difference
in standard
deviations
by pur
e
chance (0.05 = 5% rejection).
* Note that m is computed from the probit relationship given in equation (1). The
apparent inconsistency of increasing the median number of days to completion of the
fifth instar control by ignoring the end point is due to an artifact in estimation
from the probit equation.
** End point of retarded individuals ignored.
-------
m
D
o
cr
LJ
CD
|X
20
18
CO
IE
LJ
0-
CL.
O
I
CO |C
CO l6
IE
IT
LJ
H
14
12
Control
\\ 18 pphm S02
4 8 12 16 20 24 28 48 12 16 20 24 28 I 5
FEBRUARY MARCH APRIL
Figure 17.1. Grasshopper survival through five nymphal instars to adult
under control and S02 exposure. A = preliminary trial,
B = second trial.
For adult dry weight no significant treatment effect was found (P =
0.05); however, as expected, there was a highly significant (P = 0.001)
difference between sexes. No significant interaction occurred. Apparently
none of the parameters of food intake rate, digestion, and assimilation
efficiency were substantially affected.
Egg-laying success for adult females was poor compared with data from
Pfadt et al. (1979). They found that for adult females held at 30°C and fed
a diet similar to that used in this study, an average of 6.7 eggs were
produced per female per day. Smith (1966) found an even higher average
(9.4) for a wild strain of M. sangu-inipes. In this study, eggs laid per
female per day in the preliminary trial was 0.90 for both control and S02-
exposed females. In the second trial, there was an increase to 2.3 and 2.0
for the control and S02-treated females, respectively. The low egg production
resulted from fewer eggs per pod rather than a reduction in the number of
pods per female. The fact that egg-laying in both trials was not carried
out to completion for all females may have influenced the above calculations.
No significant (P = 0.05) reduction in egg production was found in either
trial. The S02 exposure apparently did not confound or add to other, undeter-
mined causes for the overall reduced egg production.
The results of this study help in part to resolve the reason for reduced
populations of M. sanguinipes in the field study by McNary et al. (submitted).
However, the implications are such that, unless S02 has greater effects on
182
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subsequent generations, the increased mortality found here would eliminate
only the marginally fit individuals from a population, leaving the healthy
individuals to produce an egg supply not substantially different from an
unexposed population. In addition, the reduction of some individuals could
decrease the intraspecific competition for the remaining individuals. The
net result is the maintenance of a field population under regional air
pollutant impact much the same as in a similar, unpolluted area. The critical
question left unanswered by this study is whether S02 can have cumulative
effects over numerous successive generations of M. sangui-nipes or any other
insect species.
CONCLUSIONS
Low-level S02 exposure of the migratory grasshopper M. sanguinipes
from egg hatch through egg-laying adult did not cause large effects on most
of the parameters of the life cycle measured, including egg-hatch success,
rate of nymphal development, adult weight, and adult egg production. Sulfur
dioxide did cause a reduction in the variability around the mean developmental
time for instars three, four, and five, possibly by increasing mortality in
physiologically marginal nymphs. An apparent increase in mortality rate was
observed in both trials. Except for egg-laying success, the general rates of
the parameters measured in this study agree with those of other life history
studies of the same species.
REFERENCES
Code of Federal Regulations, Title 40, Protection of Environment. Part
50.11, July 1, 1975. Office of the Federal Register, General Services
Administration, Washington, DC.
Finney, D. J. 1971. Probit Analysis. Cambridge University Press, Cambridge.
333 pp.
Ginevan, M. E., and D. D. Lane. 1978. Effects of Sulfur Dioxide in Air on
the Fruit Fly Drosophila melanogaste?. Environ. Sci. Technol., 12:828-
831.
Hillman, R. C., and A. W. Benton. 1972. Biological Effects of Air Pollution
on Insects, Emphasizing the Reactions of the Honey Bee (Apis mellifera
L.) to Sulfur Dioxide. J. Elisha Mitchell Sci. Soc., 88:195.
Kanaga, E. E. 1956. An Evaluation of the Use of Sulfur Dioxide in Fumigant
Mixtures for Grain Treatment. J. Econ. Entomol., 49:723-729.
Lebrun, P., T. Wauthy, C. LeBlanc, and M. Goossens. 1977. Ecologic Test
of the Tolerance to S02 Toxicity in the Oribatid Mite EimeTobates
Tostrolamellatus (Grandjean, 1936) (Acari:0ribatei). Annales Soc. R.,
Zool., Belg., 106:193.
Leetham, J. W., W. Ferguson, J. L. Dodd, W. K. Lauenroth. Design and
Construction of a Simple, Continuous-flow Sulfur Dioxide Exposure
Chamber. (Submitted.)
183
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McNary, T. J., D. G. Milchunas, J. W. Leetham, W. K. Lauenroth, and J. L.
Dodd. The Effect of Controlled Low Levels of SC>2 on Grasshopper
Densities on a Northern Mixed-grass Prairie. (Submitted.)
Pfadt, R. E., Y. Afzali, and J. S. Cheng. 1979. Life History and Ecology
of the Nondiapause Strain of the Migratory Grasshopper in the Laboratory.
Univ. Wyo. Agric. Exp. Sta. Sci. Monogr. 39, Univ. Wyoming, Laramie.
31 pp.
Pickford, R. 1960. Survival, Fecundity and Population Growth of Melanoplus
b-ilituratus (Wlk.) (Orthoptera:Acrididae) in Relation to Date of
Hatching. Can. Entomol., 92:1-10.
Pickford, R., and R. L. Randell. 1969. A Nondiapause Strain of the Migratory
Grasshopper Melanoplus sanguinipes (OrthopterarAcrididae). Can. Entomol.,
101:394-396.
Smith, D. S. 1966. Fecundity and Oviposition in the Grasshoppers Melanoplus
sangu-inipes (F.) and Melanoplus b-Lvittatus (Say). Can. Entomol.,
98:617-621.
Stinner, R. E., J. W. Jones, C. Tuttle, and R. E. Caron. 1977. Population
Mortality and Cyclicity as Affected by Intraspecific Competition. Can.
Entomol., 109:879-890.
Taylor, F. 19.80. Timing in the Life History of Insects. II. Magnitude and
Hedging Factors. Ecology. (In press.)
Weedon, F. R., A. Hartzell, and C. Setterstrom. 1939. Effects on Animals
of Prolonged Exposure to Sulfur Dioxide. Contrib. Boyce Thompson
Inst., 10:281-324.
184
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POTENTIALLY USEFUL BIOINDICATORS AND BIOMONITORS
OF COAL-FIRED POWER PLANT EMISSIONS
SECTION 18
OBSERVATIONS ON TWO LICHEN SPECIES
IN THE COLSTRIP AREA, 1979
S. Evers?nan
ABSTRACT
There was a significant increase in sulfur content
(p<0.05, linear regression ANOVA) in Usnea hirta (L.)
Wigg. samples in 1979 and 1977 as distance from
Colstrip decreased. Other observations on U. hirta
and Parmelia ohlorochroa Tayl. — respiration rates,
chlorophyll content, percentage of plasmolyzed algal
cells, rate of photosynthesis — had no significant
linear or logarithmic relationship (p> 0.05, linear
and logarithmic regression) to distance from Colstrip.
INTRODUCTION
Lichens are used throughout the world as bioindicators of air quality
(Ferry, et al., 1973; Hawksworth, 1975-1978; Hawksworth and Henderson, 1978,
1979; Henderson, 1979, 1980). Two primary techniques have been used in iden-
tifying and delineating polluted areas: mapping lichen communities near urban
and industrial areas, and observing specimens transplanted into polluted areas.
Symptoms induced by pollutants in the field are compared with symptoms in
laboratory-treated specimens.
The Colstrip lichen study used a slightly different approach since (1)
the study began before a pollution source was present and (2) epiphytic
lichens are sparse in the Colstrip area. Two native lichen species (Usnea
hir>ta (L.) Wigg, an epiphyte on ponderosa pine, and Parme'i'ia chloTOc'hToa,
a soil lichen) were observed over a period of years at varying distances and
directions from the Colstrip coal-fired power plants 1 and 2. The plants
began operation in September, 1975 and June, 1976, respectively. Observations
of anatomical and physiological states were compared with specimens treated
with S02 in a field fumigation system in Powder River County (Eversman, 1978,
1979) 100 km southeast of Colstrip. This report summarizes observations in
1979, and compares 1979 results with those of previous years.
-------
MATERIALS AND METHODS
Usnea hirta samples were collected from 19 ponderosa pine sites 1-70
km from Colstrip (Figure 18.1, Table 18.1). Monitoring sites within 10 km
of Colstrip support lichen communities only on north and east-facing trunk
bases, so ponderosa pine branches containing U. h-ivta were transplanted
to sites P1-P9 and P17-P19 in September 1975 or April 1976. All observations
1976-1979 were from these transplanted specimens. Source of transplants
was Site P10, an east-facing slope 51 km southeast of Colstrip. All sites
in Custer National Forest, P10-P16, and site P8 on the Northern Cheyenne
Indian Reservation have sufficient native population to use. Sites P8,
P15, and P16 received transplants for observation of effects of transplant-
ing. Native P. chloTOchTOa was collected from four grassland sites
near EPA exclosures (G1-G4), three other sites near ponderosa pine sites
(P3, P4, P18), and the control grassland site (G7) .
Samples were washed with distilled water and stored in air-dry conditions
in the dark at room temperature for no more than 3 weeks before laborato-
ry observations. Respiration rates were determined manometrically for
250 mg samples at 20°C in saturated condition in the dark.
Chlorophyll extracts were made in two ways. (1) 300-mg samples were
extracted with 10 ml boiling methanol, filtered, returned to 10 ml levels,
then read at 665 run on a Beckman DU spectrophotometer. (2) Total pigment
and percentages of chlorophyll and phaeophytin were obtained using the
chromatography methods and formulas of Brown and Hooker (1977) .- This was
an attempt to decrease chlorophyll degradation to phaeophytin by lichen acids.
Plasmolysis of algal cells was determined by making wet mounts of
thallus tips, then recording the number of yellow, plasmolyzed algal cells
out of 100 cells on each of three slides (300 cells per sample were
counted)- Handling of specimens in this way also allowed for close ob-
servation of color and integrity of thallus. (S02~exposed lichens became
crumbly and yellow; untreated specimens were firm and green.)
Sulfur contents were determined by the Montana State University Soil
Testing Laboratory, using a dry ash and turbidometric procedure (pers.
comm.).
Photosynthesis rates were determined by drying samples to 50 percent of
saturated weight, as determined by drying curves, then placing them in
flasks in a Gilson respirometer at 10°C in light for 1 hour. Atmosphere
samples (10 ml) were removed from the flasks with syringes, and injected
into an infrared gas analyzer to determine amount of C02 consumed. Controls
were respirometer flasks under the same temperature and light conditions,
with no lichen samples.
Statistical analysis was through the Montana State University Statistical
Center. Programs used were multiple regression; one-way analysis of
variance; Newman-Keuls Q, comparison of means; and log transformations
(Lund, 1980).
186
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Northern Cheyenne
Reservation
Figure 18 .1.
Map showing lichen collection sites. P1-P16 are,
ponderosa pine sites with Usnea lii-vta; G1-G7 are
grassland where Papmelia chloroehroa was collected.
Sites P8, P15, and P16 have both native and trans-
planted U. hirta. Site P7 has two buttes. U. hirta
was collected from the top and bottom of a hill at
P14. P10* = U. hirta transplant source. TC =
Taylor Creek fumigation sites (ZAPS).
187
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Table 18.1. LOCATIONS AND DESCRIPTIONS OF LICHEN COLLECTION SITES.
ROSEBUD, POWDER RIVER, AND BIG HORN COUNTIES, MONTANA;
1975-1979
Number
Name
Distance, Direction from
Colstrip, Exposure
Description
PI Sarpy Creek
P2 Castle Rock
P3 Kluver NE1
P4 Kluver El
P5 D. McRae
P6 Kluver West trees
P7 Diamond Buttes
P8 Morning Star View,
N. Cheyenne Res.
P9 A Kluver, near P3
P10 East Otter Creek
(transplant source)
Pll SEAM 1
P12 SEAM 2
P13 Home Creek Butte
P14 Three Mile Butte
P15 Ft. Howes
P16 Poker Jim Butte
PI 7 BNW//1
PI 8 BNW//2
P19 BNW//3
48 km Wl ENE exposure
16 km W; E
7 km ENE; SW
5 km E; W
7 km S; NNW
10 km SE; NW
20, 21 km NE; SW
26 km SSE; N
7 km NE; SW
51 km SE; ESE
45 km SE; NW
45 km SE; NW
55 km SE; NW
58 km SE; NW
70 km SSE; N
66 km SSE; NW
1 km S; N
2 km SE; N
1 km NW; SE
T2N, R37E, Sec. 36 (BH)
TIN, R41E, Sec. 36 (R)
T2N, R42E, Sec. 16 "
T2N, R42E, Sec. 29 "
TIN, R42E, Sec. 36 "
TIN, R42E, Sec. 2 "
.T2N, R43E, Sec. 22 "
T2S, R41E, Sec. 12
T2N, R42E, Sec. 16 "
T2S, R46E, Sec. 24 (PR)
T2S, R46E, Sec. 22 "
T2S, R46E, Sec. 22 "
T2S, R46E, Sec. 4 "
T4S, R47E, Sec. 10 "
T6S, R45E, Sec. 19 "
T6S, R44E, Sec. 17 (R)
T2N, R41E, Sec. 34 "
TIN, R41E, Sec. 3 "
T2N, R41E, Sec. 28 "
Gl
62
G3
G4
G5
G6
G7
Hay Coulee
McRae Knolls
Kluver West
Kluver North
Kluver East
Abandoned
Field, Taylor
Creek
11
km
SE
TIN,
R42E.
Sec.
28
(R)
Site abandoned "
12
5
21
98
km
km
km
km
SE
E
SE
SE
TIN,
TIN,
TIN,
T7S,
R42E,
R43E,
R43E,
R47E,
Sec.
Sec.
Sec,
Sec.
2
6
15
3
"
ii
"
(PR)
*
BH = Big Horn County; R = Rosebud County; PR = Powder River County
188
-------
RESULTS AND DISCUSSION
Respiration Rates
Observations from the field 862 fumigation sites (ZAPS plots,
Eversman, 1978, 1979) indicated that respiration, rates of the lichens
could be expected to rise when samples were slightly stressed and to fall
significantly when subjected to continuous higher S02 stress. Therefore,
either of these responses was watched for, particularly in samples within
a few km of Colstrip.
Variation in respiration rates of U. hirta among sites was significant
in 1979 (ANOVA, P<0.05), but was generally not related to distance from
Colstrip with one possible exception (Figure 18.2). U. hivta transplanted
in 1976 to one of the sites 1 km from the Colstrip power plant (BNW//1) had
a significantly elevated respiration rate July, 1979 (ANOVA, P<0.05). The
same phenomenon was observed at this site in September, 1977, and at site
P18 (BNW//2, 2 km from the power plant) September, 1977 and September, 1979.
It appears that these two sites may be affected by the power plants.
1000
Q 800
UJ
CO
z
8 600
M
O
f
1
=0.0001
10 20 30 40 50
KILOMETERS FROM COLSTRIP
60
70
Figure 18.2.
Linear regression of respiration rate of Usnea hirta,
July, 1979, with distance from Colstrip. Each point
represents the mean of three to six samples. Regression
line was computed using all readings comprising means.
Distance from Colstrip was not significant (P>.05,
linear and log transformation), although point with
* (Site BNW#1, 1 km from Colstrip) was significantly
higher (P<.01, ANOVA) than other sites.
189
-------
Regression lines comparing 1976-1979 (July respiration rates only)
are presented in Figure 18.3. In 1977 and 1978, respiration rates showed
a significant linear relationship to distance from Colstrip (regression
analysis of variance P<0.05, both linear and with log transformation),
however, since the slopes of the lines are opposite, the meaning of this
relationship is unclear. In 1979, when impact would be expected to have
been greater than in 1977 or 1978, the relation between respiration rates
and distance was not significant (P<0.05, linear regression and log trans-
formation, with analysis of variance). Sample size was greatest in 1979;
sample sizes may account for some differences.
All analyses of respiration rates of P. chloTOchroa gave results that
were not significant; sites were not significantly different from each other
(ANOVA) and respiration rates were not related to distance from Colstrip (re-
gression) . According to ZAPS experiments in 1976, P. chlopochroa was almost
as sensitive to S02 as U. hirta when they were placed side by side about
50 cm above the ground (Eversman, 1978, 1979). It could be that S02 was
not reaching ground level at the grassland sites, either because it is being
filtered out by grasses and forbs above ground level, or because the plume
from Colstrip was above even grass and forb level at the grassland sites.
1000
o
UJ
80°
g 600
CM
O
/I978:
0.02
1
"1979: r2= 0.02
^1977: r2 = 0.30
M976: r2=0.06
10 20 30 40 50
KILOMETERS FROM COLSTRIP
60
70
Figure 18.3. Linear regressions of U. hirta respiration rates, 1976-1979,
month of July only, with distance from Colstrip. Distances
were not significant in 1979, but were significant in 1978
and 1977 (P<0.05, regression analysis of variance). All
recorded respiration readings during July of each year
were used for regression computations. Sample size was
greatest in 1979 (100 data points) vs. 80 in 1978, 26
in 1977, and 35 in 1976. Variation in sample size may
account for some of the differences.
190
-------
Sulfur Content
The MSU Soil Testing Laboratory determined percentage of sulfur content
of lichen samples in 1975, 1977 and 1979. During these years, the laboratory
changed their analysis methods so comparisons between years are invalid
until intercalibrations are available. Within-year comparisons are possible
(Figure 18.4). In 1979, U. hirta from the two closest sites to the Colstrip
plant had higher sulfate content than samples from other sites, but dif-
ferences were not significant (ANOVA, Newman-Keuls Q test). However, sulfate
content of U. hi-rta was related linearly to distance from Colstrip in 1979
(regression analysis of variance, P<.05).
In 1977, with seven sites sampled for sulfur content (P1-P6, P10-P13),
there was also a significant linear relationship between distance from
Colstrip and sulfur content of U. hirta tissue (r2 = 0.18), P<.05, linear
regression analysis of variance). In 1975, before power plant operations
began, no linear relationship existed. Usnea hirta samples seemed to be
accumulating more sulfur in the Colstrip vicinity than in sites farther
away (in Custer National Forest).
(7)
2 0.15
0.10
0.05
CO
3*
<0.05
10 20 30 40 50
KILOMETERS FROM COLSTRIP
60
70
Figure 18 .4.
Regression of percentage sulfur (as sulfate) of U. h-ir>ta
samples, 1979. Each point is the mean of three to 12 samples.
Regression was computed using all readings comprising
means. Increased sulfur content was linearly related
to distance from Colstrip (negative slope, P<.05, re-
gression analysis of variance).
191
-------
Parmelia chlovochvoa samples all had a sulfate content between 0.06
and 0.08 percent in 1979, regardless of location. There were no significant dif-
ferences among sites and there was no linear relationship between sulfur
(as sulfate) content and distance from Colstrip in 1975, 1977, or 1979.
Plasmolysis of Algal Cells
Counting plasmolyzed algal cells gave an immediate impression of total
thallus health and integrity, as well as a quantitative measure of
presumably viable photosynthesizing algal cells (Figure 18.5). It is
perhaps a less objective method than chlorophyll content determination or
apparent photosynthetic rate, but it does not consume much sample material
nor does it require careful manipulation of temperature, light conditions
and water content.
- 75|
a.
o
x
o
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o
cc
UJ
0.
CO i
ujT
I CT
L_ *
£ ^
guj
" ^
a.
13
50
25
200
UJ
8 -200
-400
-4-
25 50 75 100
PER CENT PLASMOLYZED ALGAL CELLS
Figure 18.5. Linear regressions of plasmolysis with
percentage chlorophyll (acetone wash
technique), and plasmolysis with net
photosynthesis of U. hirta. Both re-
lations were highly significant (P<0.01,
regression analysis of variance). Data
from ZAPS samples (points without
circles) were used in computing the
regressions.
192
-------
Regression lines of plasmolyzed algal cells of U. hirta versus distance
from Colstrip had negative slopes in both 1979 and 1978, but relationships
were not significant (Figure 18.6.).
Plasmolysis of algal cells in P. chloTOchvoa was not related to
distance from Colstrip, nor were there significant differences in mean
plasmolysis among sites in 1979 (ANOVA) .
CO
«
o
5
to
40
30
<
_l
<
20
UJ
o
10
1978: r2 =0.31
10
Figure 18.6.
20 30 40 50
KILOMETERS FROM COLSTRIP
60
70
Linear regression of percentage of algal plasmolysis
of U. h'lTta with distance from Colstrip. Points re-
present means of three to nine samples collected during
1979. Regression lines were computed using all data points
comprising means. Plasmolysis was not significantly
related to distance from Colstrip in either 1978 or
1979 (regression analysis of variance).
Chlorophyll Content
Until September, 1979, chlorophyll content of lichen samples was
determined by a simple extraction in which lichen materials were boiled
in methanol for a few minutes, filtered, brought back up to 10 ml with
methanol, then read turbidometrically in a Beckman DU spectrophotometer.
Several wavelengths were read, but results were reported as relative
absorbance at 665 nm. This method actually included chlorophylls, plus
phaeophytin and any methanol-soluble materials.
It was suggested that the presence of lichen acids destroys chlorophyll,
removing magnesium and forming phaeophytin, thus giving distorted readings
on effects of S02 on chlorophyll (Brown and Hooker, 1977; Nash, pers. comm.)
193
-------
Successive washes in acetone would remove the acids and cause less degrada-
tion of chlorophyll due to acid presence. The biggest advantage I found of
successive washes in acetone was to allow better separation of pigments
(chlorophyll and phaeophytins) during chromatography, leading to ability to
quantify amounts of each pigment present. The acetone washes did not com-
pletely remove acids, as detected by addition of paraphenylenediamine
to the last wash, nor was there any apparent beneficial effect on chlorophyll
degradation.
The two methods gave similar gross estimates of effect of SC>2 on
chlorophyll content Cas judged by ZAPS samples). There were no significant
relations between distance from Colstrip and percentage of chlorophyll in
extracts (Figure 18.7), or between distance and relative absorbance of
methanol extracts (Figure 18.8).
80
i
a.
o
g 60
_i
i
o
UJ
o
cc
UJ
0.
40
20
r"=0.04
P> 0.05
10 20 30 40 50
KILOMETERS FROM COLSTRIP
60
70
.Figure 18.7.
Regression of percentage of chlorophyll in pigment
extracts of U. hirta with distance from Colstrip.
Points are means of three samples. Regression line
was computed using all observations during July,
1979. This method of chlorophyll extraction (ace-
tone washes, chromatography) gave results similar
to methanol extraction method (Figure 18.8).
194
-------
in 1.00
(0
CO
o
a: 0.80
i-
X
UJ
I
o
UJ
H
UJ
GC
0.60
UJ
o 0.40
CD
0.05, regression
analysis of variance).
CONCLUSIONS
The only measured parameter that showed significant relationship (P<0.05,
regression analysis of variance) to distance from Colstrip was sulfur content
of Usnea h-irta in 1977 and 1979. Its significantly higher level in the
immediate Colstrip vicinity as compared with previous years and with Custer
National Forest sites indicated some power plant effects but still very slight
at this time. Samples collected from all sites appeared "normally" healthy
and appropriately green in 1979 as in earlier years. Any possible impact
on lichens has been very slight to date, as was expected because of reported
low concentrations of S02.» ca- 0.10-0.34 pphm monthly averages, at electronic
monitoring stations (Ludwick, e~t al. , 1980) .
At the end of the 1979 field season, new fresh material was trans-
planted onto many sites (P2-P7, P18, P19) in anticipation of possible
future lichen biomonitoring in the Colstrip area as power plant construction
in the entire study area increases.
195
-------
Value of Lichens as Bioindicators or Biomonitors
Lichens are not an economically important plant in eastern Montana
ponderosa pine and grassland communities; their ecological importance is
probably not fully understood. Their role in invertebrate animal communi-
ties may be important, and there are many anecdotal incidents of deer
browsing on epiphytic lichens during winters.
The ubiquitous use of lichens as air quality indicators in
European countries, Canada and Japan must be considered. In these
countries where sulfur oxides and acid rains are prevalent, conditions of
lichens and lichen communities are carefully documented.
Perhaps the line of reasoning is this. Lichens are green plants,
therefore they have the same basic photosynthetic enzymes and chemical
pathways that higher plants have. In fact, most of the details of plant
photosynthesis have been elucidated using ChloTella, a green algal very
similar to Trebouxia, a major green algal component of lichens (including
U. hirta and P. Ghlorochroa).
Lichens have some anatomical and physiological differences that
appear to make them more sensitive than most vascular plants, including:
1) Absorbance of water, nutrients, and gases, directly from the atmosphere
with no soil and/or substrate filtering such as that occurring with
vascular plants. 2) Absence of stomata and protective waxy cuticle on
outer surfaces to prevent absorption of materials. If the lichen thallus
is moist and if pollutants are present, they will probably be absorbed by
the lichen. 3) Absence of deciduous parts; materials accumulate in-
definitely. 4) No dormant season of the year; their activity depends on
available moisture, including dew and melting snow, which means they can
accumulate materials throughout the year, not just during a growing season.
5) The symbiotic system, a balance between the alga and the fungus, may
be more delicate than the conventional tissue system of vascular plants.
If the processes are being impaired in lichens it should serve as a
warning that increasing levels of pollutants will probably cause the same
problem in economically important vascular plants when their protective
mechanisms have been overcome.
Lichens are an inexpensive bioindicator of air quality, and as such
it seems reasonable to include their use in the monitoring of air quality.
REFERENCES
Brown, D. H. and T, N. Hooker. 1977, The Significance of Acidic Lichen
Substances in the Estimation of Chlorophyll and Phaeophytin in Lichens,
NewPhytol., 78:617-624.
Eversman, S. 1978. Effects of Low-Level S02 on Usnae hirta and Parmelia
ahlorochroa, Bryologist, 81 (3);367-377.
196
-------
Eversman, S. 1979. Effects of Low-Level S02 on Two Native Lichen Species.
In: E. M. Preston and T. Gullett, eds. The Bioenvironmental Impact of
a Coal-Fired Power Plant, 4th Interim Report. EPA-600/3-79-044. U.S.
Environmental Protection Agency. Corvallis, Oregon, pp. 642-672.
Ferry, B. W., M. S. Baddeley, and D. L. Hawksworth. 1973. Air Pollution
and Lichens. University of Toronto Press.
Hawksworth, D. L. 1975. Literature on Air Pollution and Lichens II.
Lichenologist, 7(1):62-66.
Hawksworth, D. L. 1975. Literature on Air Pollution and Lichens III.
Lichenologist, 7(2):173-177 -
Hawksworth, D. L. 1976. Literature on Air Pollution and Lichens IV.
Lichenologist, 8(1):87-91.
Hawksworth, D. L. 1976. Literature on Air Pollution and Lichens V.
Lichenologist, 8(2):179-182.
Hawksworth, D. L. 1977. Literature on Air Pollution and Lichens VI.
Lichenologist, 9(1):77-82.
Hawksworth, D. L. 1977. Literature on Air Pollution and Lichens VII.
Lichenologist, 9(2):147-152.
Hawksworth, D. L. 1978. Literature on Air Pollution and Lichens VIII.
Lichenologist, 10(1):95-100.
Hawksworth, D. L. and A. Henderson. 1978. Literature on Air Pollution
and Lichens IX. Lichenologist, 10(2):227-230.
Hawksworth, D. L. and A. Henderson. 1979. Literature on Air Pollution and
Lichens X. Lichenologist, 11(1):91-96.
Henderson, A. 1979. Literature on Air Pollution and Lichens XI.
Lichenologist, 11(2):313-319.
Henderson, A. 1980. Literature on Air Pollution and Lichens XII.
Lichenologist, 12(1);145-148.
Ludwick, J. D., D. B. Weber, K. B. Olsen and S. R. Garcia. 1980. Air
Quality Measurements in the Coal-Fired Power Plant Environment of Col-
strip, Montana. In: E. M. Preston and D. W. O'Guinn, eds. The Bioenvi-
ronmental Impact of a Coal-Fired Power Plant, 5th Interim Report. EPA-
600/3-80-052. U.S. Environmental Protection Agency, Corvallis, Oregon.
(In Press) pp. 1-19.
Lund, R. E. 1980. A User's Guide to MSUSTAT: An Interactive Statistical
Analysis Package. Technical Report. The Statistical Center, Department
of Mathematical Sciences, Montana State University, Bozeman. 74 pp.
197
-------
SECTION 19
EFFECTS OF LOW-LEVEL S02 ON TWO NATIVE
LICHEN SPECIES: 1979 ZAPS OBSERVATIONS
AND PROJECT SUMMARY
S. Eversman
ABSTRACT
Significant reduction in respiration rate
occurred in Usnea hi-vta (L.) Wigg. after about 100
days in a fumigation plot at 2-3 pphm. S02. At this
same S02 level, significant reductions in pigment
content and increases in plasmolyzed algal cells '
occurred within 90 days. At about 5 pphm S02, there
were significant increases in plasmolyzed algal cells
within 60 days, decrease in respiration rates within
60 days, and decrease in pigment content within 90
days. Responses of Parmel-ia ohloTOohfoa Tayl. col-
lected from soil surfaces in the same plots were
slight and generally insignificant.
INTRODUCTION
The primary objective of this study was to establish S02 dose-response
curves for two native lichen species, Usnea hivta (L.) Wigg. and Parmelia
ohloTOchToa Tayl.; i.e., to establish anatomical and physiological responses
of the lichens to given dosages of S02 in the ZAPS fumigation plots.
Laboratory tests of many researchers have established responses to large
doses of S02 in short periods of time (LeBlanc and Rao, 1975). I have
attempted to determine responses of U. hirta and P. chloTookpoa to the various
S02 doses of the ZAPS plots.
MATERIALS AND METHODS
Usnea hirta samples were transplanted to four posts in ZAPS plots A, B,
and C in June, 1979 (the northwestern post in each plot was not used) by
moving entire ponderosa pine branches containing lichen growth from the
East Otter Creek site in Custer National Forest, 30 km NE of Ashland (site
P10), as in previous years. Collections were made 28, 56, and 90 days after
transplanting.
198
-------
Plot D (High) was not observed in 1979; in previous years within
30-60 days lichens in plot D showed nearly 100 percent mortality of algal cells,
complete thallus bleaching, significantly reduced respiration rates, and
no photosynthesis. Since findings in the ZAPS sites have been used for
comparisons with possible responses to S02 in the Colstrip power plant
vicinity, the dosages in D plot were unrealistically high. Observations in
1979 concentrated in plots A/B and C, ZAPS I and II.
Parmel-La chloTOclrroa samples from a nearby field were placed at the
base of one post per plot (the most northeastern one) and collected only
in September (90 days of treatment)-
The major moss in the ZAPS plots (Polytrichum piliferum) and two
Cladonia species were collected from each of the plots to check cell
condition.
Respiration rates were determined manometrically for 250-mg samples at
20 C in saturated condition in the dark. Chlorophyll extracts were made
in 1979 according to the method of Brown and Hooker (1977) described in
Section 18. Plasmolysis of algal cells was determined by counting cells in
wet mounts (Section 18). Sulfur contents were determined by the Montana
State University Soil Testing Laboratory, using a dry ash and turbidometric
procedure (pers. comm.). Photosynthesis rates were determined in an infra-
red gas analyzer (Section 18).
Statistical analyses were through the Montana State University
Statistical Center programs: multiple regression; one-way analysis of
variance, and Newman-Keuls Q, comparison of means (Lund, 1980).
RESULTS AND DISCUSSION
This report summarizes 1979 field observations, and compares and
combines results from this year with previous years to establish lichen
responses to low S02 exposures over 5-month periods in a northern plains
grassland.
Respiration Rates
The pattern of respiration rates of Usnea hirta. established in previous
years was repeated in 1979. Figure 19.1 shows results after 90 days
of fumigation in 1979, and results in samples after 92 days of fumigation
in 1978 and 96 days in 1976. Ninety days of treatment were usually not
enough to establish significant differences in Usnea respiration rates
between A and B plots. Samples from plot C usually had significantly
lower respiration rates than samples from plots A and B after 90 days of
treatment (ANOVA, P<.05).
199
-------
7 1000
i.
UJ jf1
IT
0 750
£ O 500
CL O
LJ co
CE O
250
•i I
1979 (90 days)
- 1978 (92 days)
1976 (96 days)
OPC
Figure 19.1.
IA
ITA
1C
HC
IB HB
SITE
Respiration rates of Usnea hirta, 1976, 1978, 1979, ZAPS
A, B and C (means of 3-9 samples ± .95 confidence inter-
val computed as tQ.05 x standard error). Results of 90
days exposure in 1979 closely duplicated 92 days exposure
in 1978. Respiration rates of Usnea from plot C were
consistently significantly lower than samples from A and
B by 100-110 days of exposure (ANOVA, P<0.05).
OPC = off-plot control, aa. 2 km from ZAPS plots.
The assumption was made that treatment in each plot was essentially the
same from year to year. Individual respiration rate readings for Usnea
were plotted against days for each plot (Figure 19.2). Relation between
time in ZAPS I A and II A and respiration rate was not significant (re-
gression ANOVA); the relation was significant in plots B and C, ZAPS I
(regression ANOVA, P<0.05). There was a significant drop in respiration
rate in samples from plot C after 56 days of exposure and after 96 days in
plot B (ANOVA), P<0..05> Newman-Keuls Q).
Respiration rates of PaTme1i,a ehlovoehroa on the ground showed no
significant relationship with ZAPS plots though it responded in a manner
similar to Usnea when placed 50 cm above the ground (Eversman, 1978,
1979). This position effect is discussed in the following sections. As
stated previously (Eversman, 1978) the bacterial populations associated
with Parmelia probably confound true lichen respiration readings.
Sulfur Content
There were no significant differences in sulfur content among Usnea
samples from the ZAPS sites after 90 days in 1979 (Table 19.1). While the
highest individual readings (0.17 percent, 90 days) and means were from ZAPS B
and C, means were not significantly different (ANOVA, P>0.05).
200
-------
1000
75°
UJ
UJ
en
en _
LU a.
cr
500
25°
Figure 19.2,
= 0.45, P<.OI)
25
ON
100
ZAPS
125
150
175
50 75
DAYS OF S02
Regression of Usnea respiration rate with days of SC>2 in
ZAPS I. Individual respiration readings at 0, 27, 28,
42, 56, 84, 90, 92, 96, 110, 119 and 156 days in ZAPS I
plots 1975-1979 were used in computing regression lines.
Samples from plot A were never significantly different
from day 0. Samples from B were significantly lower
than day 0 after 96 days. Samples from C were signi-
ficantly lower than day 0 readings after 56 days (ANOVA,
P<.05; Newman-Keuls Q).
Results in the Colstrip area indicated some significantly higher
sulfur content in Usnea specimens within 2 km of Colstrip (Section 18)
Perhaps the 90 days of exposure in the ZAPS plots were not adequate to
accumulate different sulfur amounts. Or perhaps as the Usnea samples
became less viable they were unable to metabolically accumulate sulfur
as healthier specimens do. Gilbert (1969) demonstrated less sulfur
accumulation by killed Usnea samples than by living ones.
Sulfur contents were not determined for Parmelia samples from the
ZAPS sites. In previous years differences across plots were not significant.
Plasmolysis of algal cells
The only analytical method that consistently showed significant differen-
ces in Usnea and Parmelia samples between ZAPS plots A and B 1976-1979 was
counting plasmolyzed algal cells. Differences became apparent at 30 days
and were always significant by 60-90 days (Figure 19.3; ANOVA, P<0.05).
Results from Parmelia samples taken from the soil surface showed less clear
responses; significantly higher plasmolysis rates generally occurred only
in plot D after 60 days of exposure (1975, 1976).
201
-------
TABLE 19.1. SULFUR CONTENT OF USNEA HIRTA, ZAPS I AND II, 1979 (MEAN
PERCENTAGE ± ONE STANDARD DEVIATION FOR THREE SAMPLES)
IA
IIA
IB
I IB
1C
IIC
July
28 days
0.10±0.03
0.09 ±0.01
0.10±0.02
0.12±0.02
0.11 ±0.02
0.11 + 0.02
August
56 days
0.11 + 0.01
0.11 + 0.02
0.11 ± 0.01
0.11 ± 0.02
0.11 ± 0.02
0.10 ± 0.01
September
90 days
0.11 ± 0.01
0.08 ± 0.02
0.13 ± 0.04
0.11 ± 0.06
0.12 + 0.05
0.12 + 0.05
OPC
EOC
0.09 ± 0.02
0.12 ± 0.02
0.10 ± 0.03
0.09 ± 0.01
0.11 ± 0.01
* t
OPC = Off-plot control EOC = East Otter Creek, transplant
source. ANOVA, P = 0.72.
Q
LJ
M
100
75
cn_i
< LJ
^°
°- 50
LJ
O
CC
LJ
Q.
25
J-
i__i
•I »•
= 1979 (90 days)
= 1978 (92 days)
= 1976 (96 days)
OPC IA IEA IB HB
ZAPS PLOTS
1C HC
Figure 19.3.
Percentage of plasmolyzed algal cells in Usnea h-irta at
90 days (1979), 92 days (1978) and 96 days (1976 S02
exposure, ZAPS I and II. Differences between A, B and
C are highly significant (ANOVA P<0.01). Each bar is
mean ± .95 confidence interval (computed as to.05 x
standard error). OPC = off-plot control.
202
-------
Figures 19.4 and 19.5 illustrate relationships between time in ZAPS I
plots and plasmolysis rates. Usnea showed a more significant response than
Pcametiaj however Usnea was about 50 cm above the ground and Parme'L'La was
on the soil surface. Sulfation plate studies (Eversman, 1978; Preston and
Gullett, 1979) showed significantly less S02 reaching 5-10 cm above the
ground (with presumably even less at ground level) than was detected 50-100
cm higher. Reduced Parmel-ia responses, compared with Usnea, were assumed to
be partly a result of lesser 862 levels at the soil surface. Parmelia
appeared to be slightly less sensitive than Usnea (Eversman, 1978), and since
it was directly on the soil, substrate buffering may have occurred. Parmetia
control samples tended to exhibit slightly increased plasmolysis throughout
the summer, from spring to autumn, that perhaps contributed to the slope
line in Figure 19.5.
Taylor, Leininger, and Hoard (pers. comm.) observed significant reduction
of lichen cover in the ZAPS plots C and D since 1976. I collected Polytr-iohum
piliferum (moss) and two sterile Cladonia (lichen) species from all ZAPS
plots in 1979 for cell observations. Cladonia plasmolysis means ranged
between 10-14 percent in ZAPS A and B, 15-18 percent in C, and over 30 percent
in ZAPS D. It seems likely that some S02 effects were felt by lichens on the
soil surface particularly in plots C and D.
Moss cells appeared "normal" in all specimens and there were no visible
differences in moss plants among plots.
CO
UJ
o
Q
UJ
N
Q.
LJ
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O
a:
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100
75
50
25
= 0.26, P<.OI)
I, P=0.32)
20 40 60 80 100 120
DAYS OF S02 ON ZAPS I
140
160
Figure 19.4.
Regressions of percentage plasmolyzed algal cells of U.
hirta with time on ZAPS I, plots A, B and C. Individual
plasmolysis readings from 0 (day of transplanting), 27, 28
33, 47, 56, 57, 90, 92, 110 and 119 days in ZAPS I plots
in 1975-1979 were used in computing regression lines.
Probabilities are from regression analysis of variance.
203
-------
0
Ul
N
o
_l LJ
a. o
UJ -J
O
o:
LU
CL
50
25
Figure 19.5,
20 40 60 80 100 120 140
DAYS OF S02 ON ZAPS I
160
Regressions of percentage plasmolyzed algal cells of P.
chloTOchroa with time in ZAPS I, plots A, B and C. Samples
were taken from the soil surface. Individual plasmolysis
readings from 0 (day of transplanting), 33, 47, 60 and 90
days were used in computing regressions. Probabilities
are from regression analysis of variance.
Photosynthesis
Usnea samples from plot C, ZAPS I and II, had a significantly reduced
photosynthesis rate after 90 days of exposure in 1979. Again, there were
no significant differences between Control samples, samples from plot A and
most of the B samples (Figure 19.6). However, one set of three Usnea samples
from one position in plot B illustrated a common occurrence in samples from
this exposure plot. Samples taken from the most southwestern post tended
to show greater response to S02 than did samples taken elsewhere in plot B.
Samples from plot B, when averaged together, consistently exhibited the
greater variances in every characteristic measured (respiration rate,
plasmolysis, etc,), indicating that the various amounts throughout the plot
seemed to be threshold between slight effect and pronounced.
Pigment and chlorophyll determinations
Removal of all pigment from the lichen samples has been very difficult
using either boiling methanol or acetone. Therefore, determinations of
pigment and chlorophyll content have been relative between Control and
S02~treated samples, not absolute. Regardless of method used, differences
in total pigment content between samples from plots A and B were usually
not significant (Figure 19.7a); variability in samples from plot B (ZAPS I
and II) was pronounced. Samples from plot C had significantly lower total
pigment amounts (P<0.05, ANOVA) than samples from A and Control plots.
Stressed chlorophyll degrades to phaeophytin, therefore higher levels of
chlorophyll were expected in healthy specimens and higher levels of phaeo-
phytin were expected in stressed specimens. However, samples treated with
acetone and chromatographed (Brown and Hooker, 1977) showed no significant
differences across treatments (Figure 19.7b). Simple pigment extract
204
-------
LU
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h- O
400
200
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I
EOCOPC IA HA IB HB
Usnea hirta
icnc F IA IB ic
Parmelia chlorochroa
Figure 19.6. Apparent photosynthesis rate of Usnea hi-rta and Parmel'ia'
olorochroa after 90 days in ZAPS plots, 1979. Each bar
is the mean ± .95 confidence interval (computed as t.os
x standard error) of three samples. Rates were deter-
mined with an infrared gas analyzer at 50 percent saturation.
Usnea samples from one position in ZAPS IB and plots C
had net respiration.
procedures appeared to be somewhat more informative in this case than the
more complex chlorophyll/phaeophytin determination methods.
Parmel-ia samples from the ground in plot C had a significantly higher
total pigment content, and a slightly higher chlorophyll content than
other samples (Figure 19.7). These results were similar to those obtained
in 1977. It appeared that presence of low amounts of S02 was stimulating
pigment production.
When regressions were computed between percentage of plasmolyzed algal
cells and apparent photosynthesis rate for Usnea (Figure 19.8), the relation-
ship was highly significant (regression ANOVA, P< 0.01). The relation between
plasmolyzed algal cells and percentage of chlorophyll (Figure 19.9) was
slightly less significant (P<0.05). Counting plasmolyzed algal cells gave
not only an observation of gross anatomical appearance, but also an estimate
of amount of chlorophyll and photosynthesis rates for Usnea.
Results for Paimeti-a were less definitive, with no apparent relation
between plasmolysis, chlorophyll content, and photosynthesis.
205
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P. ch/orochroa
Figure 19.7a. Total pigment content for Usnea and Parmelia after
90 days in ZAPS plot 1979. Underlines indicate
samples not significantly different from each other.
Figure 19.7b. Percentage of chlorophyll in pigment extracts of
Usnea and Parmelia after 90 days in ZAPS plots,
1979. Underlines indicate no significant differen-
ces across treatment plots. Bars are means ± .95
confidence interval (t. x. standard error) for three
samples. EOC = East Otter Creek (transplant source).
OPC = off-plot control 2 km from ZAPS. F = field,
source of Parmelia transplants.
CONCLUSIONS
Usnea hirta consistently exhibited better-defined responses to fumigation
than did Parmelia chloroehroa. There are two possible reasons: 1) The
growth form of Usnea is bushy (fruticose) and its usual position (tufts on
bark of trees) give more surface area for exposure to and absorption of
S02. Parmelia is leaf-shaped (foliose) with proportionately less surface
exposed to air. 2) Parmelia inhabits soil; Usnea is an epiphyte on ponderosa
pine. The elevated position of Usnea may expose it to more S02 and the
acidic bark (pH less than 5.0) offers little buffering potential. Parmelia
was exposed to less S02 probably because of vegetational scrubbing of taller
plants around it. Limestone-derived soils in many places offer buffering
capacity that would decrease S02 effects. When Parmelia was elevated on
ponderosa pine branches with Usnea in 1977 ZAPS observations, it was
nearly as sensitive as Usnea (Eversman, 1978, 1979).
LeBlanc and Rao (1975) suggested that long-range average concentrations
of S02 above 3 pphm would probably cause acute injury to epiphytes in the
Sudbury, Ontario area. Results from the ZAPS sites, C (oa. 5 pphm) and D
(ca. 8 pphm, geometric means) supported this statement. Within 60-90 days
206
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25 50 75 100
PER CENT PLASMOLYZED ALGAL CELLS
Figure 19.8.
Regression between percentage plamolyzed algal
cells of Usnea hi-rta and apparent photosynthesis
rate. Each dot represents the mean of three
samples from ZAPS plot A, B and C. I and II
after 90 days, 1979. P and F values are from
regression analysis of variance.
thalli were bleached and there were significant decreases in vital processes
(photosynthesis, respiration), in pigment content and cell viability,
especially in Usnea h-ipta.
LeBlanc and Rao also suggested that long-term SC>2 exposure of 0.6 to
3.0 pphm could cause chronic injury to lichens. After 60 days in plot B
(ca. 2-3 pphm), Usnea had elevated plasmolysis rates, reduced photosynthesis
and pigment contents, erratic respiration rates, and visibly bleached thalli.
After 90-100 days in plot B, all of these characteristics showed significant
differences when compared with samples from Control sites and ZAPS plot A.
Depending upon definitions of "acute" and "chronic", samples of Usnea and
elevated Pavmelia, always exhibited adverse effects after two to three
months in B plots.
ZAPS plot A has been recorded having about 1 pphm S02 average (geometric
mean)(Preston, pers. comm.). The longest lichen testing period was 156 days
207
-------
100
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75
50
LJ
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LU
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25 50 75 100
PER CENT PLASMOLYZED ALGAL CELLS
Figure 19.9. Regression between percentage plasmolyzed algal cells and
percentage of chlorophyll in pigment extracts for Usnea
hirta. Each dot represents the mean of three samples from
ZAPS I and II, plots A, B and C after 90 days, 1979. P and
F values are from regression analysis of variance.
in 1976; the shortest was 84 days in 1978. Within these time periods, there
were no significant differences between sampling time and the day of trans-
planting. The conclusion from plot A is that it would take more than 156
days of constant S02 exposure at that recorded level to cause detectable
adverse effects in Usnea h-Lrta in this climatic regime.
When Parmel-ia was extensively sampled from the soil most detectable
responses were from plot D with mostly statistically insignificant responses
from plots B and C. The. responses that did occur would have been at SC>2
levels less than the monitored amounts since monitoring devices were placed
above ground level where S02 amounts appear to be higher than directly
on the soil surface.
REFERENCES
Brown, D. H. and T. N. Hooker. 1977. The Significance of Acidic Lichen
Substances in the Estimation of Chlorophyll and Phaeophytin in Lichens.
New Phytol, 78:617-624.
208
-------
Eversman, S. 1978. Effects of Low-Level of SC>2 on Usnea hipta and
oKtovochToa. Bryologist, 81 (3):367-377.
Eversman, S. 1979. Effects of Low-Level S02 on Two Native Lichen Species.
In: Preston, E. and T. Gullett, eds. The Bioenvironmental Impact of a
Coal-fired Power Plant. 4th Interim Report. EPA-600/3-79-044 . U.S.
Environmental Protection Agency, Corvallis, Oregon pp. 642-672,
Gilbert, 0. L. 1969. The Effect of S02 on Lichens and Bryophytes around
Newcastle upon Tyne. Air Pollut. , Proc. Eur. Congr. Influence Air
Pollution, Plants and Animals, 1st. 1968. pp 237-272.
LeBlanc, F. and D. N. Rao. 1975. Effects of Pollutants on Lichens and
Bryophytes. In: Mudd, J. B. and T. T. Koslowski, eds. Responses
of Plants to Air Pollution. Academic Press, New York. pp. 237-272.
Lund, R. E. 1980. A User's Guide to MSUSTAT: An Interactive Statistical
Analysis Package. Technical Report. The Statistical Center, Department
of Mathematical Sciences, Montana State University, Bozeman. 74 pp.
Preston, E. M. and T. L. Gullett. 1979. Spatial Variation of Sulfur
Dioxide Concentrations on ZAPS During the 1977 Field Season. In:
Preston, E. and T. Gullett, eds. The Bioenvironmental Impact of a
Coal-fired Power Plant. 4th Interim Report. EPA-600/3-79-Q44 . U.S.
Environmental Protection Agency, Corvallis, Oregon, pp. 306-330.
209
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SECTION 20
FLUORIDE AND ARSENIC CONCENTRATIONS
IN HONEY BEES NEAR COLSTRIP
J. J. Bromenshenk
ABSTRACT
Since the coal-fired power plants were put in oper-
ation at Colstrip in 1975 and 1976, fluorides in bee
tissues have demonstrated significant increases over %
baselines at apiaries downwind and within 20 km of the
plants. In the fall of 1979, fluoride levels at sites
having concentrations significantly greater than base-
lines were similar to 1976, when some levels were more /
than twice those observed before the power plants began v
operation. Nearly all fluoride levels for early summer
of 1979 were substantially higher than any observed in
previous years. In 1977, high concentrations of fluoride
were observed in bees at two sites, one north and one
south of Colstrip. In 1979, mean fluoride levels at the
south site exceeded that of 1977 by a factor of 1.5, an
approximate 11 fold increase over baselines. No bees
were at the north site in June of 1979. At sites north-
east of Colstrip, June/July fluoride concentrations were
3 to 17 times baselines. Levels at one of these sites _,/
exceeded reported bee toxicity thresholds. Although
fluoride levels varied significantly in 1979, at none of
the sites did arsenic levels exceed baselines.
INTRODUCTION
Literature reviews and detailed rationale for selecting honey bees as
biological monitors appeared in the preceeding five interim reports of the
Colstrip project. Bees serve as bioaccumulators and magnify the levels of
many chemicals in their surroundings making it easier to detect the presence
and distribution of pollutants. They also provide information about the
potential for transfer of pollutants through food chains, especially to humans
via honey or pollen. They are manageable social insects that can inhabit
almost any biome, and they provide an abundance of sampling material (bees,
pollen, honey, and wax). Because bees are beneficial insects in terms of
210
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products (honey, wax, and pollen) and services (pollination), information
gathered using a bee monitoring system is directly applicable to human welfare.
Monitoring honey bees should serve as an early warning system of pollutant
accumulation and effects which may harm other organisms or alter ecosystem
structure and/or function. Hazards to the bees themselves and to the beekeeping
industry may also be discovered.
I emphasized fluoride in this study because it is a coal combustion emis-
sion, is relatively simple to detect analytically, has low background levels
in flora and fauna of native grasslands, and is toxic to bees. Although coal-
fired power plants emit substantially more sulfur than fluoride, my previous
attempts to examine anthropogenic sulfur in honey bees proved unsuccessful.
High background levels of sulfur in bee tissues (presumably in sulfur bonds of
proteins) tended to mask any detectable incremental increase (Hillman, 1972).
Honey bees take up large amounts of arsenic near copper smelters and have
been reported to take up arsenic emitted by coal-fired power plants (Lillie,
1972). Colstrip bees have been monitored for any long-term build-up of this
element, but 1975-78 arsenic levels in these bees, with a few exceptions, have
been low.
Air and water are the media whereby pollutants released by activities
such as the mining and burning of coal at Colstrip may readily reach bees.
Therefore, fluoride concentrations in apiary water supplies and levels of
fluoride and sulfur in the ambient air have been monitored concurrently with
levels in bees and pollen.
MATERIALS AND METHODS
In 1979, honey bees, pollen, water, and air were sampled at 16 apiaries
during late June/early July and again in mid-September. Many of the colonies
are transported to California each winter for pollination of orchards and
vineyards and are returned to Montana in April and May. Any food supplies
(pollen and honey) brought from California were likely consumed and replenished
by June. Therefore, contaminants carried back with the "migrant" colonies
should be dispersed by the June/July sampling unless fluoride is retained in
the beeswax. Also, several population turnovers should have occurred (brood
cycles are approximately three weeks). Colonies distant from Colstrip (>40 km)
are not moved out of the region and can be used as additional controls. Bees
sampled near Colstrip in June/July had collected several boxes (supers) of
surplus "sweet clover" honey. This provided further evidence that extensive
foraging and nectar gathering had occurred since their return from California.
The autumn (September) collection was performed before the apiaries were
transported back to California. The beekeeper moved the colonies from NE 3
and NE 4 to a stockpile location 1 day before they were scheduled to be
sampled. I found and sampled these before they were shipped, but it was not
clear in all cases which of the marked hives were from which of the two bee-
yards. Therefore, the results for these sites for September, 1979, are indi-
cated as NE 3/4.
211
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Bees and Pollen
Each apiary contained seven to 50 colonies; most had 20 to 30 colonies.
A high velocity, battery-powered, acrylic vacuum apparatus was used to obtain
30 gms wet weight of bees (about 300 bees) from the entrance of each of 10
hives (Bromenshenk, 1978). At each location, the samples were immediately
frozen and stored in Whirl Pacs® at -20°C.
A plastic pick was used to collect pollen from brood chamber combs of 10
colonies at each location. The pollen samples were stored in plastic vials at
room temperature. A 100 gm wet weight pooled sample (about 1,000 bees) was
obtained at the entrance of every hive in each beeyard to provide a quick
screening method of analyses, to produce an average sample from each location,
and to ensure sufficient quantities of materials for quality assurance tests,
pesticide tests, and other tests.
Sulfation and Calcium Formate Plates
Two sulfation and two calcium formate plates were mounted on posts at the
grassland canopy level (75 cm above the ground) in each apiary in June and
collected in September in order to measure ambient air concentrations of
reactive sulfurs and fluorides. All of the analyses were complete, but statis-
tical examination of the data for 1978 and 1979 was incomplete at the time of
this report.
Water
Water was sampled at each apiary, in addition to bees, pollen, and air.
All beeyards were located within a few hundred meters of easily accessible
water in streams or reservoirs. A minimum of 500 ml of water was obtained at
points where bees were landing to drink. Samples were collected and stored in
Naglene§ bottles and frozen until analyzed. Besides creeks and ponds, any
water in livestock watering tanks within 0.5 km of beeyards was also sampled.
Fluoride Analyses
Before being analyzed for fluoride, whole bees and pollen were oven-dried
at 45°C for seven days and ground in a Wiley-Mill® to pass a 40-mesh screen.
For each sample, 0.5 gm of ground and dried material was placed in a metal
crucible and slurried with distilled water with 0.05 gm of reagent grade
calcium oxide. The samples were charred under infrared lamps before being
ashed in a muffle furnace at 600°C for at least 6 hours. The ashed samples
were digested in 2 ml of perchloric acid and subsequently diluted to 100 ml
total volume with Orion Tisab® total ionic strength activity buffer. Fluoride
determinations were made using an Orion® specific ion probe inserted into the
150 ml beakers containing the dissolved samples which were stirred constantly
during analysis. Water was analyzed using the Orion probe and 150 ml of equal
parts of water and buffer solution.
212
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Arsenic
Arsenic determinations were carried out by F. F. Munshower and A. R.
Neuman, Animal and Range Sciences, Montana State University. One-g samples
were weighed into 125 ml Erlynmeyer flasks and digested in 30 ml of a 3:2
moisture of nitric perchloric acid. Samples were left overnight and then
heated slowly to solubilize. The heat was gradually increased to reduce sample
volume to one-half. The samples were cooled and 10 ml of a 1:1 mixture of
nitric:sulfuric acid added. Heat was increased until perchlorate fumes evolved
out of the flasks. Heating continued until dense white sulfate fumes occurred.
Volume was reduced by heating to 5 ml and the samples allowed to cool. The
cooled samples were transferred to 50 ml volumetric flasks containing 15 ml of
concentrated hydrochloric acid, 10 ml of water, and 1 ml of 1 percent (w/v)
potassium iodide. Cooling to ambient temperature and allowing the samples to
stand for 1 hour allowed reduction of As+^ to As+3.
Standards were prepared in 100 ml volumetric flasks which contained 30 ml
hydrochloric acid, 2 ml of potassium iodide, and 5, 15, and 20 mg of arsenic.
These were allowed to stand for 1 hour prior to analyses. The coefficients
of determination for the results of analyses for arsenic in the standards
approximates 0.997, indicating good recovery of the chemical.
All determinations were made using an atomic absorption spectrophotometer
connected to an arsine generator. The analytical procedure consisted of
placing a 20.0 ml aliquot of standard/sample into the reaction flask. The
sample was purged by bubbling 50 percent N2 through the solution. Five ml of
5 percent (w/v) sodium borohydrate was added through the septum while stirring.
Statistics
Basic parametric statistical tests (mean, standard deviation, standard
error, 95 percent confidence intervals, correlation, two-factor ANOVA; Sokal and
Rohlf, 1969) were carried out for this report. Statistical tests of data for
1974 through 1978 suggested that fluoride levels in bees may not be normally
distributed (Bromenshenk, 1979, 1980). Therefore, the 1979 data will be re-
examined via nonparametric tests such as Kruskal-Wallis and Wilcoxon two
sample tests (Conover, 1971) in order to examine possible differences in the
distributions of fluoride levels in bee populations. Also, the data obtained
since 1974 indicates standard errors for fluoride and arsenic which are a
constant function of the mean. Because of this, M. E. Ginevan, biomathematician
and entomologist at Argonne National Laboratories (personal communication, 1980)
recommends logarithmic transformations of the data prior to applying parametric
statistics. Each of the above approaches will be undertaken, the results of
which will be published in a final report covering the entire 6-year study
period. It was not possible to complete these exercises for this progress
report.
RESULTS
Fluoride levels in apiary water supplies are summarized in Appendix 20.1.
Rosebud Creek, which supplies water to 87 percent of the beeyards near Colstrip,
displayed relatively constant fluoride levels from 1974 through 1979
213
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(0.4-0.6 ppm). Water from deep wells (NE 10 and E 2) contained more fluoride
(2.0-8.0 ppm) and a reservoir (S 1) had the least fluoride (0.1 ppm). A live-
stock watering tank and a slough formed by overflow from the tank located at
NE 10 has had the highest fluoride levels (2.6-12.7 ppm). Although bees have
often been observed obtaining water at Rosebud Creek and at the stock tank and
slough at NE 10, bees were not seen at the stock tank at E 2 and appeared to
be using Rosebud Creek as their primary water supply.
Fluoride levels in pollen obtained from each beeyard are presented in
Table 20.1. From 1975 through 1978, mean fluoride levels of pollen obtained
from beeyards did not exceed 3.0 ppm (range 1.5-2.9 ppm). In 1979, 3.0 ppm
was exceeded in 43 percent of all cases (June/July and September) and 57 percent
of the June/July samples; 4.3 ppm at NE 3 in June was the high. Mean fluoride
in excess of 3.0 ppm was found in two check samples (3.4 ppm at SE 6 in Septem-
ber, 3.1 ppm at SE 12 in June/July) as well as at sites near Colstrip.
TABLE 20.1. PPM FLUORIDE IN POLLEN, 1979
Site Date X S.D. S.E.
N 1
NE 1
NE 2
NE 3
NE 4
NE 3/4
E 2
SE 1
S 4
S 5
S 1
SW 2
SW 3
SE 6
SE 12
NE 1C
Sept.
June/July
Sept.
June /July
Sept.
June /July
June /July
Sept.
June /July
Sept,
June /July
Sept.
June /July
Sept.
June /July
Sept.
June /July
Sept.
June /July
Sept.
June /July
Sept.
June /July
Sept.
June /July
Sept.
June /July
Sept.
1.5
1.9
2,9
2.0
1.8
4.3
3.1
3.6
2.7
2.4
3.8
4.0
3.2
3.4
2.5
2.4
3.2
2.7
2.2
2.6
3.2
2.0
2.7
3.4
3.1
1.6
2.8
3.8
0.69
0.56
0.72
0.57
0.69
0.92
1.43
1.04
1.04
0.84
0.39
0.85
1.06
0.75
1.07
0.74
1.13
0.96
0.42
0.91
1.25
0.55
1.01
1.00
0.78
0.24
0.61
0.51
0.23
0.18
0.28
0.18
0.22
0.29
0.54
0.29
0.33
0.26
0.12
0.27
0.35
0.24
0.34
0.23
0.36
0.30
0.13
0.29
0.40
0.17
0.34
0.32
0.25
0.07
0.20
0.16
9
10
10
10
10
10
7
13
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
214
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Mean content and 95 percent confidence intervals for fluoride in worker honey
bees collected in mid-summer and autumn of 1979 are presented in Figures 20.1
and 20.2 and Table 20.2. Figure 20.1 also presents a map of the major apiary
locations utilized since 1974. Figure 20.3 presents mean fluoride content and
95% confidence intervals for samples taken in autumn of 1975, 1976, 1977, and
1978.
Mean fluoride levels of pollen collected from each apiary in June and
September, 1979, were not significantly correlated (r> = 0.20, df 12, P >_ 0.05),
nor did mean fluoride content of bees collected at beeyards in September signi-
ficantly correlate with mean fluoride content of pollen collected in September
(r = 0.31, df 12, P _> 0.05). There was a significant, although weak, corre-
lation between fluoride levels in pollen and bees from each site (mean fluoride)
and from each hive (r = 0.59, df 11, P <_ 0.05; r = 0.3721, df 126, P <_ 0.01)
based on June/July, 1979, sampling results.
The absolute mean fluoride content of honey bees sampled in June/July of
1979 at all sites except NE 10 exceeded that of the previous 2 years
(Figure 20.1). A livestock watering tank has been a potential source of
fluoride intake by bees at NE 10 since 1974 (Bromenshenk, 1978, 1979, 1980).
It is impossible to make meaningful comparisons of 1979 to 1976 data for the
July period. A breakdown of the electric vacuum sampler in 1976 necessitated
taking bees by sweeping them from honeycombs rather than by catching them at
hive entrances. Tests conducted in September of 1976 indicated that bees
taken from inside hives contained 50 percent less fluoride than bees collected at
entrances (Bromenshenk, 1978). However, even doubling the levels of fluoride
reported for July of 1976 would still result in a mean of less than 12 ppm for
Colstrip bees at all sites. Bees collected from 17 sites in 1975 displayed a
mean fluoride content of 8.7 ppm, SE = 0.44, as compared to 27.8 ppm, SE = 8.87,
for 14 sites in the same area in 1979.
Site NE 4 had a mean fluoride content of 30.9 ppm in early summer of 1979.
A mean of 153.8 ppm fluoride in bees at NE 3 in June was the highest value
observed at any apiary during the 6 years of this project. Mean values of
53.2 ppm at S 1 in 1979 and 35 ppm in 1977 greatly exceeded the 4.9 ppm for
1978 and 5.2 ppm for 1975. In general, fluoride levels in bees at all of the
sites near Colstrip, but not at the "check" sites (SE 6 and SE 12), ranked
among the highest observed in this region.
Although water contributes fluoride to bees at NE 10, water at NE 4, NE 3,
and S 1 is unlikely to be the source of the fluoride in bees. The reservoir
at S 1 typically contains not more than 0.1 ppm fluoride (1976 through 1979
data), while NE 2 and NE 3 are supplied by water from Rosebud Creek (0.4-0.6
ppm), as are most of the other Colstrip beeyards.
Bees sampled in September of 1979 most closely resembled those of 1976,
both in terms of mean fluoride content and the geographical distribution of
"elevated" levels of fluoride. The use of two-factor ANOVA demonstrated
highly significant year and site differences among variances for 1979 versus
1975 levels of fluoride in autumn samples of worker honey bees:
Fs = 12"73 > F-001[1,22] = 6'73 (y£ars); Fs = 9'20 > F.001[24,160] = 2'13 (sites)
215
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JUNE/JULY 1979
SW-I
SW-3
SE-12
Figure 20.1. Mean fluoride in worker honey bees and 95 percent confidence inter-
vals, June/July collections 1977, 1978 and 1979.
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honey bees collected in autumn, 1979. Site E 2 was utilized in 1979
in lieu of SE 2. It falls within the forage area (flight range) and
has the same water supply as SE 2.
216
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NE-4 NE-2 S-5 SW-I SW-3 SE-12
NE-3 SE-2 S-4 SW-2 SE-6
NE-4 NE-2 SE-I S-4 SW-I SW-3 SE-12
NE-3 SE-2 S-5 S-l SW-2 SE-6
Figure 20-3. Mean fluoride content and 95 percent confidence intervals of worker honey bees collected in
autumn, 1975, 1976, 1977 and 1978. Due to occurrences such as grass fires, lack of forage,
and dry ponds, not all sites were utilized over all years.
-------
TABLE 20.2. FLUORIDE CONTENT OF ADULT WORKER HONEY BEES, 1979
Site
N 1
NE 1
NE 2
NE 3
NE 4*
NE 3/4
E 2t
SE 1
S 4
S 5
S 1
SW 1
SW 2
SW 3
SE 6
SE 12
GB 3f
NE 10
Date
Sept
June
Sept
June
Sept
June
June
Sept
Sept
June
Sept
June
Sept
June
Sept
June
Sept
June
Sept
June
Sept
June
Sept
June
Sept
June
Sept
June
Sept
Sept
June
Sept
1
10.5
23.0
15.7
18.6
14.2
175.2
32.4
21.3
16.1
22.0
10.8
20.5
16.9
14.8
6.8
14.2
9.6
43.7
11.1
14.1
14.0
10.1
11.0
14.1
12.3
7.1
7.5
11.1
13.0
10.8
97.8
36.3
2
11.0
23.2
15.3
12.7
14.3
171.8
28.8
15.3
16.9
18.1
16.7
24.1
18.7
20.0
10.2
17.3
13.0
63.5
8.0
14.5
14.3
16.2
12.9
14.5
7.9
5.4
7.1
10.8
6.6
8.3
101.3
25.3
3
10.
20.
15.
16.
13.
135.
34.
20.
17.
16.
11.
18.
16.
16.
7.
15.
5.
53.
11.
11.
13.
17.
12.
11.
10.
6.
8.
7.
7.
11.
116.
14.
7
6
6
2
0
5
6
2
2
1
6
6
3
1
7
7
6
8
2
8
3
7
1
8
5
5
3
7
6
8
4
4
4
5.7
21.2
15.2
17.6
16.1
115.9
22.6
17.1
15.9
22.2
11.3
13.2
18.4
15.0
11.0
19.2
11.0
70.0
10.7
13.5
15.0
12.0
13.9
13.5
12.1
8.5
8.5
9.1
7.8
11.7
104.5
14.9
Sample
5
6.1
30.6
19.3
19.9
14.5
137.6
33.4
19.7
15.9
23.0
12.9
16.1
16.6
13.2
6.7
20.2
7.6
39.5
6.8
13.0
13.3
12.8
12.9
13.0
12.3
7.0
7.7
7.3
9.0
12.9
100.2
7.9
Hives (ppm)
6
7.7
28.3
15.3
15.7
16.1
190.9
32.8
14.1
17.4
24.0
13.4
15.0
13.9
20.6
8.0
15.9
7.9
31.7
7.5
16.1
16.5
7.9
10.3
16.1
12.1
7.2
6.9
11.9
9.8
13.2
94.6
33.2
10
15
15
14
19
182
32
17
16
19
13
25
13
15
4
15
-
49
7
12
13
13
11
12
9
6
8
9
15
11
101
32
7
.5
.4
.2
.2
.7
.2
.0
.7
.9
.5
.7
.0
.8
.4
.9
.9
.0
.6
.3
.8
.2
.1
.3
.7
.5
.4
.8
.4
.5
.0
.7
8
5.4
18.7
18.4
16.8
12.4
109.4
-
16.1
18.7
16.9
13.5
15.5
16.8
18.6
7.8
15.3
13.4
78.1
12.7
14.0
12.4
14.5
8.0
14.0
13.2
5.2
5.3
13.1
10.9
13.4
89.1
14.8
9
6.
28.
17.
18.
13.
150.
-
18.
16.
17.
13.
25.
12.
18.
12.
12.
7.
41.
7.
15.
16.
12.
12.
15.
9.
7.
6.
9.
11.
14.
107.
16.
4
4
9
9
4
5
3
5
0
8
7
6
1
8
4
2
5
7
2
4
1
9
2
0
2
6
3
9
8
0
4
10
6.4
25.3
15.2
16.8
11.8
169.2
-
17.8
-
20.5
16.8
19.1
15.2
20.2
13.4
20.8
9.5
61.5
7.3
13.1
17.7
11.9
19.3
13.1
30.6
8.4
8.8
12.3
6.7
13.8
98.7
15.0
Combined
Sample
8.1
23.7
17.7
15.0
16.5
133.2
34.9
-
18.9
19.1
13.1
18.6
16.4
16.1
13.0
14.9
10.8
59.5
10.9
_
18.0
12.2
15.0
15.0
11.7
6.3
10.3
7.0
14.0
14.3
85.4
32.1
X
8.0
23.5
16.3
16.7
14.6
153.8
30.9
-
17.3
19.9
13.4
19.3
15.9
17.2
8.9
16.7
9.4
53.2
9.1
13.8
14.7
12.8
12.4
13.7
13.0
6.9
7.5
10.2
10.0
12.2
85.4
32.1
S.D.
2.3
4.8
1.6
2.2
2.3
28.3
4.1
-
1.8
2.8
2.0
4.4
2.0
2.6
2.8
2.7
2.7
14.8
2.1
1.3
1.7
2.8
2.9
1.3
6.4
1.1
1.1
1.9
3.1
1.8
7.3
9.9
S.E.
0.74
1.50
0.50
0.69
0.73
8.96
1.29
-
0.57
0.88
0.64
1.41
0.64
0.76
0.88
0.85
0.84
4.68
0.66
0.41
0.59
0.89
0.93
0.41
2.02
0.35
0.34
0.30
0.96
0.58
2.31
3.13
95% Confidence
Interval
6
20
J5
15
12
133
27
16
17
11
16
14
15
6
14
7
42
7
12
13
10
10
12
8
6
6
8
7
10
80
39
.4 -
.1 -
.2 -
.2 -
.9 -
.6 -
2 _
.5 -
9 —
.9 -
. 1 -
.5 -
.3 -
.9 -
.8 -
.5 -
.6 -
.5 -
.8 -
.4 -
.8 -
.3 -
.8 -
.4 -
.2 -
.7 -
.9 -
.8 -
.9 -
.2 -
.2 -
9.7
26.9
17.4
18.3
16.0
174.1
34.7
18.2
21.9
14.9
22.5
17.4
19.1
10.9
18.6
11.3
63.8
10.6
14.7
15.8
14.8
14.6
14.7
17.6
7.7
8.3
11.6
12.2
13.5
90.6
19.80
* Only seven colonies in beeyard.
t Located north of SE 2, within same forage area.
Located at Billings, Montana.
-------
Two-factor ANOVA for 1979 versus 1976 levels of fluoride in bees from August/
September revealed highly significant differences in variances among sites but
not years:
Fs = i'11 < F.05[l,23j " 4'28 (^ears)' Fs = U'14 > F.001[20,144] = 2'51 (sites)
Comparing absolute mean fluoride content of bees (95 percent confidence intervals)
for autumn of 1975 through 1979, levels in 1979 and 1976 were generally higher
than in 1975, 1977, and 1978.
In both 1979 and 1976, fluoride levels were lowest at sites directly south
of Colstrip and at the "checks" distant from Colstrip. The 1979 mean fluoride
content of bees samples in September exceeded 1975 baselines and equalled or
exceeded levels observed in 1977 and 1978 at sites northeast, east, southeast,
and southwest of Colstrip (Figure 20.3). The 1979 fluoride levels in bees
collected in September were lower than those of June/July at many Colstrip
sites, although fluoride levels at apiaries southwest of Colstrip and at the
check sites were essentially the same for both the early and late summer periods.
Fluoride Values for Pooled Samples Versus Mean of Separate Samples
Values for fluoride in pooled samples versus the mean of independent
samples are presented in Figures 20.4 and 20.5. Figure 20.4 summarizes data
from all sites from 1974 through 1979. The correlation coefficient (r = 0.99)
indicates a highly significant relationship, P <_ 0.001. The coefficient of
determination (r2 = 0.98) indicates that only slightly over 2 percent of variation
in fluoride indicated by the mean of independent observations is due to vari-
ation not associated with "fluoride content" as displayed by the pooled samples.
Figure 20.5 shows cases in which fluoride content was less than 20 ppm.
Here r2 = 0.80 signifies that 80 percent of fluoride variation in the mean of
independent observations is associated with fluoride, as indicated by pooled
samples. Again, the association is significant; less than 3 percent of obser-
vations would be expected to fall outside the 30 confidence intervals.
Figure 20-5 includes mean values based on as few as four independent colonies.
I recomputed the correlation coefficient and coefficient of determination for
only those values based on eight to 10 observations and obtained values of
T = 0.94, r2 = 0.883.
Arsenic
The results pf 63 arsenic determinations demonstrated levels equivalent
to those of baseline at all sites during 1979. The highest recorded value was
0.51 ppm at E 2 in September; the lowest value was 0.13 ppm at SE 12 in
September. The data indicated somewhat lower values in bee samples ground in
a Wiley Mill® before analysis, compared with the levels in whole bees. It was
concluded by the chemist performing the analyses that the ground tissues
remained damper than the whole bees after oven-drying.
219
-------
ho
O
(f)
LU
_l
Q.
\-
LU
140-
120-1
100-
UJ
Q_
UJ
o
so
60
40
UJ
| 2°
0
Q_
O.
0 20 40 60 80 100 120
PPM FLUORIDE (POOLED SAMPLES)
CO
LU
_l
Q.
CO
LU
Q
-z.
LU
Q.
LU
Q
O
I
LU
Q
tr
o
CL
Q_
20-
18-
16-
14-
12-
10-
8-
6-
4-
2-
0
r2 = o.so
n = 9i
0 2 4 6 8 10 12 14 16 18 20
PPM FLUORIDE (POOLED SAMPLES)
Figure 20 .4.
Comparison of fluoride content of
worker honey bees as determined by
pooled samples and by the mean of
independent observations over a 5
year period.
Figure 20 .5.
Comparison of fluoride content of
worker honey bees as determined by
pooled samples and by the mean of
independent observations over a
5 year period for values less
than 20 ppm. The circled values
were based on four observations.
-------
DISCUSSION
Fluoride and arsenic are released by coal-fired power plants. Measured
stack concentrations of Colstrip Unit 2 indicate a level of 2,130 ± 400 (SD)
ppm for fluoride and 221 ± 20 (SD) ppm arsenic (Crecelius et at,, 1978).
Munshower (personal communication, 1978) has detected increased postoperational
arsenic levels in pine needle sheaths from trees near Colstrip. His analysis
of 1977 bee samples showed arsenic levels in bees from NE 2 were 2 to 3
times higher than baseline and check site levels. Other investigations have
shown substantially higher fluoride levels in mice and pine foliage from sites
near Colstrip compared to more distant sites (Gordon et al. , 1978, 1979). My
own studies have repeatedly demonstrated significant postoperational fluoride
changes in bees from apiaries downwind and as far as 20 km from Colstrip
(Bromenshenk, 1976, 1978, 1979, 1980).
Fluoride in honey bees was used in this study as a tracer in an attempt
to determine the distribution patterns of the power plant plumes. The 1979
fluoride data in bees is consistent with previously reported wind patterns
(Van Valin et al. , 1980; Ludwick et al. , 1980; Bromenshenk, 1979).
Although summer winds are usually westerly during the daytime, night
surface winds are variable and typically light. Furthermore, the plumes from
the power plants are affected by the underlying terrain topography being
diverted from the direction of the prevailing winds by as much as 20°. Plumes
follow valleys and are deflected around higher terrain (Van Valin et al., 1980).
It appears that plumes may be trapped in valleys and then flow southward with
northerly winds mixing at times to ground levels (Ludwick et al., 1980).
Plumes have been tracked as far as 50 km from Colstrip. Therefore, although
several years of wind tower data gathered by the Montana State Department of
Health suggest that prevailing winds in order of importance are E-SE, W-NW,
and E-NE, this may be only a crude approximation of plume dispersion patterns.
The data obtained from the sulfation and formate plates which were set out in
1978 and 1979 at each apiary should prove useful in determining whether fluoride
is reaching the apiaries via the air. Unfortunately, the analytical results
were not returned in time for incorporation into this report.
As in previous years, the highest fluoride levels in bees in autumn of
1979 were at apiaries located in a "downwind" or easterly direction from '
Colstrip (NE, SE, E). Apiaries directly south and north of Colstrip ancj the
more distant checks exhibited levels essentially identical to baselines. Also,
as in other years, sites located just west of directly south displayed inter-
mediate levels of fluoride somewhat higher than baselines. The results from
autumn of 1979, both as regards levels and the geographic distribution of
fluoride in bees, were similar to those of 1976.
At all locations including the checks, the June/July fluoride content of
bees was as high or higher than those observed in 1977 and 1978. A prolific
flowering of yellow sweet clover in June provided plentiful supplies of nectar
and undoubtedly stimulated foraging activity. This may have contributed to
the generally higher fluoride levels because of more flight activity, greater
probability of contact, more materials brought back to the hives, etc. However,
221
-------
mean fluoride content of bees obtained at each of two "check" apiaries in 1979
(approximately 40 and 80 km from Colstrip) was lower than that of the bees
from the Colstrip locations.
Disregarding NE 10, the highest fluoride concentrations for 1979 occurred
at NE 3, NE 4, and S 1 during June and July. Only once before were levels
greater than 30 ppm observed—at sites N 1 and S 1 in June/July, 1977. Dumping
mine waste waters into Arnell's Creek (the apiary's water supply) may have
caused high levels of fluoride in bees at N 1 (Bromenshenk, 1980). No source
of the fluoride at the other sites is known. It was not in any of the water
supplies sampled. These fluoride levels are cause for concern. Levels
observed at NE 3 could poison bees based on literature reports and my own
observations (Bromenshenk, 1980).
Long-term studies are needed to determine how serious these fluoride
levels in bee systems may be and how the bees are taking up the fluoride. The
weak correlation of fluoride levels in bees and pollen collected in June/July
suggests the airborne fluoride may be reaching the bees via the food. However,
one would expect the highest levels to occur in nurse bees or pupae if bioaccu-
mulation is via pollen and in foragers if from nectar. My previous studies
(Bromenshenk, 1978, 1979, 1980) showed little if any fluoride accumulation in
pupae and hive bees (mainly nurse bees) and very low levels of fluoride in
floral parts, which provides indirect measure of fluoride in nectar. It is
possible that the high fluoride concentrations observed in field bees came
about as a result of exposure to airborne contaminants either via penetration
of the cuticle, which seems unlikely, or via the tracheal system. This is an
attractive hypothesis since food correlations (pollen) are so low, r2 < 14 percent
However, there are possible explanations for such a weak correlation
between levels in pollen and bees. P. Tourangeau, who carried out all of the
fluoride analyses, suggested that the levels of fluoride in pollen are near
the lower limits of detectability of the Orion probe, using our present
methods, and the sensitivity may not be good enough in this range to reliably
separate the signal from the noise. We currently are investigating this
possible source of error.
There is always the possibility that the fluoride seen in early summer
got into the bee systems before the colonies were set out in the Colstrip area.
However, it is likely that the fluoride accumulation was actually caused by
exposures in Montana. Any fluoride carried back in colonies exposed at
apiaries in California during the winter should be diluted and "cleaned out"
by June/July for the following reasons:
1. Preoperational studies did not detect any fluoride carried back
from California.
2. Except for the brood boxes, none of the equipment is taken to
California. Brood cells are lined by bees with a "papery" material
which effectively isolates the brood from the wax.
3. Bees are returned to Montana with marginal food stores which are
rapidly consumed.
222
-------
4. The bees had been at the Colstrip locations since late April and
early May which is sufficient time for replenishment of stores and
for several population turnovers.
5. All of the sampled colonies had a considerable amount of surplus
honey in the "honey supers" at the time of collection, indicating
they had been at the beeyards long enough to build up food reserves.
6. Marked hives taken to California and located upon their return
indicated that the boxes become well mixed and more or less
randomized while being stockpiled, inspected for disease, split
to form new colonies, and transported via truck.
Also, one would not expect to see all colonies within a given Colstrip
apiary displaying- similar levels of increased fluoride, &-<3- , NE 3 samples
were all greater than 100 ppm, those of S 1 were all greater than 31 ppm but
less than 70 ppmseta. Fluoride brought back from California should show up
as a more random pattern—some colonies at a given location displaying high
levels, others intermediate and some very low—since the colonies at any
Colstrip beeyard probably came from several California beeyards.
As in all previous years, the fluoride in bees at NE 10 appeared to be
associated with the fluoride in water in a nearby stock tank. These colonies
were moved farther from the tank and closer to Rosebud Creek in 1979 in an
attempt to change their water supply. Fluoride levels in these bees was still
high in June/July but for the most part were below 100 ppm. They declined to
less than 36 ppm by September. Although high, these levels, especially in
September, are lower than those observed in individual colonies at this site
during previous years and are below the 120-130 ppm levels, which seem to
"definitely" indicate acute poisoning. The colonies at this apiary have always
been characterized by a lack of vigor, poor brood laying, and low honey
production. In 1979, the beekeeper reported that this was one of his best
apiaries, producing more than twice as much honey per colony as in previous
years. This suggests at least a partial solution to this specific problem and
tends to confirm the assumption that fluoride levels exceeding 100 ppm were
affecting these colonies. However, the data from 1979 indicate that merely
moving the bees farther from a "contaminated" water supply and closer to a
"clean" water supply does not guarantee that the bees will utilize the
preferred one.
The fluoride levels in water at sites other than NE 10 were almost
identical to previous levels, and water does not appear to be the source of
the high fluoride levels in the bees during either of the 1979 sample periods.
A stock watering tank about 0.3 km from E 2 contained more fluoride than
Rosebud Creek, which was very close to the apiary. The fluoride concentration
in bees at E 2 was higher in June/July than in September, but that of the water
was considerably higher in September. Thus, it is improbable that the stock
tank in this case was a major contributor of fluoride.
It is possible that fluroide in water may be a contributing factor. Many
ranchers in the area have complained of wells going bad since the mining
activities began. Also, mine waste waters apparently have sometimes been
223
-------
discharged into streams. However, most apiaries are located near Rosebud Creek
and relatively distant from other water supplies; fluoride levels in the
Rosebud have remained almost constant since 1974.
It is becoming apparent that fluoride either via air and/or water is
reaching apiaries near Colstrip and at levels which may pose potential hazards
to bees and beekeeping. The fluoride data from 1979 raises many questions and
only suggests possible answers.
Although arsenic appeared at relatively high levels at an apiary northeast
of Colstrip in 1977, this was not observed in 1979. If arsenic were to sporad-
ically impact the apiaries, longer term monitoring would be needed to adequately
address potential for buildup of this toxic material. One might expect to see
correlations between levels of arsenic and fluoride in bees if these materials
are being inserted into their environs by the combustion of coal or by coal-
mining activities. The one instance of "elevated" arsenic in 1977 was at a
site that over the years has tended to display higher fluoride levels.
According to Crecelius et at. (1978), 10 times more fluoride than arsenic is
emitted by the power plants. Thus, it is not surprising that arsenic levels
in bees are low in comparison to fluoride. In addition, the two contaminants
are dissimilar in physical and chemical aspects which may affect factors such
as dispersion and transport in the plume, uptake routes into bee systems,
chemical forms encountered, and biochemical/physical chemical interactions.
CONCLUSIONS
Honey bees collected in 1979 at apiaries within 20 km of Colstrip,
Montana, failed to show any arsenic levels above baselines but continued to
show significant postoperational fluoride changes compared with preoperational
levels. Unusually high mean fluoride levels in bees, ranging from 2 to 17
times baselines, were found at several beeyards sampled in early summer. These
levels in bees did not correlate with levels in water supplies. A significant
correlation (P _< 0.05) was obtained for fluoride in pollen and bees, although
the correlation coefficient was weak (r = 0.37).
Bees and pollen from a site northeast of Colstrip in June of 1979 had the
highest mean fluoride content (154 and 4.3 ppm, respectively) ever recorded
from southeastern Montana. According to literature reports and my own obser-
vations, this level indicates poisoning. Levels greater than 30 ppm were
observed at sites northeast and south of Colstrip during June/July, 1979.
Very high fluoride values were observed in June/July of 1977 at two sites.
One of these was S 1, which in 1979 had 1.5 times the "high level" of 1977 or
53 ppm versus 35 ppm; baselines averaged 5 ppm for this site.
Fluoride concentration in the autumn, 1979, collections did not demon-
strate the unusually high levels of the earlier sample period, although mean
fluoride was approximately double that of baselines at sites northeast and
southeast of Colstrip and somewhat higher than baselines at sites southwest of
Colstrip. The patterns of fluoride concentration and distribution were very
similar to significant postoperational increases of fluorides in these bee
systems in 1976. Fluoride concentrations in bees from the "check" sites
approximated baselines, and levels in September were the same as those in July.
224
-------
APPENDIX 20.1. PPM FLUORIDE IN APIARY WATER SUPPLIES, 1979
Dates
June/
July
Sept.
NE 10 NE 4 NE 3 NE 2 NE 1 N 1* E 2t E 2
0.6 0.6 0.6 0.6 0.4 - 2.6 0.6
0.5 0.4 0.6 0.6 - 0.4 6.3 0.6
SE 1 S 5 S 4 S l:j: SW 1 SW 2 SE 3 NE lOt
June/
July 0.6 0.6 0.6 0.1 0.4 0.4 0,6 8.3
Sept. 0.6 0.5 0.6 0.1 0.4 0.6 0,6 10.4
Rosebud
Sites
X
S.D. =
S.E. =
N
Creek
(1977)
0.51
0.03
0.01
10
Rosebud
Sites
X
S.D. =
S.E. =
N
Creek
(1978)
0.55
0.10
0.03
10
Rosebud
Sites
X
S
S
N
Creek
(June/July 1979)
=
.D. =
,E. =
^
0.55
0.09
0.03
11
Rosebud Creek
Sites
X
S.
S.
N
(Sept
_
D. =
E. =
=
. 1979)
0.54
0.08
0.03
10
* Arnell's Creek (a dry creek most of the summer).
t Livestock Watering Tank.
t Reservoir.
225
-------
Regression analyses of the results of fluoride determinations for samples
"pooled" at the time of collection versus the mean fluoride content of four to
10 "individual colonies" revealed significant, P ^ 0.001, correlations.
Pooled samples appear to be adequate and reliable for use in a rapid screening
procedure to locate "hot spots" of pollutant contamination. Observations of
values for individual hives increases information content but may not be
necessary for an initial monitoring effort.
The 1979 data raises critical questions, while only suggesting answers.
It is apparent that fluoride is impacting apiaries near Colstrip at levels
which may harm bees and beekeeping. Whether the fluoride is coming from air
or water or both media is unclear. Patterns of fluoride in pollen and the
geographical distribution of fluoride buildup with respect to Colstrip and the
prevailing winds suggest airborne fluoride.
REFERENCES
Bromenshenk, J. J. 1976. Investigations of the Effects of Coal-Fired Power
Plant Emissions Upon Insects, Report of Progress. In: The Bioenviron-
mental Impact of a Coal-Fired Power Plant, Second Interim Report,
Colstrip, Montana. R. A. Lewis, N. R. Glass, and A. S. Lefohn, eds.
EPA-600/3-76-013, U.S. Environmental Protection Agency, Corvallis,
Oregon. pp. 112-129 and 286-312.
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Plant Emissions Upon Insects. I. Entomological Studies in the Vicinity
of Colstrip, Montana. II. Entomological Studies at the Zonal Air
Pollution System. In: The Bioenvironmental Impact of a Coal-Fired
Power Plant, Third Interim Report, Colstrip, Montana. E. M. Preston and
R. A. Lewis, eds. EPA-600/3-78-021, U.S. Environmental Protection Agency,
Corvallis, Oregon. pp. 146-312 and 473-507-
Bromenshenk, J. J. 1979. Honeybees and Other Insects as Indicators of
Pollution Impact from the Colstrip Power Plants. In: The Bioenviron-
mental Impact of a Coal-Fired Power Plant, Fourth Interim Report,
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U.S. Environmental Protection Agency, Corvallis, Oregon. pp. 215-239.
Bromenshenk, J. J. 1980. Accumulation and Transfer of Fluoride and Other
Trace Elements in Honeybees Near the Colstrip Power Plants. In: The
Bioenvironmental Impact of a Coal-Fired Power Plant, Fifth Interim
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Ames, Iowa. pp. 9-33.
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Ponderosa Pine Near Colstrip. In: The Bioenvironmental Impact of a
Coal-Fired Power Plant, Fourth Interim Report, Colstrip, Montana. E. M.
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Protection Agency, Corvallis, Oregon, pp. 141-214.
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Dioxide. Ph.D. Thesis, The Pennsylvania State University, University
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Animals—A Literature Review. U.S.D.A. Agriculture Handbook No. 380.
109 pp.
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Quality Measurements in the Coal-Fired Power Plant Environment of Colstrip,
Montana. In: The Bioenvironmental Impact of a Coal-Fired Power Plant,
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of Statistics in Biological Research. W. H. Freeman and Company,
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Plume and Aerosol Properties Near Colstrip. In: The Bioenvironmental
Impact of a Coal-Fired Power Plant, Fifth Interim Report, Colstrip,
Montana. E. M. Preston and D. W. O'Guinn, eds. EPA-600/3-80-052, U.S.
Environmental Protection Agency, Corvallis, Oregon, pp. 20--48.
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SECTION 21
BASELINE HISTOLOGY OF SELECTED ORGANS OF THE DEER MOUSE,
PEROMYSCUS MANICULATUS, IN ROSEBUD COUNTY, MONTANA
M. D. Kern and R. A. Lewis
ABSTRACT
The normal histology of selected organs of the
deer mouse (Peromysous manieulatus) is presented. We
believe that these organs are especially sensitive to
long-term insults by low levels of pollutants such as
those produced by coal-fired power plants. We also
suggest that abnormalities in their structure due to
such stress will be readily perceived by gross and
histological examination. The organs include the male
accessory reproductive glands, ovary, uterus, vagina,
adrenal gland, spleen, liver, and kidney. Descrip-
tions of the testis, epididymis, and heart of the deer
mouse appear in an earlier report (Lewis et al..,
1978).
INTRODUCTION
This portion of our investigation of the deer mouse (Peromyscus man-
i-Qulatus ) provides quantitative descriptive information concerning the
normal histology (central tendency and variation) of selected organs.
Anatomical and histological abnormalities may provide useful indicators of
long-term pollution impacts. The organs studied include the male accessory
reproductive glands, ovary, uterus, vagina, adrenal gland, spleen, liver, and
kidney. A basic understanding of these structures will help us to assess or
predict trends and impacts of pollutants from coal-fired power generation
(Lewis et al . , 1978; Lewis and Lewis, 1979). These data are, in any event,
essential to the interpretation of impacts that may occur in the future.
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MATERIALS AND METHODS
Mice were trapped in southeastern Montana and transported alive to our
field laboratory (Lewis et> al. , 1978) where they were sacrificed and dissected
in random rotation. Organs were immediately placed in 10 percent buffered
neutral formalin or Bouinrs solution. They were later weighed and examined in
the laboratory, and then dehydrated, embedded in paraffin, and sectioned for
histological study. Representative sections were stained with haematoxylin
and eosin and evaluated.
Accessory Sexual Glands
The growth and maintenance of the accessory sexual glands depend directly
on androgen production by the testis and in some cases characterize a male's
reproductive condition better than the testis itself (Ewel, 1972). Accord-
ingly, they are useful indicators of general reproductive condition, and
specific androgen production.
We examined the vesicular gland, coagulating gland, and ventral prostrate
glands for general structure, size, and seasonal changes. In addition, we
used an ocular micrometer to determine the average diameter of each gland in
section. We also measured the maximum diameter of this fusiform-shaped vesi-
cular gland. Average tubule diameter is based on 10 independent measurements1,
of the width of each vesicular gland. Measurements of 10 separate tubules
(or acini) were made in the case of the ventral prostrate and coagulating
glands.
Examination of the ovary is probably the most reliable method of deter-
mining the reproductive condition and maturity of female deer mice. For
example, mature females have ovaries that contain corpora lutea (endocrine
glands that develop from ovulated follicles), but immature animals do not.
The number of corpora lutea in the ovary is also a measure of fecundity
(number of eggs ovulated) and may be used to determine the egg production of
mice (Coutts and Rowlands, 1969). The number of sets of corpora lutea, number
of degenerate (atretic) follicles, abundance and appearance of interstitial
tissue (which produces steroidal hormones), and the number and size of ovarian
follicles within the ovary provide information about previous and current
reproductive activity. Since follicular development is regulated by gonado-
trophins produced by the anterior pituitary gland, ovarian histology can also
be used to assess pituitary function (e.g., Clarke and Kennedy, 1967).
Fixed ovaries (prior to imbedding) were examined with a dissecting micro-
scope for grossly visible follicles, corpora hemorrhagica (follicles that have
just ovulated), and corpora lutea. The following criteria were used to dis-
tinguish among the three: 1. Corpora hemorrhagica.--Small, punctate blood
spots on the surface of the ovary; 2. Corpora lutea.--Round protrusions of
variable size (classified as small, medium, or large) on the surface of the
ovary; always solid and curdlike in appearance; 3. Mature (Graafian)
follicles.--Small (always!) round protrusions on the surface of the ovary;
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hollow, and in this way different from small corpora lutea with which they
might otherwise be confused—a liquid-filled center (antrum) is visible
through the wall of the follicle.
Serial sections of at least half of each ovary were examined for the
number and size of (1) follicles at various stages of development, (2) corpora
hemorrhagica, (3) corpora lutea, (4) atresias, and (5) scara (corpora albican-
tial). We also noted the amount and functional state (based on histological
considerations) of interstitial tissue.
Uterus and Vagina
Uterus and vagina are two of the major regions of the reproductive duct
of female deer mice. The vagina is especially useful for assessing the repro-
ductive condition of an animal because there are obvious changes in its struc-
ture at various stages of the cycle, during pregnancy, and during sexual
development. In contrast, changes in the structure of the uterine horn are
subtle and difficult to interpret. It is nevertheless very useful for identi-
fying newly pregnant females or those whose reproductive tracts contain
embryos (blastocysts) that have not yet implanted.
We examined the uterine horn and vagina for changes associated with the
principal reproductive states of female deer mice — reproductive inactivity,
stages of the cycle, pregnancy, parturition and the immediately postpartum
state, and lactation. We concentrated on epithelium, glands in the uterine
horn, contents and size of the lumen, and characteristics of the connective
tissue (lamina propria) and muscle (tunica muscularis). In the uterine horn,
we quantified the height, mitotic activity, and number of inflammatory cells
within the endometrial epithelium; the number of uterine glands, their dia-
meter, and contents; the vascularity and width of the lamina propria and the
degree to which it was infiltrated with inflammatory cells; and the width of
the tunica muscularis. These characteristics were measured with an ocular
micrometer or evaluated on a scale of 0 (none present) to 5 (very many or very
high concentrations present). Averages are based on at least 10 measurements
of each structure.)
Adrenal Gland
The adrenal gland of the deer mouse, like that of mammals generally,
consists of two distinct and functionally independent glands: an inner
medulla which produces catecholamines (e.g., epinephrine), and an outer cortex
which produces steroidal hormones (e.g., corticosterone). Both parts respond
to internal and external stressors. Epinephrine, for example, prepares the
body to deal with immediate emergencies, increasing respiratory and cardio-
vascular activity, elevating blood sugar, and-redirecting blood flow. As a
biomonitor, the adrenal gland has the advantage of high sensitivity to exter-
nal stressors. There is the further advantage that this gland responds non-
specifically to stress and therefore integrates all sources. The condition of
the adrenal gland thus provides a basis not only for evaluating the degree of
stress chronically recently experienced, but also provides information regard-
ing tolerance or potential resilience to additional insults.
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Its involvement with reproduction and certain other activities (see
below) may complicate structural interpretation. However, knowledge of its
normal structure in field populations of deer mice is essential to any evalua-
tion of changes that may occur at sites of coal combustion.
To assess adrenal activity, we measured the cross-sectional areas of its
major regions and the lipid content of the cortex. We also noted degenerative
and pathological changes.
To determine cross-sectional areas of regions of the gland, we treated
them as a series of concentrically arranged ellipsoids in cross-section. Four
such ellipsoids exist in the normal gland. The innermost is the medulla.
This is surrounded by the three major regions of the adrenal cortex: the zona
reticularis (ZR) , zona fasciculata (ZF) , and zona glomerulosa (ZG) , in that
order. Accordingly, the other three ellipsoids are combinations of (1)
medulla and surrounding ZF, (2) medulla, ZR, and surrounding ZF, and (3)
medulla, ZR, ZF, and surrounding ZG. Because the area of ellipsoid is TT ab/4,
where a and b are its major and minor diameters, respectively, cross-sectional
areas of each ellipsoid were readily obtained from measurements made with an
ocular micrometer. However, it was necessary to combine the ZR and ZF for
analysis because the boundary between them was frequently indistinct.
Mice that are either immature or have never been pregnant sometimes have
additional cortical zones with unknown functions (Howard, 1927; Jones, 1957;
Delost and Delost, 1954; Quay, 1960; Christian and Davis, 1964; Tahka, 1979).
Having only two immature animals, we did not include this zone in our analy-
sis. It is thus possible that some areas classified as ZR in females whose
reproductive condition was not accurately known are in error in this regard.
We also assumed that the sections of adrenal gland examined were from the
center of each gland. Actual variation in the total cross-sectional areas of
all glands examined supports this assumption.
The lipid content and the presence of degenerative areas in the adrenal
cortex have been frequently used as indicators of adrenal activity (e.g.,
Andersen and Kennedy, 1932; Allen, 1960; Dawson et al. , 1961; Christian and
Davis, 1964). When the gland is functioning at "normal" levels of activity,
there is a large amount of cortical lipid in the form of large droplets; at
higher levels of activity (e.g., under conditions of moderate stess), the
amount of lipid diminishes and only small droplets may be seen. When the
gland is overtaxed, cortical lipid may be absent, and degenerative areas are
seen.
Although the tissues were not prepared by procedures designed specifi-
cally to preserve lipids, we attempted to roughly quantify the lipid in the
cortex and the form in which it occurred. Observed areas of degeneration do
not appear to be fixation artifacts.
Data on male and female deer mice were treated separately because estro-
gens promote cortical growth in small mammals, whereas androgens retard corti-
cal development. Hence, the adrenals of female mice tend to be larger than
those of males. We also grouped females according to reproductive state
(immature, reproductively inactive, cycling, pregnant, or lactating) because
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the cortex exhibits periodic fluctuations in size and activity associated with
reproductive condition. The cortex is large and highly active during estrus
and lactation, but smaller and less active during diestrus and pregnancy
(Andersen and Kennedy; 1932, 1933).
Social and population characteristics also influence the adrenal struc-
ture of rodents. Dominant individuals have smaller glands than subordinates;
mice in small populations have smaller adrenals than those in larger popula-
tions (Christian, 1955; Andrews, 1970). These sources of variation are prob-
ably small in deer mice because of their relatively nonaggressive nature
(Christian and Davis , 1964).
The mammalian spleen has many functions, including production of red and
white blood cells and platelets; destruction of old or damaged red cells;
storage of blood; production of antibodies; and the removal of foreign bodies
from the body fluids that circulate through it. It can thus be expected to
detoxify or in some cases accumulate pollutants from coal combusion that occur
systemically.
Our measurements in the spleen were designed to estimate its role in the
production and/or storage of blood cells and platelets, destruction of erthro-
cytes, immune responses, and the removal of foreign materials from the body
fluids. We suggest that this can be ascertained by measuring changes in
absolute and relative numbers of the various cell types within the spleen.
Accordingly, we identified 40 cells in the red pulp of each spleen by use of a
reticule with 0.1-mm divisions. More specifically, we identified the cell at
each 0.1-mm mark along the reticule in four areas of red pulp selected
randomly.
We also determined: (1) The relative abundance of red and white blood
cells after scanning the entire section at low magnification. Each spleen was
assigned to one of five categories — red cells far less, less, equally, more,
or far more numerous than white cells; (2) The amount of hemosiderin present,
determined by scanning the section and then rating the concentration of pig-
ment on a scale of 0 (none) to 5 (extremely high); (3) The number of germinal
centers in the white pulp. We counted the number present and noted the phago-
cytic and mitotic activity within each; (4) The number of megakaryocytes and
hemocytoblasts in section. In many cases we counted the number of such cells
in the section. Since this number depends somewhat upon the area of the
section, we also estimated the abundance of each on a scale of 0 (none) to 5
(very numerous).
Our rationale for using the above as measures of splenic activity are:
1. The relative and absolute abundance of megakaryocytes are estimators
of the organ's role in platelet formation since megakaryocytes produce plate-
lets.
2. The relative abundance of red and white blood cells and of erythro-
cytes, normoblasts, and hemocytoblasts in the red pulp are estimators of the
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organ's role in the formation and/or storage of erythrocytes. (Hemocytoblasts
and normoblasts are immature stages of erythrocytes).
3. The hemosiderin content of the spleen is a measure of the organ's
role in the destruction of erythrocytes.
4. The relative abundance of red and white blood cells and of medium-
and large-size lymphocytes, hemocytoblasts, myelocytes and metamyelocytes in
the red pulp are estimators of the organ's role in white cell production.
(Lymphocytes of these sizes, hemocytoblasts, myelocytes and metamyelocytes are
immature white cells).
5. The number, size, and mitotic activity of germinal centers in the
white pulp of the spleen reflect its role in antibody production.
6. The relative abundance of macrophages, plasma cells, medium- and
large-sized lymphocytes, and neutrophils in the red pulp indicate how much
foreign material is removed from the body fluids as they perfuse the spleen,
since the recruitment of these cells is induced by such material. (Macro-
phages and neutrophils phagocytize foreign materials; plasma cells produce
antibodies; lymphocytes produce antibodies and cytotoxic substances that
destroy foreign bodies on contact).
Liver
The vertebrate liver has many functions. For example, it produces bile;
stores and/or synthesizes lipids, glycogen and plasma proteins; and stores
vitamins and minerals. It also detoxifies or removes from the blood numerous
foreign and endogenous substances including organic pesticides, poisons,
hormones and ammonia. It is thus very likely to be directly affected by air
pollutants and to mediate many of the animal's specific responses to pollutant
stress. Knowledge of the normal structure of this organ in deer mice is prob-
ably essential to the assessment of pollutant-related changes that may develop
at sites of coal-fired power generation.
We thus examined the liver to determine its normal structure and to
identify seasonal changes, particularly in the incidence of: (1) autolysis of
hepatic tissue accompanied or not by cirrhosis; (2) glycogen depletion of
liver cells (hepatocytes); (3) fatty degeneration of hepatocytes; (4) fat
storage in hepatocytes; (5) foreign materials and pigment in Kiipffer cells; (_6)
invasion of hepatic parenchyma by foreign bodies and/or inflammatory cells.
For purposes of this study, we used the following criteria to distinguish
among glycogen depletion, autolysis and fatty degeneration: (1) Glycogen
depletion--Hepatocytes more or less empty and unstained; only the cytoplasm
surrounding the nucleus and along the margins of the cell is stained (in
contrast to normal cells in which the cytoplasm is uniformly sprinkled with
well-stained granules); (2) Autolysis--Hepatocytes as above, but also with
pycnotic nuclei; (3) Fatty degeneration--Hepatocytes with unstained cyto-
plasmic vacuoles of variable size (usually small).
Mild autolysis (with little nuclear change) could not be distinguished
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from giycogen depletion. Such livers were arbitrarily placed in the latter
category.
The above items, together with hepatic blood flow, were each rated on a
scale of 0 (none) to 5 (heavy or pronounced). The validity of these measure-
ments depends critically on rapid fixation. Tissue was thus invariably placed
in the fixative within a few minutes following sacrifice of each specimen.
The kidney (in addition to the liver and spleen) is a principal site of
detoxification in the vertebrate, and thus is likely to be directly affected
by inverse pollutants.
We examined the kidney primarily to determine its normal structure and
associated pathologies. Examples of the latter are precipitated protein or
concretions in the nephrons, dilatation of the nephrons, changes in their
epithelial lining, degeneration of renal parenchyma, and the presence of
hemosiderin, ascites fluid, inflammatory cells, or foreign bodies in the renal
tissue.
RESULTS AND DISCUSSION
Accessory Sexual Glands of Male Deer Mice
Histology
The histology of the accessory sexual glands of several murine rodents
(e.g., house mice, voles, and to a limited extent, Peromysous') has been
described (Snell, 1941; Anthony, 1953; Lecyk, 1962; Arata, 1964; Clarke and
Forsyth, 1964; Hrabe, 1970; Ewel, 1972). Our observations are generally
similar to those presented in these earlier studies. Major differences among
the glands occur in the epithelium and in the characteristics of the secre-
tion. All have a wall that consists of a mucosal epithelium that borders the
gland's lumen, an underlying connective tissue (the lamina propria), beyond
this a tunica muscularis or coat of smooth muscle, and finally an outer sero-
sal covering.
Vesicular Gland (Seminal Vesicle).--The mucosal epithelium of the active
vesicular gland is a high, crowded, simple columnar layer. The cells are
uniformly basophilic with a basal, vesicular nucleus. They contain a supra-
nuclear vacuole of approximately nuclear size. This vacuole contains one to
few prominent, large, deeply stained granules that resemble those in the
gland's lumen. Many cells can be found discharging these granules into the
lumen. Such supranuclear vacuoles do not occur in the other accessory glands.
The mucosa is thrown up into primary folds of variable length. They are
frequently very long and narrow, consisting of little more than two layers of
epithelial cells situated back to back. Secondary folds occasionally occur.
Primary and secondary folds sometimes intersect forming a reticulum of epi-
thelial-lined pockets near the margins of the gland's lumen.
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The tunica muscularis is thick and heavy in the active vesicular gland,
especially in comparison with the muscle layer of the other accessory sexual
glands. In many places, it appears to be a single, longitudinal layer.
Elsewhere, it consists of an inner circular and outer longitudinal or oblique
layer.
This is the largest accessory sexual gland. It is comprised of com-
pletely or partially separated, adjoining compartments. These are separated
from each other by inward extensions of the mucosa and underlying muscularis.
The lumen is capacious and characteristically filled with intensely eosino-
philic (bright red) secretion in preparations stained with haematoxylin and
eosin. The secretion is homogenous or granular. So much is present that it
fills each chamber and exhibits fractures.
When inactive, the gland is small with a slit-like empty lumen which is
lined by an inactive, low columnar epithelium. Epithelial cells are mostly
filled with the nucleus and exhibit vacuolar degeneration. Mucosal folds
still penetrate the lumen and compartmentalize the gland, and the reticulum
formed by intersecting primary and secondary folds may fill the entire lumen.
The lamina propria is densely cellular connective tissue, relatively wider
than that of the active gland. Two layers of highly cellular and dedifferen-
tiated smooth muscle form the tunica muscularis.
Coagulating Gland (Anterior Prostate Gland)--This compound tubular gland
nests in the lesser curvature of the vesicular gland and is somewhat smaller
than the latter. When active, its lumen is lined by a simple cuboidal to
columnar layer consisting of cells with well defined cell membranes, basal
vesicular nuclei, and much apical cytoplasm filled with fine eosinophilic
granules. The epithelial cells are brick red, in contrast to those of all
other accessory glands. Each cell is distinctly rounded on the luminal sur-
face which thus appears scalloped.
Small, widely spaced mucosal folds jut into the lumen of the active
gland. They rarely have secondary folds. They tend to be thick and round, in
distinct contrast, for example, to the delicate narrow folds in the vesicular
gland. A thin capsule of circular smooth muscle (tunica muscularis) surrounds
each tubule and frequently abuts on the epithelium. The secretion of the
active gland is granular and only moderately eosinophilic. Small, bright red
droplets are dispersed throughout.
Wh-n the gland is inactive, its lumen is tiny, empty and lined by an
epithelium comprised of (1) inactive cuboidal cells with large dense nuclei,
and (2) larger cells that protrude into the underlying lamina propria. The
latter occur in small groups, commonly adjacent to mucosal folds. They have
considerable and poorly stained cytoplasm and central dense nuclei. The
lamina propria is thick and highly cellular. The tunica muscularis consists
of two layers of highly cellular dedifferentiated smooth muscle.
Dorsal Prostate Gland.--When active, this gland is a cluster of large
acini, each surrounded by a thin capsule of smooth muscle (tunica muscularis).
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Each acinus is lined by a simple cuboidal or low columnar epithelium. Nuclei
in these cells are vesicular and basal. The apical cytoplasm is finely
strippled and eosinophilic. This gland resembles the coagulating gland, but
has fewer folds of the mucosa and smaller tubules. Some acini have no mucosal
folds. The secretion is only faintly eosinophilic and frothy, but is
sprinkled with bright red droplets.
The inactive gland is similar to the inactive ventral prostrate and
ampullary glands and will be described with them below.
Ventral Prostrate Gland.--This compound tubular gland also consists of a
cluster of tubules, each surrounded by a thin tunica muscularis. When active,
each tubule is lined with a crowded simple cuboidal to columnar epithelium,
the cells of which have basal vesicular nuclei and poorly stained, basophilic
cytoplasmic granules in haematoxylin and eosin preparations. The height of
the epithelium is inversely related to the volume of secretion in the lumen of
the tubule. Each tubule is distended with an avidly eosinophilic (bright red)
homogenous secretion similar to that of the vesicular gland. A layer of
vesicles frequently separates the secretion from the epithelium. The eosino-
philia, together with the small cluster of tubules that comprise the ventral
prostate gland, are diagnostic. Few mucosal folds occur in the tubules and
those present are extemely small and round.
Our description of the ventral prostate differs from that presented by
Snell (1941) for the house mouse but instead resembles his description of the
ampullary gland.
Ampullary Gland.--The numerous tubules of this compound gland are notably
large and polygonal in section when the gland is active. The height of the
epithelium varies considerably both among and within tubules, consisting of
low cuboidal to crowded high columnar cells similar to those described above
in the dorsal and anterior prostate glands. Nuclei are vesicular and basal.
The apical cytoplasm is uniformly eosinophilic. Many tubules lack mucosal
folds, but others have delicate folds of variable length. There is almost no
lamina propria, and the thin circular layer of smooth muscle that constitutes
the muscularis abuts on the epithelium of each tubule. Lumens, even in highly
active glands, are often empty. The secretion, when present, consists of
large masses of a highly vacuolated homogenous material, moderately stained
with eosin and confined to the center of the lumen. It resembles that illus-
trated by Snell (1941) for the ampullary gland of the house mouse, but differs
considerably from his description of the gland (ibid).
Inactive prostate and ampullary glands consist of tiny acini with minute
lumens that are lined by a simple layer of cuboidal cells containing dense
nuclei. Little and poorly stained cytoplasm is present. The epithelium is
surrounded by a thick capsule of smooth muscle (tunica muscularis) which is
generally highly cellular, circularly oriented, and dedifferentiated.
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Seasonal Changes in the Accessory Sexual Glands of Male Deer Mice
Quantitative information concerning seasonal changes in the dimensions of
the accessory reproductive glands of deer mice appears in Table 21.1.
Morton (in Lewis et at . , 1978) has shown that the annual reproductive
period of male deer mice near Colstrip extends from mid-March through mid-
September. Testicular and seminal vesicle weights are highest between March
and August, suggesting maximal performance of the male reproductive system
during this period. Kern in the same report (Lewis et al. , 1978) found active
leydig cells in the testis between December and the following August and
progressive increases in the diameter of tubules in the epididymis between
March and June, followed by a brief decline in July and recovery in August.
Seminiferous tubules in the testes were also enlarged between March and
August. Development of the epididymis and seminiferous tubules depends on
androgens produced by Leydig cells. The data suggest that the testes begin to
secrete substantial and increasing amounts of androgen in February or March,
and continue to produce enough to maintain the reproductive apparatus through
August.
These findings concerning the steroidogenetic activity of the testis are
consistent with the additional data presented here for the seminal vesicle,
coagulating gland, and ventral prostate gland of the same mice. During 1974,
the diameter of the vesicular gland was high in July and August, then dimin-
ished; diameters of the coagulating gland and ventral prostate were similarly
large between July and September, then shrank abruptly. During 1975, the
seminal vesicle was enlarged between April and August; the other glands
between March and August (compare diameters during 1975 with those shown for
October and December of 1974).
Fluctuations in the diameters of accessory glands during April, May,
July, and/or August suggest that cyclic discharges of androgen from the testes
occur during the breeding period. Changes in the head and tail of the epidi-
dymis of these mice (Lewis et al-, 1978) support this interpretation, although
no peaks in size of the epididymis occur early in the breeding period.
Our specimens tended to have larger testes and seminal vesicles in May
and June than in July and August (Lewis et al . , 1978) . This trend is not
present in the histometry of the seminal vesicle, coagulating gland, or ven-
tral prostate (Table 21.1). The trend is observed, however, in the diameter
of the seminiferous tubules of immature deer mice; diameter of tubules in the
head of the epididymis of immature mice; and in the diameter of tubules in the
tail of the epididymis of adults (see Tables 6.24-6.29, p. 248-253, of Lewis et
al . , 1978).
Our data generally support Morton's suggestion (ibid} that immature males
born early in the reproductive season breed later in the same season. The
size of acini in the ventral prostate gland of immature males in June averages
360.0 [jm (n = 2). This value is clearly within the size range of reproduc-
tively active adults (March-August interval in Table 21.1). Also, the dia-
meters of the anterior prostate gland and seminal vesicle of immature males
are within or just below adult size and sperm are frequently present in the
seminiferous tubules and the epididymis of these males (Lewis et al. , 1978).
237
-------
TABLE 21.1. SEASONAL CHANGES IN THE SIZE OF SELECTED ACCESSORY SEXUAL GLANDS OF ADULT MALE Peromyscus
maniculatus COLLECTED AT COLSTRIP DURING 1974 AND 1975
00
Month - Year
Diameters of the Vesicular Gland*
Average Diameter
(Urn)
Maximal Diameter
(Mm)
Diameter
of the Anterior
Prostate Gland
(Mm)
Values in the table are Means ± SEM (n).
Diameter
of the Ventral
Prostate Gland
(Mm)
Jul
Aug
Sep
Oct
Dec
Mar
Apr
May
Jun
Jul
Aug
1974
1974
1974
1974
1974
1975
1975
1975
1975
1975
1975
1968.0
1656.5
1268.6
1398.0
635.8
1380.0
2300.6
1893.4
2034.0
2287.9
2183.3
± 139.1
± 106.1
± 368.6
± 75.1
± 162.1
± 150.2
± 139.7
± 153.1
± 265.7
( 1)
( 5)
( 8)
( 5)
( 4)
( 2)
( 7)
(10)
(18)
(10)
( 7)
2880.0
2265.6
1587-0
1761.6
756.0
2196.0
2904.0
2575.2
2596.0
2800.8
2842.3
± 310.8
± 125.1
± 366.0
± 103.9
± 172.0
± 210.4
± 198.4
± 202.0
± 325.1
( 1)
( 5)
( 8)
( 5)
( 4)
( 2)
( 7)
(10)
(18)
(10)
( 7)
300.0
374.8
300.7
180.6
78.7
366.5
380.9
396.0
314.6
336.6
381.9
±39.1
± 31.3
±47.4
±48.3
±26.8
± 55.5
±85.3
( 2)
( 5)
( 7)
( 3)
( 2)
( 2)
( 7)
( 8)
(12)
( 9)
( 7)
323.8 ± 40.1 ( 4)
252.8 ± 37.8 ( 4)
202.3 + 27.0 ( 8)
119.8 ± 47.1 ( 4)
90.5 ( 2)
272.7 ( 3)
254.3 ± 39.3 ( 7)
356.1 + 31.4 ( 7)
287.3 ± 27.2 (10)
311.4 ± 18.5 (10)
280.1 ± 39.9 ( 7)
-------
Histology-Preliminary Introduction
Ova undergo limited growth and two meiotic cell divisions while in the
ovary. More conspicuous concurrent changes take place within the follicles in
which the ova mature. In the early developmental stages, the ova are near the
surface of the ovary and are surrounded by one to few layers of flattened
"follicle cells" which, together with the immature egg, are called primordial
follicles. As the ovum grows and divides, the surrounding follicle cells
proliferate and become many layers deep and a connective tissue capsule
(theca) forms on the outer surface of the mass. Thus, a "primary follicle" is
formed. Liquid-filled spaces now appear among the follicle cells, increase in
size, and eventually coalesce to form a single large cavity (the antrum)
within the follicle. Follicle cells now form a layer several cells in depth
around the antrum. This layer is called the zona granulosa or membrane granu-
losa and the cells are now called granulosa cells. The theca is now thicker
and consists of an inner vascular layer (theca internal) containing clusters
of hormone-producing gland cells, and an outer layer (theca external) of
connective tissue and smooth muscle. In contrast to the condition in most
other rodents that have been studied, the theca of the deer mouse is usually
thin and not well differentiated into layers. It usually consists of several
to many layers of vascular connective tissue with few gland cells and little
muscle.
We refer to follicles in which cavities have appeared and/or coalesced to
form a small antrum as "antral follicles", and those with large antra into
which the oocyte and an investing layer of granulosa cells project as "mature"
or "Graafian follicles". There is considerable ambiguity in the literature
concerning the names of follicles at various stages of development and defini-
tions are thus particularly important. Pedersen and Peters (1968) have pro-
posed a uniform system of nomenclature to resolve the problem. Wherever
possible, we have included their designations for clarity.
After ovulation, the ruptured follicles collapse and more or less fill
with blood from the ruptured thecal blood vessels and are then termed corpora
hemorrhagica. The blood is rather quickly replaced by granulosa cells which
increase in size and obliterate the original antrum, transforming the
follicles into an endocrine gland, the true corpora luteum (TCL) which pro-
duces progesterone.
True corpus lutea are generally nonfunctional, transient structures in
small rodents such as PeTomysous unless the female becomes pregnant, in which
case they remain active for some time. Additional follicles in pregnant mice
develop into accessory corpora lutea (ACL) by a process similar to that
described for TCL except that the ova simply degenerates within the follicle.
We find ACL to be uncommon in the ovaries of deer mice.
Eventually, TCL and ACL degenerate. Cells in the antrum diminish in size
and number, undergo fatty degeneration, die, and are replaced by connective
tissue. Ultimately, all that remains of them are small nonvascular clumps of
scar tissue called corpora albicantes. Additional masses of cells that
secrete progesterone occur in the ovarian stoma. They may be of thecal or
239
-------
granulosa origin (Harrison and Weir, 1977; personal observations). At any
rate, they are called interstitial tissue and are especially prevalent during
late gestation in many rodents.
Ovarian follicles may degenerate at any stage of development to form
atresias which are viable endocrine glands that produce progesterone
(Richards, 1978).
The endocrine component of the ovary is thus potentially large, consis-
ting of estrogen-producing tissue (thecal gland cells and granulosa cells),
androgen-producing tissue (thecal gland cells), and tissues that produce
progestins (the granulosa layer of viable follicles; corpora lutea; artesias;
interstitial tissue) (Bjersing, 1978; Richards, 1978).
Histology of the Deer Mouse Ovary
The following histological characteristics are diagnostic of the various
stages of follicular development in the deer mouse ovary:
Primordial follicles of deer mice consist of an ovum and one to few
enveloping layers of squamous (always!) follicle cells, average 24.67 ± 303 |Jm
(x ± SEM; n = 18) in diameter, and characteristically occur in clusters near
the ovary's surface.
Primary follicles consist of the ovum and one to several layers of sur-
rounding follicle cells. If only one layer is present, the follicle cells are
cuboidal or columnar (not squamous!). A theca of vascular connective tissue
may be present. The egg itself is frequently surrounded by a well-defined,
conspicuous zona pellucida. Mitotic figures commonly occur in the mass of
follicle cells and in the theca. Primary follicles of deer mice are readily
separated into small, intermediate, and large categories (Table 21.2).
TABLE 21.2. DIAMETER OF PRIMARY FOLLICLES OF DEER MICE
Average Diameter Range of
(|jm ± SEM) (n) Diameters
Primary follicles of small size 39.4 ± 3.6 ( 9) <50
Primary follicles of intermediate size . 82.3 ± 3.2 (27) 50 - 105
Primary follicles of large size 150.8 ± 7.9 (18) 106 - 240
The primary follicles of deer mice correspond structurally to follicles of
types 3-5 of Pedersen and Peters (1968).
240
-------
Antral follicles of deer mice are generally larger than primary
follicles. Many layers of follicle cells surround the egg. Among these are
small fluid-filled spaces or a small, frequently slit-like antrum. If an
antrum is present, the cumulus oophorus is broadly attached to the granulosa,
i.e.j it barely projects into the antrum and most definitely not by a stalk.
A conspicuous, but frequently thin theca is also present. "Early" antral
follicles contain several small fluid-filled cavities, whereas "late" antral
follicles contain no antrum. The diameters of the two types differ (see
Table 21.3).
TABLE 21.3. DIAMETER OF ANTRAL FOLLICLES
Average Diameter
(|Jm ± SEM) (n)
Range of
Diameters (|Jm)
Early antral follicles
Late antral follicles
204.2 ± 14.2 (16)
262.8 ± 8.8 (29)
112 - 304
167 - 352
The antral follicles of Peromysaus correspond structurally to follicles of
types 6 and 7 of Pedersen and Peters (1968).
Mature (Graafian) follicles of deer mice are generally so large that they
bulge from the ovary's surface. The antrum is large with a cumulus oophorus
that projects from a stalk of granulosa cells. The membrana granulosa sur-
rounding the antrum is many cells deep. The theca is broad and sometimes
divisible into internal and external layers. We have divided these mature
follicles into three size classes which presumably represent successive stages
of growth (see Table 21.4).
TABLE 21.4. FOLLICLE SIZES REPRESENTING SUCCESSIVE STAGES OF GROWTH
Average Diameter
(|jm ± SEM) (n)
Range of
Diameters (Mm)
Graffian follicles of small size 269.3 ± 5.8 (17) 204 - 296
Graafian follicles of intermediate size . 343.8 ± 5.9 (24) 310 - 391
Graafian follicles of large size 534.3 ± 20.0 ( 6) 462 - 592
241
-------
The mature follicles of deer mice correspond structurally to follicles of type
8 of Pedersen and Peters (1968).
Corpora hemorrhagica of the deer mouse are generally ruptured and
collapsed (or at least laterally compressed or irregular in shape) and filled
with erythrocytes. Granulosa cells more or less crowd the erythrocytes into
the center of the antrum and obliterate it.
Corpora lutea of deer mice form when the original antrum is filled with
large cells, presumably of granulosa origin (Brambell, 1956). This "luteal"
tissue is highly vascular, divided into small compartments by connective
tissue, and is encapsulated by a stretched and thin theca in which there is
little distinction between interna and externa.
We are able to classify the corpora lutea of deer mice into several func-
tional categories based on cytological characteristics (see also Long and
Evans, 1922; and Boling, 1942). Active corpora lutea (at peak secretory
function) contain large round luteal cells with well-defined cell membranes;
abundant, homogenous, deeply eosinophilic cytoplasm; and large vesicular
nuclei with prominent nucleoli. Corpora lutea that have begun to regress (as
defined by Brambell, 1956) contain vacuelated luteal cells. As regression
progresses, the number and size of the vacuoles (lipid) increases, as does the
number of cells with vacuoles. Nuclei are unchanged, but cell membranes
become ill-defined. When completely regressed and nonfunctional, the corpora
lutea contain small luteal cells that are vacant; with shrunken, dense and
pycnotic nuclei; and ill-defined cell membranes. Neutrophils and macrophages
commonly occur among the cells and there is an increase in connective tissue.
They are also less vascular than active or regressing corpora lutea.
The corpora lutea of the deer mouse are readily classified into active,
regressive, and nonfunctional types on the basis of size, as well as the
cytological characteristics presented above (see Table 21.5).
TABLE 21.5. DIAMETER OF CORPORA LUTEA
Average Diameter Range of
(|Jm ± SEM) (n) Diameters (|jm)
Active corpora lutea 643.9 ±15.6 (123) 296 - 1221
Regressing corpora lutea (Table 21.6) 314 - 1036
Dysfunctional corpora lutea 337.0 ± 27.4 ( 14) 185 - 574
We have also been able to distinguish regressing corpora lutea at several
stages of involution on the basis of their vacuolation and size (see
Table 21.6).
242
-------
TABLE 21.6.
IDENTIFICATION OF REGRESSIVE CORPORA LUTEA BASED ON VACUOLATION
AND SIZE
Degree of Regression
Very mild
Mild
Mild to moderate . . .
Moderate
Moderate to heavy . . .
Vacuole
Size
0
Tiny
Small
Small to
Large
Small or
Large
Variable
Number of
Vacuoles
per Cell
0
1-2
1-f ew
1-f ew
Several
1
Many
Number of
Vacuolated
Cells
0
Few and
Scattered
Many and
Scattered
Most
Most or
All
Most or
All
Average Diameter
(Urn ± SEM) (n)
(Range of
Diameters, (J
0
602.7 + 21.4
(329 to 1036
626.7 + 24.9
(314 to 925)
687.9 ± 28.6
(536 to 832)
659.8 + 22.7
(444 to 925)
578.8 ± 28.5
(500 to 722)
(50)
(40s)
(11)
I"? 71
( 7)
Two sets of corpora lutea were frequently found in a single ovary. We define
those belonging to a single set as those at the same stage of development. Of
the 89 adult ovaries that we examined, 13.5 percent contained two sets of cor-
pora lutea. Tn each case, these appeared to be of successive sets. If one was
fresh and active, the other consisted of regressing corpora lutea. On the
other hand, if one set was regressing, the other was old and dysfunctional.
Corpora albicantes are spindle-shaped collagenous scars of variable size
that frequently occur in large numbers in the ovaries of deer mice.
Atresias of all sizes occur in the ovary of the deer mouse. They exhibit
numerous signs of degeneration such as shrunken oocytes with pycnotic nuclei,
granulosa cells that are sloughing into the antrum, a collapsed antrum or one
that contains connective tissue cells or inflammatory cells.
Interstitial tissue consists of eosinophilic cells free in the stroma of
the deer mouse's ovary. The cells in these irregular well-vascularized masses
have small dense nuclei, abundant lipoidal or poorly stained cytoplasm, and
indistinct cellular membranes. They resemble the luteinized cells in dysfunc-
tional corpora lutea.
Seasonal Changes in the Histology of the Ovary in the Deer Mouse
Seasonal changes in the ovary of adults during 1974 and 1975 appear in
Table 21.7. Primary follicles were numerous in most ovaries throughout the
year. Antral and Graafian follicles were also present in a high percentage of
cases. Active corpora lutea were found in all months except the December-
243
-------
TABLE 21.7. SEASONAL CHANGES IN THE OVARY OF ADULT Peromysaus maniculatus
Month and Year
Item in Ovary
n
Primary Follicles:
% Females Possessing
Females (Number) with
Small 1°F
Intermediate 1°F
Large 1°F
Females Examined
Antral Follicles:
% Females Possessing
Females (Number) with
Early A.F.
Late A.F.
Females Examined
Graafian Follicles:
% Females Possessing
Females (Number) with
fe Small G.F.
*~ Intermediate G.F.
Large G.F.
Females Examined
Corpora Hemorrhagica:
% Females Possessing
Corpora Lutea:
% Females with Active C.L.
% Females with Regressing C.L.
% Females with Dysfunctional C.L.
Females (Number) with C.L. in
Mild Regression
Moderate-Heavy Regression
Atresias :
% Females Possessing
Interstitial Tissue:
% Females Possessing
Jul 74
4
100.0
1
4
3
(4)
75.0
1
2
(2)
75.0
2
3
0
(3)
0.0
50.0
50.0
0.0
2
0
0.0
50.0
Aug 74
7
100.0
2
5
4
(5)
85.7
4
2
(5)
85.7
1
2
3
(6)
0.0
57.1
28.6
0.0
1
1
75.0
14.3
Sep 74
15
100.0
8
15
11
(15)
86.7
9
8
(11)
80.0
2
2
1
(3)
6.7
20.0
60.0
20.0
5
4
20.0
60.0
Oct 74
10
100.0
6
8
9
(10)
60.0
6
3
(6)
50.0
0
1
2
(3)
0.0
50.0
20.0
10.0
1
1
20,0
10.0
Nov 74
8
87.5
5
5
4
(7)
62.5
3
1
(3)
37.5
2
0
0
(2)
0.0
62.5
0.0
12.5
0
0
14.3
50.0
Dec 74
2
100.0
2
1
1
(2)
50.0
1
0
(1)
0.0
—
—
0.0
0.0
50.0
0.0
1
0
0.0
0.0
Jan 75
3
100.0
1
3
2
(3)
100.0
1
3
(3)
66.7
0
0
2
(2)
0.0
0.0
0.0
0.0
0
0
0.0
0.0
Mar 75
2
100.0
1
2
1
(2)
50.0
1
1
(1)
100.0
1
1
1
(2)
0.0
0.0
100.0
0.0
1
1
0.0
0.0
Apr 75
7
100.0
1
7
6
(7)
71.4
2
5
(5)
85.7
1
0
4
(5)
14.3
42.9
57.1
0.0
3
1
28.6
28.6
May 75
6
100.0
2
6
5
(6)
100.0
1
5
(5)
66.7
0
0
1
(1)
16.7
66.7
50.0
33.3
3
0
66.7
16.7
Jun 75
8
87.5
2
7
5
(7)
50.0
2
4
(4)
87.5
3
1
7
(7)
0.0
62.5
50.0
0.0
2
3
12.5
0.0
Jul 75
8
87.5
5
7
3
(7)
62.5
0
4
(4)
75.0
1
2
5
(6)
0.0
50.0
50.0
12.5
2
2
0.0
37.5
Aug 75
9
100.0
4
7
5
(9)
66.7
2
5
(6)
88.9
0
2
6
(8)
11.1
55.5
44.4
0.0
3
1
11.1
0.0
Supplemental Information:
% Females Examined That Vfere
Pregnant or Postpartum
% Females Examined That Had
Uterine Scars
75.0
0.0
71.4
0.0
28.6
0.0
57.1
0.0
10.0 12.5
0.0
0.0
0.0 100.0 55.6
0.0
0.0 57.1
66.7 77.7
0.0 37.5
40.0 40.0
25.0 22.2
-------
March interval (NB--we lack data for February). The distribution of corpora
lutea suggests that the breeding season extended from March through December
in 1974, but was suspended during winter. Late autumn breeding is probably
unusual for populations of deer mice in southeastern Montana since it was not
repeated in 1975, and in 1974 was accompanied by an unusually wet fall (Lewis
e~t at., 1978). However, deer mice breed year-round elsewhere (Brown, 1945;
Asdell, 1964).
The ovary during January (including the absence of corpora lutea), is
similar to that of voles during their nonbreeding (winter) season (Lecyk,
1962; Clarke and Forsyth, 1964; Clarke and Kennedy, 1967; Coutts and Rowlands,
1969), even though voles seem to be induced ovulators, and deer mice ovulate
spontaneously (Asdell, 1964).
According to Brown and Conaway (1964), the corpora lutea of Peromysaus
persist for about 2 months postpartum with little or no discernable change in
structure. Morton (in Lewis et at., 1978) found pregnant females among deer
mice captured as late as October during 1974. If corpora lutea persist for at
least 2 months before showing marked regression, then one would expect to find
them in females as late as December 1974, which is the case (Table 21.7). The
finding of two sets of corpora lutea in a substantial number of females in our
samples also suggests that corpora lutea persist for some time. Even in unmated
house mice, they may persist for two to four successive estrous cycles (Snell,
1941).
The amount of interstitial tissue in the murine ovary increases during
late gestation (Brambell, 1956). Thus, interstitial tissue is abundant in
reproductively active mice. It was also common in our specimens between April
and September, but conspicuously absent through December and March
(Table 21.7). Atresias were also absent in the latter months, yet abundant at
times (June and August 1974) when interstitial tissue was not. In other
words, the ovary of the deer mouse apparently contained progesterone-secreting
structures only throughout the breeding season.
It would seem, then, that the atretic follicles and interstitial tissue
of deer mice supplement the corpora lutea in the production of progesterone,
which may be important in sustaining pregnancy after the corpora lutea begin
to regress. We are surprised, however, that the incidence of atresia in any
one deer mouse ovary is as low as it is (usually confined to one or at most a
few follicles) given the fact that 75-77 percent of all ovarian follicles become
atretic in the ovaries of rats and house mice (Arai, 1920; JMandl and Shelton,
1959; Jones and Krohn, 1961).
Additional results of the histological survey are the following:
1. Accessory corpora lutea rarely occur in the ovaries of deer mice. We
found only one clear-cut example in the 89 adult ovaries that we examined.
2. The number of ovaries of immature deer mice available for study (11) was
too small to permit us to generalize (Table 21.8). Notable, however, was the
absence of atretic follicles and interstitial tissue in all 11. All contained
primary follicles, frequently of large size and 10 also contained antral
follicles. Six had Graafian follicles, some of which were of advanced age.
245
-------
TABLE 21.8. RELATIONSHIP BETWEEN OVARIAN HISTOLOGY, OVARIAN WEIGHT, AND THE REPRODUCTIVE STATE OF ADULT AND IMMATURE Peromyscus maniaulatus*^
Reproductive
State
Immature
Adult:
Proestrus
Estrus
Sfetestrus
Diestrus
Anestrus
Pregnant
Immediately
Postpartum
Lactating
Anestrus
N
11
0
4
4
5
15
26
4
11
1°F
90.
100.
100.
100.
100.
96.
100.
90.
.9
,0
,0
.0
.0
.2
,0
9
A.F.
90.9
75.0
75.0
60.0
53.3
65.4
75.0
81.8
G.F.
54.5
100.0
100.0
60.0
26.7
84.6
100.0
81.8
At CH
0.0 0.0
50.0 0.0
0.0 0.0
40.0 0.0
6.7 0.0
26.9 3.8
25.0 0.0
9.1 0.0
Active
0.
50.
50.
40
20
46.
75
27
.0
.0
.0
.0
.0
.2
.0
.3
Very
Mild
0.0
25.0
0.0
40.0
0.0
15.4
25.0
0.0
Corpora Lutea
Regressing
Mild- Mod-
Mild Mod Mod Heavy
0.0 0.0 0.0 0.0
0.0 25.0 0.0 0.0
0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0
13.3 0.0 0.0 0.0
26.9 3.8 15.4 3.8
0.0 0.0 0.0 0.0
18.2 18.2 36.4 0.0
Int.
Heavy Dys Tiss.
0.0 0.0 0.0
0.0 25.0 0.0
0.0 0.0 25.0
0.0 0.0 40.0
0.0 0.0 20.0
0.0 7.7 19.2
0.0 0.0 25 . 0
9.1 36.4 54.5
Ovary Weight
of Body Weight
Gng/g)*
0.372 ± 0.048
0.425 ± 0.131
0.277 (3)
0.337 ± 0.121 (4)
0.287 + 0.034
0.313 ± 0.028
0.257 + 0.117
0.228 ± 0.051
* All values except those for ovary weight are the percent of the females in each group that exhibit each item.
t Abbreviations in the table: AF = antral follicles; At = atresias; CH = corpora hemorrhagica; Dys = dysfunctional corpora lutea; GF = mature
(Graafian) follicles; Int. Tiss. = interstitial tissue; Mod = moderate; 1°F = primary follicles.
^ Ovary weights are presented as x ± SEM. Numbers in parentheses indicate sample size when it differs from that given on the left side of the
table.
-------
Changes in the Histology of the Ovary of Deer Mice During the Estrous Cycle
Data (1974-1975) in Table 21.8 are grouped according to reproductive
state at time of capture (we will amplify later on the method we used to
determine the mouse's reproductive state).
Primary follicles were well represented at all stages of the estrous
cycle, and also in the ovaries of immature, pregnant, postpartum, and lactat-
ing mice. Atretic follicles were also common in the ovaries of reproductively
active females. Curiously, active corpora lutea occur in many females at all
stages of the estrous cycle. This suggests that corpora lutea do not immedi-
ately involute even when the female fails to become pregnant and is consistent
with Snell's (1941) finding that the corpora lutea of house mice persist for
two to four successive cycles.
We were quite surprised to find active corpora lutea in the ovaries of
reproductively inactive (anestrus) mice, especially since few of these females
had Graafian follicles. However, we expected to find corpora lutea at many
different stages of regression in pregnant and lactating females. This is
probably related to the fact that individuals were collected at various stages
of gestation (which lasts 22-27 days) or lactation (which lasts up to 4
weeks), intervals in which the corpora lutea normally degenerate.
The average number of medium to large corpora lutea in the ovaries of
pregnant deer mice (5.76 ± 0.01, n = 34) is slightly (0.02 < P < 0.05; t-test
using square root transformations) higher than the number of embryos implanted
in the uteri of the same mice. (5.09 ± 0.04), but is very similar to the
overall mean litter size of deer mice collected at Colstrip during 1975 (5.75)
(Lewis &t al• > 1978). These findings suggest that fecundity and fertility are
on the average the same, but that fecundity frequently exceeds fertility in
individual mice. The latter is typical of the bank vole, C. glareolus ,
(Coutts and Rowlands, 1969) and has also been reported in two subspecies of Per-
omysous, P. m. gracilis and P. m. bairdii (Asdell, 1964),
Uterine Horn and Vagina
Histology
The uterus and vagina of deer mice are structurally similar to those of
other species of murine rodents as described by Parkes (1956). In both
regions, the wall of the reproductive duct consists of three layers: endo-
metrium, myometrium and perimetrium.
Changes in the Histology of the Vagina of Deer Mice During the Estrous Cycle
Diagnostic changes in the structure of the reproductive duct, particu-
larly of its endometrium, occur during the 4 or 5 day estrous cycle of
deer mice. These changes are similar to those exhibited by vaginas of rats and
mice (Asdell, 1964). Vaginal smears are also similar (described for the house
mouse by Long and Evans, 1922). This provides an accurate method of assessing
the reproductive status of an individual deer mouse and is the rationale for
247
-------
including detailed studies of the vagina in the baseline data for Peromyscus.
In addition, very early stages of pregnancy are easily missed during gross
inspection of the uterus, as are unattached blastocysts in the uterine horns.
Both are readily apparent in histological section making it possible to more
closely define a female's reproductive state. This is the rationale for
including the uterine horn in the baseline data.
The diagnostic characteristics of the vagina at each stage of the estrous
cycle of the deer mouse are presented in Table 21.9. These are generally
reliable for determining reproductive state. To these, we add descriptions of
the histology of the vagina in pregnant and anestrous females. The descrip-
tion for pregnant animals is based on only three mice and is therefore tenta-
tive. In all cases, diagnostic changes occur primarily in the endometrium.
During pregnancy, the endometrium is similar structurally to that of a
female in anestrous (Table 21.9) except that the epithelial cells may be muci-
fied, as in house mice. The structure of the vagina of immature deer mice and
of reproductively inactive adults is essentially the same. The endometrial
epithelium is only one to three cell layers in depth (NB—this is the most re-
liable criterion for assigning individuals to the anestrous category). The most
superficial layer consists of squamous or cuboidal cells with poorly stained
cytoplasm, well defined cell membranes and large oval nuclei. The innermost
layer blends into the highly cellular lamina propria. The epithelium lacks
mitotic figures and rarely contains leukocytes. The underlying and lightly
vascularized lamina propria consists of dense, highly cellular connective
tissue, and also lacks inflammatory cells. The tunica muscularis has the two
characteristic layers, but is thin and undifferentiated. Its undifferentiated
state is the most reliable characteristic for distinguishing between diestrous
and anestrous adults.
During lactation anestrous, the histology of the vagina is similar to the
above, except that (1) the endometrial epithelium consists of three to five
cell layers and contains some leukocytes (neutrophils and lymphocytes), and
(2) the tunica muscularis is thick and well developed.
We cannot emphasize enough the necessity of routinely including sections
of the vagina in all future studies of Peromysous in Montana (Table 21.10).
Vaginal characteristics appear to provide the most reliable information con-
cerning reproductive state.
Changes in the Histology of the Uterine Horn of Deer Mice During the Estrous
Cycle
The histological structure of the uterine horn is not a reliable indi-
cator of reproductive condition in deer mice. The relevant data are presented
in Tables 21.9 and 21.11. One stage grades almost imperceptibly into the
next. As in the vagina, most of the changes in the wall occur in the endome-
trial epithelium.
Because most of the slide material at our disposal consisted of uterine
horns and so few vaginas were included (Table 21.11), we had to make infer-
ences about stages of the estrous cycle for many of our animals from field
248
-------
TABLE 21.9. HISTOLOGICAI CHARACTERISTICS OF THE UTERINE HORN OF Peromysaus manicutatus
Reproductive
State
Endometrial Epithelium
Uterine Glands
Lamina Propria
Tunica Muscularis Lumen
Immature Simple crowded cuboidal or columnar Cuboidal to low columnar lining
layer; nucleus fills most of cell
Tend to be small in diameter and
Few mitotic figures inactive in appearance
Few Leucocytes in lining (only
lymphocytes) Lumens usually small and contain
traces of secretion
No mitotic figures in lining
Dense, cellular connective Densely cellular Tiny
tissue
Undifferentiated,
No mitotic figures except for layer
No leucocytes in stroma of numerous and
large blood
Lightly vascularized vessels between
muscle layers
Adult:
Proestrus1
Estrus
Metestrus
Pseudostratified layer
Traces of vacuolar degeneration
Moderate numbers of mitotic
figures
No leucocytes in lining
High pseudostratified layer
Cell membranes well defined
Moderate numbers of mitotic
figures
Variable, but small numbers of
leucocytes in lining
High columnar to pseudostratified
cells, frequently of unequal
height, their rounded borders
projecting unevenly into lumen
Pillar cells (= discharged goblet
cells) prominent and numerous
Prominent vacuolar degeneration:
vacant cells to cells with a
supranuclear vacuole and above
that a heavily stained eosino-
philic layer facing the lumen
Occasional mitotic figures
Lined by truncated columnar cells
Lumens small and empty
Occasional mitotic figures in
lining
Lined by high columnar cells
Lumens of variable size and contain
traces of secretion
Individual glands highly coiled
(tortuous)
Mild-to-moderate numbers of mitotic
figures in lining
Lined by truncated columnar cells,
some showing vacuolar degeneration
Lumens of variable size with only
traces of secretion
No mitotic figures in lining
Neutrophils and pillar cells in
lining
Dense, cellular connective
tissue
Occasional mitotic figures
No leucocytes in stroma
Lightly vascularized
Highly cellular connective
Occasional mitotic figures
Variable infiltration of
stroma with leucocytes (neu-
trophils and lymphocytes)
Highly vascularized
Dense, cellular connective
tissue
No mitotic figures
Variable, but large infil-
tration of neutrophils in
stroma
Highly vascularized by
numerous hyperemic
capillaries
Densely cellular Tiny
Just beginning to
differentiate
Well-developed
Highly vascular
Hyperemic
Well developed
Highly vascular
Hyperemic
Variable, but
large infiltra-
tion of neutro-
phils in and
between muscle
layers
Small and
slitlike
Variable:
small to
large in
size
(continued)
-------
TABLE 21.9. (continued)
Reproductive
State
Endometrial Epithelium
Uterine Glands
Lamina Propria
Tunica Muscularis Lumen
Metestrus Conspicuous and numerous leuco-
cytes in lining (ueutrophils and
lymphocytes)
Diestrus Variable:
Low crowded columnar lining with
cells of equal height in which
most of the cell is filled with
the oval nucleus
Lined by cuboidal or columnar cells
Diameter of glands frequantlv small
Commonly lack a lumen or have
minute lumen
Epithelium of irregular height with
rounded peaks and wide or narrow
valleys, made up of many cells in
which cell membranes are
indistinct; nuclei are crowded and
basal in the lining; superficial
region of epithelium is eosino-
philic cytoplasm
No mitotic figures
Occasional lymphycytes in lining
Anestrus Simple crowded cuboidal or low
columnar layer
Nuclei fill most of epithelial
cells; little cytoplasm
No mitotic figures
Occasional lymphocytes in lining
Dense, cellular connective
tissue
Small numbers of leucocytes
infiltrate the stroma
High vascularity; numerous
hyperemic capillaries
Generall well
developed, but
beginning to
differentiate in
some cases
Little
leucocytosis
Lined by inactive low columnar
cells; sometimes almost cuboidal
Diameter characteristically small
Lumens tend to be small and to
contain traces of secretion
Dense, cellular connective
tissue
No infiltration of
leucocytes
Lightly vascularized
As in immature
mice
Small and
slitlike
Tiny
Pregnant
(areas other
than in the
sites of
implanta-
tion)
High columnar to pseutostratified
layer
Epithelial cells sometimes of
equal height, but sometimes groups
of cells with basal nuclei and
eosiuophilic apical cytoplasm
collectively drawn out into sharp
high peaks that protrude into the
lumen
Vacuolar degeneration common
Occasional mitotic figures
Occasional lymphocytes in lining
Lined by secretorily active
columnar cells
Lumens of variable, but frequently
large size; empty or contain
traces of secretion
No mitotic figures in lining
Vacuolar degeneration in lining
Dense, cellular connective
tissue
Variable leucocytosis
As in metestrus
(continued)
-------
TABLE 21.9. (continued)
Reproductive
State Endoraetrial Epithelium
Uterine Glands
Lamina Propria
Tunica Muscularis Lumen
Immediately High columnar to pseudostratifled
Postpartum lining
Vacuolar degeneration common
No mitotic figures
Large numbers of leucocytes in
lining (neutrophils and
lymphocytes)
Lining of high truncated columnar
cells
Lumens empty
Lining exhibits vacuolar degenera-
tion and contains variable numbers
of neutrophils
Dense, cellular connective As in metestrus
tissue
Marked leucocytosis (neutro-
phils and lymphocytes)
Extravasation, in some
places extreme
Very highly vascular with
numerous small capillaries
and extreme hyperemia
Large and
distended
Lactation Simple crowded columnar lining
Anestrus with cells of unequal height
extending unevenly into the lumen
Nucleus occupies most of each
cell; between it and lumen is a
conspicuous, but small vacuole
No mitotic figures
Occasional lymphocytes in lining
Lined by active truncated, high
columnar cells
Lumens of variable size and contain
traces of secretion
No mitotic figures in lining
Dense, very cellular
connective tissue
Variable, but small
infiltration of leucocytes
in the stroma (neutrophils
and lymphocytes)
Highly vascular
Well developed
Highly vascular
Variable leuco-
cytosis in and
between muscle
layers
Small or
moderate
in size
* This description is based on the uterine horns of two immature mice that were entering proestrus for the first time.
-------
TABLE 21.10. H1STOLOGICAL CHARACTERISTICS OF THE VAGINA OF Peromyscus
manioulatus AT VARIOUS STAGES OF THE ESTROUS CYCLE
State of the Estrous Cycle Diagnostic Histological Features of the Vagina
Proestrus Epithelium consists of a cornified stratified
squaraous layer under the surface and a super-
ficial layer of mucified cuboidal or columnar
cells with pycnotic nuclei
Epithelium thick: 14-18 cell layers. Many
mitotic figures in the epithelium. Few to no
leucocytes in the epithelium
Estrus Epithelium is a cornified stratified squamous
layer
Epithelium thick: 20-25 cell layers. Few mi-
totic figures in the epithelium. No leucocytes
in the epithelium
Metestrus Initially epithelium is a stratified layer with
a sloughing cornified surface; the cornified
layers disappear during late metestrus
Epithelium thin: 9-12 cell layers. No mitotic
figures in the epithelium. Leucocytes appear
and gradually increase in number in the lamina
propria under the epithelium; later, they become
numerous in the epithelium
Diestrus Epithelium is stratified, but not cornified
Epithelium thin: 5-6 cell layers. No mitotic
figures in the epithelium. Mild numbers of
leucocytes in the epithelium
252
-------
TABLE 21.11. RELATIONSHIP BETWEEN THE HISTOLOGY OF THE UTERINE HORN AND REPRODUCTIVE STATE OF FEMALE Peromyscus maniaulatus *t
Endometrial Epithelium f
Reproductive
State N
Immature 26
Type of
Epithelium
Cu-Co (12)
Co (12)
Co-Ps ( 2)
Number of Presence of
Height Mitotic Imflammatory
(|Jm) Figures Cells
10
0
. 02± 0.40±0.20 0.08 ± 0.06
.53
Type of
Inflammatory
Cells
None
L
(24)
( 2)
Appearance
of Basement
Membrane
D
D-F
F
( 2)
( D
(18)
Vacuolar
Degeneration Number
+ ( 5)
± ( D
- (20)
2.17 ±
0.23
Endometrial Glands
Diameter
(k"n)
22.23 +
0.89
Type of
Epithelium
Cu-Co
Co
( 2)
(22)
Mitotic
Figures in
Epithelium
+ ( 2)
- (24)
Adult:
Proestrus 0 ' ' ' ' ' ' ' ' ' ' ' " ' ' ' ' ' ' ' ' '
Estrus 4-5
Metestrus 5
Diestrus 12
Anestrus 35
Pregnant 25
Immediately 4
Postpartum
Lactation 14
Anestrus
Co-?s ( 2)
Ps ( 3)
Co ( 1)
Co-Ps ( 4)
Cu-Co ( 2)
Co ( 6)
Co-Ps ( 3)
Ps ( 1)
Cu-Co (11)
Co (23)
Co-Ps ( 1)
Cu-Co ( 2)
Cu-Ps ( 1)
Co ( 7)
Co-Ps (10)
Ps ( 5)
Pillar
cells(ll)
Co ( 1)
Co-Ps ( 2)
Ps ( 1)
Cu-Co ( 4)
Cu-Ps ( 1)
Co ( 5)
Co-Ps ( 2)
Ps ( 2)
23
2
22
2
18
1
10
0
18
1
29
3
18
1
.66± 3.1010.71 1.20 ± 0.73
.51
.70± 1.2010.58 3.10 ± 0.78
.34
.50± 0.33±0.26 1.38 ± 0.26
.47
.77± 0.00 0.39 ± 0.08
.45
.82+ 1.02±0.28 1.64 ± 0.30
.13
.75+ 0.7510.48 4.00 1 0.71
.91
.111 0.2510.21 1.57 ± 0.16
.26
None
L
PMN
None
L
PMN
None
L
None
L
None
L
PMN
None
L
PMN
None
L
PMN
( 2)
( 2)
( 1)
( 0)
( 4)
( 3)
( 1)
(11)
(18)
(17)
( 6)
(18)
(10)
( 0)
( 4)
( 4)
( 0)
(14)
( 3)
D
D-F
F
D-F
F
D
D-F
F
D
D-F
F
D
D-F
F
D
F
D-F
F
( D
( D
( 2)
( 3)
( 2)
( 2)
( 5)
( 5)
( 6)
( 7)
(22)
( 7)
(10)
( 7)
( D
( 3)
( 5)
( 8)
+ ( 2)
- ( 2)
+ ( 5)
+ ( 5)
± ( 3)
- ( 4)
+ ( 3)
1 ( 7)
- (25)
+ (14)
± ( 7)
- ( 3)
+ ( 3)
- ( D
+ ( 2)
± ( 3)
- ( 8)
4.40 1
0.24
4.00 1
0.55
3.83 ±
0.34
2.93 1
0.20
3.22 ±
0.26
2.75 +
0.75
4.25 1
0.24
42.20 1
4.82
48.48 +
5.17
34.94 1
2.46
21.53 1
0.76
35.32 1
2.20
46.55 1
4.83
38.38 ±
1.58
Co
HiCo
Co
HiCo
Cu-Co
Co
HiCo
Cu-Co
Co
( 2)
( 2)
( D
( 4)
( D
( 4)
( 4)
( 4)
(31)
Co (14)
Co-HiCo(l)
HiCo ( 9)
Co
HiCo
Co
HiCo
( D
( 3)
( 5)
( 8)
+ ( 4)
+ ( 2)
- ( 3)
- (12)
- (35)
+ ( 3)
- (21)
- ( 4)
+ ( D
- (12)
* Abbreviations in the table: AcS = acellular secretion; An = anestrus; C = cells; Co = columnar; Cu = cuboida; D = distinct; Drv = debris;
D-F = distinct in some places, but faded elsewhere; Df = differentiated; Di = diestrus; EC = epithelial cells; Es — estrus; F ~ faded; I = lumens
of intermediate size; L = lymphocytes; L-D = lumens that are large and distended; Met = metestrus; Muc = mucified; PMN = neutrophils;
Pro = proestrus; Ps = pseudostratif led; RBC = red blood cells or erythrocytes; S-S1 = lumens that are small and slitlike; St = stratified;
T = lumens that are tiny; Tr = just beginning to differentiate; UDf = undifferentiated or dedifferentiated; (+) = present and obvious;
(1) = rare; (-) = absent.
t Values in parentheses in the table indicate the number of mice exhibiting the characteristic (i.e., sample size); other values are
Means t SEM.
£ Mitotic activity of the endometrial epithelium, presence of inflammatory cells in the endometrial epithelium, the number of endometrial
glands, the amount of secretion in the endometrial glands, the vascularity of the lamina propria, the degree of infiltration of the lamina
propria with inflammatory cells, and the amount of material or fluid in the lumen of the uterine horn were each rated on a scale of 0 (non) to 5
(very marked or very numerous).
(continued)
-------
TABLE 21.11. (continued)
Endometrial
Glands*
(continued) Lamina Propria of Endometrium*
Amount of Infiltration Type of
Reproductive Secretion of Inflamma- Inflamma-
State Present Vascularity tory Cells Cells
Immature 0.4810.15 1.96 1 0.21 0.12 1 0.08 None
L
Adult:
Proestrus
Estrus 2.0010.77 2.80 ± 0.49 1.60 + 0.68 None
L
PMN
Metestrus 0.4010.24 3.20+0.58 2.40+0.24 None
L
PMN
Diestrus 1.75+0.33 2.67+0.33 1.08 1 0.34 None
L
Anestrus 1.0910.15 2.27+0.18 0.06 1 0.06 None
L
Pregnant 1.48+0.22 3.52+0.10 2.02 1 0.28 None
L
PMN
RBC
Immediately 1.0010.41 3.25+0.25 4.00 1 0.41 None
Postpartum L
PMN
Lactation 1.5010.27 3.21 1 0.21 1.96 1 0.29 None
Anestrus L
PMN
(24)
( 2)
( 2)
( 1)
( 3)
( 0)
( 2)
( 5)
( 6)
( 6)
(34)
( 1)
( 4)
(19)
(15)
( 1)
( 0)
( 3)
( 4)
( 1)
(13)
( 7)
Width
Myometrium*
Width
(|Jm) (|Jm)
91.29
± 5.
278.
+34.
215.
+48,
202
+29,
106
+ 6,
205.
126,
145,
+40
236
+25
.63
.06
.20
.00
.76
.09
.73
.33
.23
.88
.50
.62
.64
.75
.95
62.
1 2.
134.
115.
162.
+15.
141.
110.
73.
+ 3.
225.
114.
285,
+59
205
+17
31
82
.22
.70
.20
,83
.24
.34
.54
.65
.15
.52
.50
.37
.24
.60
Degree of
Differen-
tiation
UDf (25)
Tr ( 1)
Df ( 4)
Df ( 5)
UDf ( 5)
Tr ( 2)
Df ( 5)
UDf (34)
Tr ( 1)
Df (24)
Df ( 4)
UDf ( 1)
Df (12)
Lumen*
Contents
Size (Amount)
T 0.19 1
0.08
S-S1 0.40 1
0.28
S-S1 ( 3) 0.40 +
I ( 1) 0.40
L-D ( 1)
S-S1 (11) 0.08 +
I ( 1) 0.08
T (32) 0.34 +
S-S1 (3) 0.08
2.00 +
0.38(23)
L-D (4) 1.50 1
0.29
S-S1 ( 6) 0.57 ±
I ( 6) 0.14
L-D ( 1)
Nature of
Contents
None (21)
AcS ( 2)
Deb ( 4)
C ( 1)
None ( 3)
AcS ( 2)
None ( 4)
EC ( 1)
None (11)
AcS ( 1)
None (22)
AcS ( 9)
Deb ( 4)
C ( 5)
PMN ( 1)
RBC ( 1)
None ( 5)
AcS (18)
Deb ( 2)
C ( 4)
PMN (10)
RBC ( 7)
L ( 2)
Sperm (1)
Pigment(l)
None ( 0)
AcS ( 3)
C ( 2)
PMN ( 3)
RBC ( 3)
L ( 1)
Sperm ( 1)
None ( 6)
AcS ( 8)
Deb ( 1)
C ( 1)
Uterine
Weight as
a Function
of Body
Weight
(mg/g)
1.366 1
0.398
2.651 +
0.352
4.317 ±
0.286
2.722 1
0.340
1.173 +
0.114
14.425 1
3.548
16.030 1
3.529
4.195 1
0.536(13)
Repro-
ductive
State of
Vagina
Pro
Di
An
Es
Met
Di
Di
An
Pro
Met
Di
An
Pro
Met
Di
An
( 1)
C 1)
(13)
( 1)
( 2)
( 80
( 1)
(17)
( 1)
( 1)
( 2)
( 1)
( 1)
( 1)
( 2)
( 2)
-------
data on: (1) body and uterine weight; (2) the uterus — scarring, distension
with fluid, implanted embryos; (3) the vagina--perforate or inperforate at
capture; (4) condition of the mammary glands — conspicuous grossly or lactat-
ing. Guidelines used to establish reproductive state in questionable cases
are these:
1. Small rodents such as Pepomyscus with short estrous cycles and
spontaneous ovulation do not have a luteal phase during a typical estrous
cycle. Hence, the presence of well developed corpora lutea probably indicates
pregnancy.
2. Reproductively immature mice do not ovulate. Hence, a mouse of
unknown age whose ovary contains corpora lutea is an adult.
3. Deer mice have a postpartum estrus during which they ovulate. If
fertilized, the ova develop into blastocysts, which may remain free in the
lumen of the uterine horn for 15-20 days while the female is actively lactat-
ing (Asdell, 1964). Furthermore, females are in a state of anestrous while
lactating. Hence, a deer mouse whose uterus contains free blastocysts is
probably in a state of lactation anestrus.
4. Mammary glands are enlarged and conspicuous only during lactation.
Hence, those females with conspicuous mammary glands are probably in a state
of lactation anestrus.
5. The vagina of the deer mouse is perforate only at copulation and
parturition. Thus, individuals of unknown reproductive state with perforate
vaginas are either in estrus or immediately postpartum.
6. Mice weighing 14 or more grams were classified as adults, whether
their ovaries contained corpora lutea or not (see also Lewis et al. , 1978).
Those weighing less than 14 g, but having corpora lutea or uterine scars were
also classified as adults, their small body weights notwithstanding.
7. Vaginal histology is a reliable indicator of the reproductive state
of a deer mouse.
Using the above criteria, the deer mice in our sample sort out into the
various reproductive classes described in Table 21.9 and 21.11. A few addi-
tional notes are in order:
1. Determination of the reproductive state of deer mice cannot be made
on the basis of distinctness of the basement membrane of the endometrial
epithelium, nor vacuolar degeneration in the epithelium, although these cri-
teria have been used in studies of other murine rodents (e.g., Parkes, 1956).
2. The uterine horn of the anestrous adult and the immature deer mouse
are essentially the same and represent the basal, undifferentiated state of
the tube.
3. The endometrial epithelium, and the glands derived from it, are
especially proliferative (mitotically active) during estrus.
255
-------
4. The acellular secretion in the lumen of the gravid and immediately
postpartum uterus is similar to that in the uterine glands and is likely
derived from them.
5. The state of the vagina in pregnant females varies considerably. In
our limited sample of five vaginas, mudification of the vaginal epithelium was
consistently present, although such is reportedly characteristic of pregnant
house mice (Gorbman and Bern, 1962).
6. Changes in height of the uterine epithelium of small rodents during
the estrous cycle are usually subtle, small, or inconsistent (Allen, 1922;
Allen, 1931; Clauberg, 1931). However, (see Table 21-9), such changes are
among the more reliable discriminating characteristics of the uterine horn of
deer mice. The epithelium is low (10-11 |Jm) during anestrus and in repro-
ductively immature deer mice; intermediate in height (18-19 |Jm) during anes-
trus pregnancy, and lactation anestrus; and high (23-30 pm) during estrus
metestrus and immediately postpartum.
7. Uterine weight is a function of body weight and may also be useful
in determining the reproductive state of female deer mice. This criterion
allows clear separations of the following groups, which are listed in order of
increasing uterine weights: (immature and anestrus adults) < (estrous and
diestrous adults) < (adults in metestrus or lactation anestrus) < (pregnant
and immediately postpartum adults).
8. The maternal and fetal placenta of the deer mouse is similar to the
respective portions of the placenta of the house mouse (described by Rugh,
1968).
Adrenal Gland
Histology
The adrenal gland of the deer mouse is structurally similar to that of
mammals generally (Bloom and Fawcett, 1975) and murine rodents specifically
(Snell, 1941; Jones, 1957).
It has a core of medullary (chromaffin) tissue surrounded by a ring of
cortical (interrenal) tissue. The medulla consists of cords of large poly-
gonal cells with vesicular nuclei and abundant finely strippled cytoplasm.
The cords are one or two cells across and separated from one another by large
irregular venous channels, which are typically filled with erythrocytes. Even
in preparations stained with haematoxylin and eosin, it is possible to distin-
guish two types of cells in the medullary cords. Most are a light burgundy
color. However, other scattered cells are a deep purple color. The medulla
is separated from the cortex by a thin capsule of connective tissue which is
frequently difficult or impossible to identify in section.
The adrenal cortex exhibits typically mammalian zonation. Adjacent to
the medulla is a zona reticularis (ZR) of loosley packed, polygonal cells,
laced with small vascular channels (sinusoids). The irregular packing of
cells in this zone is diagnostic. Cells tend to be small relative to those
256
-------
elsewhere in the cortex. They have vesicular or dense (sometimes pycnotic)
nuclei. The cytoplasm contains moderately eosinophilic granules. Also found
in this zone are scattered cells filled with a golden brown pigment (ceroid).
External to the ZR is a wider zona fasciculata (ZF) which is composed of
narrow palisade-like columns of cells, radially arranged with respect to the
medulla and separated from one another by fine vascular channels (sinusoids).
Each column is one to three cells across (most commonly only one cell). The
cells are generally large and square. Nuclei are quite irregular in size and
vesicular. The cytoplasm is abundant and filled with eosinophilic granules
giving the cells a diagnostic uniform red appearance.
The outermost and narrowest region of the cortex is the zona glomerulosa
(ZG) . It is not always clearly demarcated from the ZF. It consists of small
clusters of cells surrounded by narrow vascular channels. The cells them-
selves are small and round with vesicular nuclei and little cytoplasm.
Sexually immature and nulliparous adult female mice are reported to have
additional cortical zones, the most common of which is the X zone (Howard,
1927; Jones, 1957). These disappear in adult males and in females during
pregnancy. Such zones are most often juxtamedullary. So few immature mice
were available for study that we largely ignored the X zone. However, our
limited data indicate that an X zone persists in reproductively active cycling
female deer mice (n = 3) until they become pregnant. It is absent in pregnant
(n = 6), postpartum (n = 1), and lactating mice (n = 1), as is a ZR. In its
absence, the ZF is separated from the medulla by a broad hyperemic layer of
loosely arranged connective tissue. Although no ZR is present, cells near the
inner face of the ZF frequently have nuclei that are smaller than those in
more peripheral areas of the cortex. In contrast, the ZR of reproductively
active male deer mice is well-developed, but also frequently separated from
the medulla by a hyperemic region of loosely organized connective tissue. (We
had no immature male deer mice for comparison with adults.) Structurally, the
X zone is similar to the ZR of adults.
Cortical cells frequently contain lipoidal vacuoles of variable size and
number. Vacuoles also occur in medullary cells in some cases.
The entire gland is encapsulated by several closely applied, thin layers
of connective tissue.
Seasonal Changes in the Adrenal Gland of Deer Mice
Seasonal changes in the histology of the adrenal gland are compiled in
Table 21.12, from which we draw the following conclusions:
1. The adrenal gland rarely exhibits pathological changes in these
populations of deer mice. In our sample of 72 mice, only three had clearly
diseased adrenal glands. One exhibited extensive fatty degeneration of the
medulla. Two others contained infiltrations of neutrophils in the ZR. (Small
infiltrations of lymphocytes do occasionally occur in the ZR and medulla.)
257
-------
TABLE 21.12.
SEASONAL CHANGES IN THE HISTOLOGY OF THE ADRENAL GLAND OF Peromyscus mamculatus.
THE TABLE ARE MEANS ± SEM
VALUES IN
Age/Sex
Group
Adult
Males
Reproductive
Status
Reproductively
Active
Month
Year
Jul
Aug
Sep
Oct
Nov
Dec
Jan
Mar
Jun
74
74
74
74
74
74
75
75
75
n
7
7
7
7
3
4
1
3
2
Total X-sec.
of Adrenal
Sec.
2.12
1.99
2.03
1.78
1.73
1.72
2.10
2.79
1.44
Percent of Total
Cross-Sectional Area Occupied
. (mm2)
± 0.
± 0.
± 0.
± 0.
± 0.
19
17
12
12
06
7
9
8
9
10
15
8
6
16
ZG*
.7 ±
.4 ±
.5 ±
.8 ±
.9
.3 ±
.6
.6
.4
ZF-ZR(X)*
0.4
1.1
1.0
1.4
3.4
79.9 ±
77.9 ±
78.1 ±
74.1 ±
72.6
65.6 ±
88.6
80.0
69.4
3.2
3.9
4.8
2.4
3.7
by
M*
12.4
12.7
13.4
16.1
16.5
19.0
2.9
13.4
14.2
+
+
+
+
+
3.6
3.7
4.9
1.7
1.4
Adrenal
Weight as a
Function of Body
Weight
0.540
0.491
0.552
0.411
0.549
0.602
0.643
0.961
1.000
± 0
± 0
± 0
± 0
± 0
(mg/g)
.093
.044
.055
.038
.084
(5)
M Immature Reproductively
^ Females Inactive
1 1.56
10.3
77.6
12.2
0.677
Adult Anestrus
Females (inactive)
Cycling
Pregnant
Lactating
Anestrus
2
4
18
6
2.16
2.46 ± 0
2.39 ± 0
2.81 ± 0
.16
.10
.18
8.0
8.8
10.7
9.9
± 1.4
± 1.6
± 1.8
78.7
79.5 ±
75.3 ±
78.5 ±
4.3
2.2
1.9
13.4
11.7 ±
14.0 ±
11.6 ±
0.424 (1)
3.0
1.6
2.1
0.667 ±
0.696 ±
0.713 ±
0.064
0.078
0.075 (5)
* ZG = zona glomerulosa; GF = zona fasciculata; ZR = zona reticularis; M = medulla.
t The degree of vacuolation, degeneration, or infiltration of cortical tissue was quantified according
to the following scale:
Symbol
0
Definition
None
Traces or rare
Some, several, few, or mild
Moderate numbers or amount
Many, numerous, or heavy
Very many, very numerous, or very heavy
Assigned Numerical Value
0.00
1.
2.
3.
4.
5.
00
00
00
00
00
(continued)
-------
TABLE 21.12. (continued)
to
t-n
Cortical Vacuolation
Age/Sex
Group
Adult
Males
Immature
Females
Adult
Females
Number of
Mice with
Cortical
Vacuoles
(%)
28.6
42.9
28.6
28.6
0.0
75.0
100.00
33.3
50.0
100.0
0.0
0.0
61.1
66.7
Degree of Cortical Vacuolation*,!
Large Vacuoles in
Cortical Cells
0.00
0.29 ± 0.29
ZF (2)
0.00
0.00
0.00
0.00
0.00
0.00
0.00
2.00
ZF
0.00
0.00
0.72 ± 0.30
ZG(1) ZF(5)
0.00
Small Vacuoles in
Cortical Cells
1.14 ± 0.74
ZF(2)
1.43 ± 0.92
ZF(2)
0.86 ± 0.59
ZF (2)
0.86 ± 0.59
ZF(1) ZR(1)
0.00
3.00± 1.00
ZF(3)
4.00
ZF
1.33 ± 1.33
ZF(1)
2.00
ZF(1)
4.00
ZF
0.00
0.00
2.11 ± 0.49
ZF(8) ZR(2)
3.17 ± 1.01
ZF(4) ZR(1)
Degree of
Cortical Degeneration*,!
Pycnotic
Nuclei
2.86 ± 0.59
ZF(2) ZR(4)
0.00
2.57 ± 0.92
ZR(4)
1.14 ± 0.74
ZF(1) ZR(1)
2.00
ZR(2)
3.25 ± 1.11
ZR(3)
0.00
1.67
ZF(1)
0.00
0.00
1.00
ZR(1)
1.50 ± 0.96
ZF(1) ZR(1)
0.22 ± 0.22
ZF(1)
0.33 ± 0.33
ZR(1)
Degenerate
Cells
0.57 ± 0.57
ZR(1)
0.00
0.00
0.00
0.00
0.00
0.00
1.67
ZF(1)
0.00
0.00
0.00
1.00 ± 1.00
ZR(1)
0.00
0.00
Degree
of Lymphycyte
Infiltration* , t
0.29 ± 0
ZR(D
0.00
0.00
0.43 ± 0
ZR(1)
0.00
0.00
0.00
0.00
0.00
0.00
0.00
1.00 ± 1
ZR(1)
0.33 ± 0
ZR(2)
0.00
.29
.43
.00
.23
-------
2. Cross-sectional areas of adrenals of adult males suggest that the
medulla was more active in October-December 1974 than during other months.
3. Monthly differences in the cross-sectional area of the ZF-ZR of adult
males suggest that cortical function is relatively uniform except in early
winter (December), when it is reduced.
4. On the basis of cortical vacuolation, more than 25 percent of adult
males appear to have been under mild to moderate stress during December 1974
and perhaps in the January-August 1975 interval. In view of this and the
tabulated data concerning degeneration of cortical tissue, adult males appear
to be under mild to severe stress for much of the year.
5. Pregnancy and lactation appear to have imposed moderate stress on
more than half of the females in these reproductive states.
6. There is no correlation between the cross-sectional area of the
ZF-ZR and the lipid measurements for the same regions of the cortex.
7. Changes in the weight of the adrenal gland as a function of the body
weight correspond to changes reported for rats of similar reproductive stages.
As reported by Andersen and Kennedy (1933), adrenal weight is lowest in anes-
trous females, low during pregnancy, and highest at parturition, estrus and
during lactation. In rats, these changes are associated with similar changes
in the size of the adrenal cortex, but in deer mice this correlation is not
apparent in the percent of the adrenal section consisting of cortex. However,
it is reflected in the actual areas of the ZF-ZR in the female population
(Table 21.13).
TABLE 21.13. AREA OF THE ZONA FASCICULATA AND ZONA RETICULARIS (ZF-ZR) IN
FEMALE DEER MICE
Anestrous adults (n = 2) 1.70 mm2
Cycling adults (n = 4) 1.96 ± 0.20
Pregnant adults (n = 18) 1.81 ± 0.10
Lactating adults (n = 6) 2.21 ± 0.18
(values are means ± SEM)
8. Androgens are generally considered to inhibit the release of ACTH
from the anterior pituitary gland of mammals, whereas estrogens promote its
release. Consequently, the adrenals of male mammals tend to be smaller than
those of females of the same species. However, this trend is not clearly
shown in our data for deer mice. If androgens inhibit adrenal cortical func-
260
-------
tion and presumably size, then one might expect changes in the size of the
adrenal cortex and either testes size or the development of androgen-dependent
male accessory sexual glands to be inversely related. Comparison of the data
in Lewis e~t al- (1978) for the testes and seminal vesicles of our animals with
the data on the adrenal cortex (specifically the ZF-ZR, 'i.e.., ACTH-dependent
regions) tabulated in this report illustrates that for months in which sample
size is three to seven, the area of the ZF-ZR and the weight of testes or sem-
inal vesicles are directly related. All three are elevated in March-September,
then decline to annual lows during December.
Histology
In general, the tissue structure of the deer mouse spleen conforms with
that of mammals (Bloom and Fawcett, 1975) and murine rodents specifically
(Snell, 1941; Elaine and Conaway, 1969).
The organ is encapsulated by several layers of connective tissue and
smooth muscle, from which muscular trabeculae extend into the splenic pulp.
In section, the spleen is long and flat and trabeculae tend to be most
numerous in the thinner regions where they extend from one side of the gland
to the other. The prominence of smooth muscle in the trabeculae, and the
extension of the latter from one side of the gland to the other, suggest that
they may function to expel blood from the splenic pulp, e.g . , suggest that the
spleen of Peromysausis a blood cell reservoir.
The splenic tissue consists of red and white pulp. The white pulp
(diffuse lymphoid tissue and germinal centers) is not usually present immedi-
ately beneath the capsule, but is well represented in the organ's center.
Here, there are many arteries, each surrounded by white pulp, arranged in
tandem along the long axis of the spleen. This queue extends from one end of
the spleen to the other. Additional finer arteries and arterioles surrounded
by thin cuffs of white pulp are numerous and widely scattered throughout the
gland.
It is sometimes difficult to distinguish diffuse lymphoid tissue from
germinal centers in the white pulp because all of the lymphoid tissue around a
major blood vessel resembles one gigantic germinal center. Accordingly, we
adopted the following criteria to distinguish germinal centers: (1) they
exhibit light and dark hemispheres; and (2) are encapsulated by a dense layer
of reticular cells and small lymphocytes. Except for their occasionally large
size, the germinal centers are histologically similar to those of other
mammals (see Bloom and Fawcett, 1975: 446-561). They are occasionally packed
with large clear spaces containing macrophages (with phagocytized hemosi-
derin), cellular debris, eosinophils, and neutrophils.
The red pulp consists of (1) vascular (splenic) sinuses lined by simple
squamous or cuboidal cells, and (2) intervening splenic cords, islands of
tissue containing many small lymphocytes, reticular cells, and macrophages.
Hemosiderin, when present, is usually in macrophages within the cords, rather
than in the vascular sinuses. The cords may also house nests of plasma cells
261
-------
and medium to large sized lymphocytes, and occasionally neutrophils. They al-
ways contain hemocytoblasts and megakaryocytes (occasionally even metamyelo-
cytes and megakaryoblasts), e.g., stem cells that produce blood cells and
platelets, respectively. These stem cells commonly contain hemosiderin.
Since we did not see platelets (perhaps because of the extreme cellular den-
sity of the spleen), it is possible that some of the cells identified as
megakaryocytes, especially those with large amounts of hemosiderin, are multi-
nucleated giant cells. However, Snell (1941) notes that megakaryocytes are
conspicuous and characteristic of the spleen of house mice.
Seasonal Changes in the Histology of the Spleen of Deer Mice
The data tabulated in Tables 21.14 and 21.15 provide quantitative infor-
mation about the red and white pulp, respectively, of the deer mouse at vari-
ous times of year. From the tables, we make the following general observa-
tions :
1. Platelet formation occurred year-round, but was depressed in
December. (Observe changes in the population of megakaryocytes in the red
pulp.)
2. Erythropoiesis and/or storage of erythrocytes in the spleen was
elevated in July-August 1974. (Note rations of red to white blood cells and
the percent of the spleen cells in the red pulp represented by erythrocytes
and normoblasts.)
3. The destruction of erythrocytes was elevated in May and June, and
the August-October interval. (This conclusion is based on the hemosiderin
content of the spleen.)
4. Lymphopoiesis was elevated during September-November. (Note the
percent of the cellular population represented by medium and large sized
lymphocytes.)
5. The number of foreign bodies trapped in the spleen and the antibody
production of the organ was high in August-November and perhaps in June.
(This is suggested by the numbers of macrophages, neutrophils, plasma cells,
and medium to large lymphocytes in the red pulp, as well as the number and
mitotic activity of the germinal centers in the white pulp.)
6. On a seasonal basis, splenic weight declines during November and
December. Spleen weight also shows considerable individual variation in Per-
omysous , ranging between 1.498 and 16.337 mg/g of body weight. Such large
variation is not unexpected (see Skryja and Clark, 1970). In house mice, for
example, splenomegaly is found in subordinate males, in distinct contrast to
dominant individuals, and is accompanied systemically by anemia and histo-
logically by marked reduction in the white pulp, and increased formation of
erythrocytes and megakaryocytes in the red pulp (Elaine and Conaway, 1969).
However, if the differences in spleen weight in deer mice are a function of
social status, then we might expect to find the following in enlarged spleens:
(1) low percentages of small lymphocytes; (2) high percentages of erthrocytes,
megakaryocytes, hemocytoblasts, and normoblasts in the red pulp; (3) large
262
-------
TABLE 21-14- SEASONAL CHANGES IN THE HISTOLOGY OF RED PULP IN THE SPLEEN OF Peromysaus manioulatus. AGE AND SEX CLASSES ARE COMBINED IN EACH
MONTH. CELL POPULATIONS ARE EXPRESSED AS A PERCENT OF THE TOTAL CELLS PRESENT UNLESS OTHERWISE INDICATED. VALUES ARE MEANS ± SEM
Month-
Year
Jul 1974
Aug 1974
Sep 1974
Oct 1974
Nov 1974
Dec 1974
Jan 1975
Mar 1975
May 1975
Jun 1975
Jul 1975
Aug 1975
fa
CO T3 CO i— 1
QJ -O Q) QJ rH
4-> O N 4-1 V
>» CO -H >. CJ
U CQ U
O S 1 O CO
H A 3 OJ A B
rH O, 'H W 0. a
CO |3 -O H [3 CO
n ti S a i3 tn- ft!
12 26.7 4.6 0.0
±3.6 ±2.4
19 26.4 6.0 0.1
±3.5 ±1.0 ±0.1
19 22.2 13.9 0.1
±1.9 ±2.3 ±0.1
13 20.7 9.4 1.1
±3.3 ±2.0 ±0.4
6 19.6 14.2 0.4
±2.3 ±2.6 ±0.4
4 18.8 3.1 0.0
±5.8 ±1.6
1 22.5 5.0 0.0
6 35.0 4.6 0.0
±5.7 ±1.5
3 26.9 3.3 0.0
7 26.9 2.5 0.0
±4.3 ±1.1
1 35.0 7.5 0.0
4 19.0 4.1 1.2
6.2 ±1.1 ±1.2
* Subjective ratings concerning the
hemocytoblasts , and the amount of
been
+
++
w
QJ
t>0
(3
J3
ft
O
IH
u
a
4.2
±1.4
5.1
±0.9
7.8
±1.6
9.7
±1.6
10.0
±1.9
8.8
±4.3
7.5
9.6
±1.9
17.8
21.1
±2.7
7.5
17.6
±4.4
number of
T3
d
«
CO cfl
l-H rH
•H -H
JS Jl
& o.
0 0
u a
4-1 -rt
S co
QJ O
z w
0.4
±0.3
0.5
±0.3
2.4
±0.6
3.0
±1.0
2.5
±1.3
1.2
±0.7
0.0
0.4
±0.4
0.0
1.8
±0.9
0.0
0.0
io
QJ in
4-1 4-1
r-> CO
0 CO
O r-t
H f
ja o
h g
t? 0
W K
40.1 0.2
±2.9 ±0.2
35.0 0.0
±2.4
21.1 0.1
±2.4 ±0.1
20.3 0.0
±2.7
20.8 0.0
±3.5
26.2 0.0
±2.2
17.5 0.0
19.6 0.0
±2.6
28.4 0.0
21 . 4 0.0
±3.4
12.5 2.5
24.4 0.0
±6.9
megakaryocytes and
en
4J
CO
>
u
§
OJ
1.2
±0.6
0.5
±0.2
0.6
±0.2
0.8
±0.4
1.7
±0.8
0.9
±0.6
0.0
1.7
±0.5
0.0
1.4
±0.7
2.5
2.4
±1.0
**
hemosiderin, in each spleen have
transformed into numerical equivalents
Rating Scale
0 = none
± = traces (rare)
+ = mild (some or few)
+ = moderate
+ = heavy (many or numerous)
of this
summary, viz:
CO
H QJ
O 4-*
W U
OJ O
4-1 rH
>> OJ
U >>
o e
rH CO
QJ 4-1
£>> OJ
as
0.0
0.0
0.1
±0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Subjective
Cfl
i-H
W rH
U rH QJ
QJ rH CJ>
4-1 OJ
>-. U rH
U CO
O ^4 -H
>> CO rH
£4 rH QJ
Ctj 3 J3
jj U 4-1
CO -H O
00 4-1 TJ
QJ QJ d
0.8 14.9 6.0
±0.4 ±1.6 ±1.4
0.4 16.6 7.5
±0.2 ±2.1 ±1.1
0.4 20.4 9.2
±0.2 ±1.9 ±1.2
0.2 22.7 11.0
±0.2 ±2.3 ±1.6
0.4 17.5 12.5
±0.4 ±1.9 ±2.5
0.0 30.0 10.0
±7.9 ±1.0
0.0 37.5 10.0
0.0 21.7 6.2
±3.5 ±1.9
0.8 15.5 5.7
0.0 19.3 5.0
±2.1 ±2.2
0.0 17.5 15.0
1.1 21.4 4.0
±1.1 ±6.6 ±1.0
ratings of the ratio
*
CO
QJ QJ
rH 4-1
U !>>
CO U
3 0
a >>
u
,d co
4-1 M
O to
O CJC
S a1
0.4 1.83
±0.3 ±0 . 34
1.8 1.63
±0.4 ±0 . 22
1.6 1.68
±0.4 ±0.31
1.1 1.23
±0.5 ±0.23
0.4 1.67
±0.4 ±0.33
1.2 0.50
±1.2 ±0.29
0.0 1.00
1.2 2.33
±0.6 ±0.80
1.6 1.33
0.7 1.86
±0.5 ±0.70
0.0 1.00
4.7 3.50
±1.0 ±0.50
•i!
(0
4-1
CO
CO
r— I
,0
O
4-1
>l
U
O
B
2.67
±0.43
2.42
±0.26
2.37
±0.32
2.23
±0.38
2.83
±0.48
1.00
±0.41
4.00
4.17
±0.40
3.67
2.67
±0.71(6)
4.00
3.50
±0.50
d
>H
ti
QJ -K
TJ 4-1
•H d
CO QJ
0 4J
B d
QJ O
1.75
±0.33
2.26
±0.27
3.00
±0.30
2.27
±0.34
1.67
±0.44
1.25
±0.63
0.00
1.25
±0.25
3.83
3.36
±0.28
0.00
3.25
±0.32
of erythrocytes to leucotytes in
red pulp of the spleen have been transformed
alents for
Numerical Equivalent
++-H- = very heavy (very numerous)
0
1
2
3
4
5
£
0 S
4J ^
«
CO * — '
QJ
4-> CO
>. QJ
m u 4-1
o o >>
l-< U
0 XI 0
•H 4-1 U
4-1 >> a
(5 U OJ
ffn W 1— 1
1.33
±0.17
1.13
±0.12
1.03
±0.13
1.35
±0.16
1.25
±0.21
1.38
±0.24
0.50
1.00
±0.18
1.33
0.93
±0.07
0.50
1.12
±0.43
the
into numerical equiv-
purposes of this summary, viz:
Rating Scale
R « W
R > W
R = W
R > W
R » W
Numerical
0
0
1
1
2
Equivalent
.0
.5
.0
.5
.0
-------
TABLE 2L15. SEASONAL CHANGES IN THE HISTOLOGY OF WHITE PULP IN THE SPLEEN OF Peromyscus manieulatus.
AGE AND SEX CLASSES ARE COMBINED IN EACH MONTH
Germinal Centers in White Pulp
Month-
Year
Jul
Aug
Sep
ro Oct
ON
Nov
Dec
Jan
Mar
May
Jun
Jul
Aug
1974
1974
1974
1974
1974
1974
1975
1975
1975
1975
1975
1975
n
12
20
19
13
6
4
1
6
3
7
1
4
Total
f x "*~
16
13
18
16
25
13
14
17
12
15
14
20
.67
.00
.21
.31
.83
.75
.00
.67
.33
.57
.00
.75
Number
SEM)
± 1.91
± 1.18
±2.75
±2.02
± 4.75
+ 2.56
± 2.70
±2.26
±2.46
Number with Pronounced Spleen Weight as a
Mitotic Activity Function of Body Weight
Range and/or Lymphoblasts in mg/g (x ± SEM)
3 -
3 -
4 -
4 -
14 -
8 -
12 -
4 -
7 -
17 -
27
23
51
31
44
19
26
17
24
28
0
0
0
0
2
0
0
1
0
0
0
2
4
4
4
3
3
2
4
4
6
4
5
.545 ± 0.469
.192 ± 0.475
.966 ± 1.240
.899 ± 0.993
.376 ± 0.451
.128 ± 0.271
.521 ± 0.907
.224
.860 ± 1.433
.323
.411 ± 0.471
(14)
(11)
( 6)
-------
numbers of megakaryocytes and hemocytoblasts in the red pulp; (4) high red to
white blood cell ratios; and (5) relatively few germinal centers in the white
pulp. In only four of the 12 mice with splenomegaly did three or more of the
above characteristics occur. It appears that social status and other as yet
unidentified factors contribute to differences in the size and histology of
this organ in deer mice. For example, four of the five females with enlarged
spleens were pregnant.
Liver
Histology
The deer mouse liver is structurally similar to that of mammals (Bloom
and Fawcett, 1975) and mice specifically (Snell, 1941). It consists of large
"classical" lobules that are indistinctly separated from one another by connec-
tive tissue. Where three lobules meet, they form a space, the triad, contain-
ing branches of the hepatic artery, portal vein, bile ducts and lymphatic
channels. Radially arranged vascular channels (sinusoids) carry blood toward
the center of each lobule and anastomose there to form a central or hepatic
vein. Sinusoids tend to be narrow and are sometimes difficult to locate.
They are lined by a simple squamous endothelium and large, conspicuous Kiipffer
cells which project into the lumen. They are commonly filled with blood
cells, which were frequently refractile, poorly stained and had considerable
associated hemosiderin. This suggests that blood stood in the tissue for some
time before fixation. Characteristics of the liver cells are consistent with
this interpretation. Kiipffer cells also commonly contained hemosiderin and
occasionally black specks which we assume are particles of carbon.
Each lobule is composed of large, polygonal hepatic cells that are organ-
ized into cords or plates that radiate like the fins of a paddle wheel from
the central vein to the edges of the lobule. Each hepatic plate is one cell
across and separated from the next by a sinusoid. The liver cells or hepto-
cytes in the plates have finely strippled cytoplasm and one or two nuclei.
They are sometimes vacuolated (infiltrated with fat), have poorly stained
cytoplasm (indicating hydropic degeneration and/or glycogen depletion) or
pycnotic nuclei (indicating cellular degeneration) or contain a brown pigment.
Autolysis was frequently present in our liver samples. However, the
observed pattern varied considerably from one liver to another. These varia-
tions may indicate shifts in blood flow through the liver as a function of
processing at or near the mouse's death.
The liver was also commonly infiltrated by small focal aggregates of
lymphocytes, usually near the triads.
Several livers exhibited pathological changes such as cirrhosis (n = 1) ,
epithelioid replacement of hepatic tissue (n = 2), infiltration of the hepatic
parenchyma by giant cells (n = 1), cocci with or without inflammatory re-
sponses (n = 3), and localized hepatic degeneration accompanied by inflamma-
tory responses (n = 2).
265
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Seasonal Changes in the Histology of the Liver of Deer Mice
Seasonal changes in the histology of the liver are summarized in
Table 21.16, from which we draw the following inferences: 1. Fat storage in
the liver was uncommon; 2. In contrast, lymphocyte infiltration, usually
focal in nature and largely restricted to the triads, was common; 3. Glycogen
depletion, widespread when present, occurred during July-September 1974; 4.
Widespread autolysis was common, especially during December 1974 and June
1975; 5. Pathologies were most common during September 1974. In combination,
items 3-5 suggest that the energy demands and/or sources of stress on the mice
were unusually high during June-September and December. Results of the
adrenal survey (changes in cortical lipid) are consistent with this inter-
pretation: they suggest that the mice were under considerable stress during
December 1974 and January-August 1975.
6. Kiipffer cells contained the most hemosiderin during May and August
1975 (but not during August 1974). If this reflects erythrocyte destruction,
then the latter was high during the same months, and erythropoiesis might be
expected to rise shortly thereafter. The results of the spleen survey are
consistent with this interpretation: they suggest that erythropoiesis was
high in July and August and that erythrocyte destruction was high during
May-June and August-October.
7. Blood flow through the liver was elevated during September, October,
and December 1974, and March 1975, but reduced in May-August 1975. Whether
this reflects real differences in liver function or simply variation in pro-
cessing of the tissues (e.g., the time of day when the mice were killed or the
speed with which the liver was fixed) is unknown.
Histology
The histological structure of the kidney of deer mice generally conforms
to descriptions presented in standard references (Bloom and Fawcett, 1975).
Renal corpuscles are mammalian in type with a simple squamous Bowman's
capsule and glomerular capillaries. The juxtaglomerular apparatus is a con-
spicuous feature of the vascular pole of many of them.
The proximal portion of the renal tubule consists of convoluted and
straight regions. The latter is considered to be the descending thick limb of
the loop of Henle (Bloom and Fawcett, 1975).
1. Convoluted region—this is a round, relatively large tube, lined
with a simple high columnar epithelium that has a conspicuous brush border and
radial striations in the basal region of the cells. Their cytoplasm is avidly
eosinophilic. The nuclei are sharply defined, but variable in position in the
cell. The lumen of the tubule is irregular, either large and oval or slit-
like. Proximal convoluted tubules are especially numerous in the outer cortex
of the deer mouse kidney.
266
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TABLE 21-16. SEASONAL CHANGES IN THE HISTOLOGY OF THE LIVER OF Peromyscus maniaulatus .* VALUES IN THE
TABLE ARE MEANS ± SEM. SEX AND AGE GROUPS HAVE BEEN COMBINED
Month-
Year
Characteristics of Hepatocytes
Fatty Glycogen
Infiltration Depletion Autolysis
Pathology
Pigment Content
of KUpffer Cells
Lymphycyte
Infiltration
Hyperemia
Jul 1974 13
Aug 1974 20
Sep 1974 16
Oct 1974 14
Nov 1974 6
Dec 1974 4
Jan 1975 1
Mar 1975 6
May 1975 4
Jun 1975 7
Jul 1975 1
Aug 1975 4
Grand Mean ±
SEM (n = 12)
0.31 ± 0.13
0.25 ± 0.16
0.19 ± 0.19
0.29 ± 0.16
0.00
0.00
0.00
0.33 ± 0.21
0.00
0.00
0.00
0.00
1.62 ± 0.51 0.85 ± 0.52
0.65 ± 0.31 0.60 ± 0.17
1.62 ± 0.52 0.88+0.33
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.71 ± 0.29
0.83 ± 0.48
3.50 ± 0.87
1.00
1.33 ± 0.56
2.00 ± 0.47
3.17 ± 0.91
1.00
1.25 ± 0.63
0.46 ± 0.14
0.35 ± 0.17
0.00
0.92 ± 0.51 (13)
0.50 ± 0.22
0.25 ± 0.25
0.00
0.17 ± 0.17
3.50 ± 0.78
1.29 ± 0.61
0.00
2.75 ± 1.30
0.85 ± 0.22
0.60 ± 0.17
0.40 ± 0.20
0.79 ± 0.39
0.50 ± 0.22
0.00
0.00
0.17 ± 0.17
0.00
0.57 ± 0.57
0.00
0.25 ± 0.25
1.69 ± 0.60
2.00 ± 0.46 (19)
4.62 ± 0.20
3.57 ± 0.39
2.33 ± 0.95
5.00 ± 0.00
0.00
3.67 ± 0.67
0.75 ± 0.75
0.14 ± 0.14
0.00
0.75 ± 0.75
0.11 ± 0.04 0.32 ± 0.18 1.43 ± 0.28 0.85 ± 0.33
0.34 ± 0.09 2.04 ± 0.52
* Each histological characteristic in the table has been estimated on a scale of 0 to 5, according to
which 0 = absent; 1 = mild; 3 = moderate; and 5 = heavy or pronounded. The values in the table are
averages of values assigned to the animals in each monthly sample.
-------
2. Straight region--This region is similar structurally to the convo-
luted region, but is somewhat smaller in diameter. The epithelium is also
lower, consisting of simple cuboidal or low columnar cells which have the same
high affinity for eosin, basal striations, and brush border. The lumen is
round or oval. These tubules lie in clusters within the inner cortex and at
the junction of the cortex with the medulla.
Loops of Henle are very narrow, round tubules with a thin, simple
squamous lining.They occur in groups of 12 to 15 among thick limbs (proximal
and distal) and collecting tubules. They can be recognized by their small
diameter and the fact that they are accompanied by vasa rectae, capillaries
filled with red blood cells. (This portion of the renal tubule is tradi-
tionally called the thin limb of the loop of Henle.)
The distal portion of the renal tubule, like its proximal counterpart,
has a convoluted and a straight region, the latter being the ascending thick
limb of the loop of Henle.
1. Convoluted region—This region of the tubule is round in section and
somewhat smaller than its proximal counterpart. It occurs in the same general
area, however. Groups of these tubules are frequently interspersed with
groups of proximal convoluted tubules in the cortex. They are recognized by
their simple cuboidal to low columnar epithelium, the cells of which are
characteristically irregular in height and have poor affinity for eosin,
granular cytoplasm, and lack of brush border. The lumen of this region of the
renal tubule is slit-like and stellate-shaped in section.
2. Straight region--Histologically, this region of the tubule resembles
the distal convoluted region, but is somewhat smaller in diameter. It is
lined by a simple squamous or low cuboidal epithelium, consisting of poorly
stained, granulated cells. In contrast to the convoluted region of the distal
tubule, the lumen is round, generally empty, and has an epithelial lining of
uniform height. Straight regions of the distal tubule occur in the outer
medulla and bordering inner cortex.
Renal tubules are connected with a system of collecting tubules including
collecting and papillary ducts.
1. Collecting ducts—These occur in the medulla and extend into the
cortex as medullary rays to pick up the more peripheral renal tubules. They
are round in section and relatively small with a simple cuboidal (in some
places almost squamous) lining. The epithelium is characterized by its
irregular height, and its cells by their lack of staining (vacant cytoplasm);
they have well defined cellular membranes and contain a large, well defined
nucleus.
2. Papillary ducts--These are large, round or oval tubules that are
lined with a simple low cuboidal or columnar epithelium. Epithelial cells are
similar to those lining the collecting ducts, but may also contain a few
stained granules. The lumen is small and polygonal in shape. Papillary ducts
are confined to the medulla, especially to the papilla.
268
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Seasonal Changes in the Histology of the Kidney of the Deer Mouse
Seasonal changes in the histology of the kidney are compiled in
Table 21.17. The data suggest that proteinuria commonly occurs in the kidney
of deer mice; that concretions are rare; and, as expected, that hyperemia is a
constant feature of the kidney.
We found many cases (n = 10) in which blood had pooled in the kidney for
some time. Blood vessels were choked with poorly stained, refractile erythro-
cytes and hemosiderin. We suspect that these are fixation artifacts. Consis-
tent with these observations is the fact that many of the kidneys were poorly
fixed. Pycnosis, for example, was commonly widespread, particularly in the
deeper regions of the kidney.
The kidney frequently (44.4 percent) exhibited pathological changes. The
incidence of pathology exceeds 50 percent of the sample in September, October,
and November, 1974 and in May, 1975. The most common pathology (28.4 percent
of our sample) was focal lymphocyte infiltration. In only five cases did lympho-
cytes surround foreign bodies (bacilli). However, they were frequently accom-
panied by other types of inflammatory cells, such as macrophages, neutrophils,
and in one case plasma cells. Lymphocytic infiltration was confined to the
renal cortex and the distribution of lymphocytes there suggests that they arose
by perivascular cuffing.
Epithelioid replacement of renal tubules occurred in three mice. In
these animals, the renal tubules were solid cords of cells encased by lympho-
cytes and neutrophils. The kidneys of two other mice exhibited intertubular
edema.
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269
-------
TABLE 21.17. SEASONAL CHANGES IN SELECTED ASPECTS OF THE KIDNEY OF Peromysaus mani.cula.tus.* SEX AND AGE CLASSES ARE COMBINED IN EACH MONTH
Month-
Year
Jul 1974
Aug 1974
Sep 1974
Oct 1974
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Jan 1975
Mar 1975
May 1975
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Number of
n
13
19
15
11
6
3
1
6
4
2
1
PCT
0
7
8
7
2
1
1
2
0
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1
PCT-I
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1
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0
0
0
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Mice with Protein In*
DCT
0
1
8
5
3
2
0
3
1
1
1
DCT- 1
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0
0
0
0
0
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0
0
0
LH
0
0
2
0
0
0
0
0
0
0
0
RC
0
1
0
0
0
0
0
0
0
0
0
Number of Mice with
Concretions In*
PCT
2
0
1
0
0
0
0
0
0
0
0
PCT-I
2
0
0
0
0
0
0
0
0
0
0
LH
1
0
0
0
0
0
0
0
0
0
0
Degree of
C
2.56 ± 0.31
(9)
3.15 ± 0.31
(10)
2.46 ± 0.41
(13)
1.25 ± 0.53
(8)
2.00 ± 0.89
(5)
4.00
(2)
2.00
2.00 ± 0.82
(4)
3.00
(3)
4.00
(1)
4.00
(2)
M
2.33
(3)
2.50
(2)
1.25 ±
0.72(4)
0.00
(4)
0.00
(2)
0.00
(1)
3.00
(1)
Hyperemia In*,
(C)(M)
3.00 ± 0.58
(4)
3.78 ± 0.22
(9)
1.33
(3)
0.86 ± 0.40
(7)
0.67
(3)
4.00
(1)
2.00
(3)
4.00
(1)
2.00
(1)
2.00
(2)
,t
Whole Kidney
2.68 ± 0.28
3.45 ± 0.20
2.53 ± 0.37
1.45 ± 0.39
2.00 ± 0.73
4.00
2.00
2.33 ± 0.61
3.25 ± 0.48
3.00
3.00 ± 0.58
Mice in
Sample with
Pathological
Kidneys
5
4
10
6
5
0
0
1
3
1
1
* C = cortex; (C)(M) = cortex and medulla combined; DCT = distal convoluted tubule; DCT-I = inner (straight) region of distal convoluted tubule;
LH = loop of Henle; M = medulla; PCT = proximal convoluted tubule; PCT-I = inner (straight) region of proximal convoluted tubule; RC = renal
corpuscle.
t Hyperemia was estimated on a scale of 0 (none) to 5 (very marked). Values are means ± SEM (n).
-------
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273
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SECTION 22
SEASONAL CYCLES IN BODY COMPOSITION, ORGAN SYSTEM FUNCTION
AND ENERGETICS OF THE WESTERN MEADOWLARK IN SOUTHEASTERN MONTANA:
A REPORT OF PROGRESS
M. L. Morton, R. A. Lewis and E. Zerba
ABSTRACT
Results of a portion of a baseline investigation
of grassland birds in southeastern Montana are
reported and evaluated in relation to future energy
development.
Mensural and compositional characteristics of the
whole body and of selected organs of the western
meadowlark (_Stwcne11a neglecta} are treated. The
types of systems and functions represented are those
that reflect the condition, vigor, nutritional state,
metabolic state and resource relationships of the
study population.
PREFACE
This paper is a synopsis of a small portion of our baseline investigation
of grassland birds in southeastern Montana in relation to further energy
development. A monograph is in preparation that will more fully treat the
mensural and compositional characteristics of the whole body and of selected
organs of the western meadowlark (Sturnella negleata) and four other species
of birds. The monograph includes baseline information on postnatal development,
reproductive biology, histology, population dynamics and community structure.
Results reported herein together with those of the overall investigation
will permit evaluation of the above aspects of avian organization as a func-
tion of age, sex, season, and physiologic state in relation to the physical
and biotic environment. The present analysis treats a number of types of
systems and functions that reflect the condition, vigor, nutritional state,
resource relationships, metabolic state, and net energy balance of the sample
274
-------
population as a function of environmental information or gradients, including
potential changes in air quality and other anthropogenic disturbances related
to future energy development in southeastern Montana. The data are essen-
tially baseline in nature.
We believe that future air pollution effects research in the northern
Great Plains will rely heavily upon such results. The work in its entirety
represents a baseline evaluation of annual cycle and life cycle phenomena and
the mechanisms that regulate such functions in relation to potential vulner-
ability to air pollution induced stress. See Lewis et al. (1976), for a more
complete statement of objectives.
INTRODUCTION
During the first 3 years of the study (1974-76) large numbers of avian
specimens were collected in Rosebud and Powder River Counties. Previously we
reported preliminary information obtained on these specimens (Lewis et al.,
1976, 1978). The available data on western meadowlarks have now been consoli-
dated in the present report in order to characterize more fully the status of
this species in the southeastern Montana environment at a baseline or pre-
pollution condition.
Data on bodily composition of all vertebrate classes have powerful
utility as measures of condition, metabolic and energetic stress, and life
history strategies (Connell et al., 1960; Helms, 1968; Helms and Smythe, 1969;
McNeil, 1969; Myrcha and Pinowski, 1970; Telford, 1970; Adolph and Hegeness, 1971;
Aleksiuk and Stewart, 1971; Perkins and Dahlberg, 1971; Krulin and Sealander,
1972; Morton et al. , 1974; Morton, 1975; Fehrenbacher' and Fleharty, 1976).
Changes in body composition are also adaptive and highly predictable
measures of age and phase of the annual cycle (Morton, 1975; 1976). Thus
deviations from normal patterns that are deleterious, should be easily
detected and should provide strong evidence for presence of environmental
perturbation. Indeed, we have good evidence (Lewis et al • , unpublished data)
that during certain phases of the annual cycle (e.Q-> reproduction, growth,
molt), that at least some of the species under investigation are living close
to the limit of their resources or are under substantial environmental stress
that may condition their responses to pollutants. Special attention will be
given to these processes and their implications. We hope eventually to deter-
mine the extent of pollution-related effects on small birds in the study area
and to distinguish, to the extent possible, between direct and indirect air
pollution effects and the effects of other human activities that might other-
wise tend to confound our results (e.g., effects of coal-mining, water use,
increased human population density, use of herbicides and pesticides, eta.).
Western meadowlarks and other grassland birds are potentially valuable
monitors of environmental disturbance because they live in a relatively simple
environment wherein the effects of abiotic factors should be only slightly
buffered by the biotic community and thus easily discerned (Weins, 1973; 1974).
This relationship has been amenable to development of useful models for energy
flow in grassland populations (Wiens and Innis, 1974).
275
-------
The annual cycle is the fundamental temporal unit in most long-lived
animals. The essential processes of life such as winter maintenance, migra-
tion, reproduction, and molt occur within a highly specific schedule such that
conflict between major energy-requiring events and environmental fluctuations
and among the events themselves is minimized. The precise duration and timing
of annually occurring functions are therefore essential and comprise key loci
for selection pressures that shape evolutionary tendencies toward optimizing
an individual's fitness (Mewaldt and King, 1977).
Some of the organs examined in this study, vary appreciably in mass
during the season (spleen, liver, gonads) whereas others (heart, lungs,
kidneys) do not. Both types could have considerable worth as pollution moni-
toring systems. For example, significant deviation from normal values for
static organs could be indicative of pathology. Organs that cycle do so
within highly specific temporal boundaries. Departure from the usual schedule
might be a harbinger of environmental perturbation. In either case, precise
diagnosis of pathology will depend upon corroboration through additional
assessment of function. Such evaluations would logically involve biochemical
and histochemical characters and microstructural parameters such as cellular
size, organization, and number. Conducting evaluations of this type is
usually expensive in time and money. Highly developed techniques and special-
ized equipment are also required. Collection of organ weights, however, is a
relatively straightforward process that can be accomplished by any worker with
simple equipment and basic biological skills. Changes in organ weights signal
qualitative or quantitative changes in organ structures and function and these
indicate bases for further analysis of tissues that we have banked.
MATERIALS AND METHODS
Specimens were collected with a shotgun, sealed in plastic bags with appro-
priate labels, and retained frozen until analysis. In many specimens internal
organs were removed for weighing and/or fixation. In such cases, dissections
were performed at the field vehicle immediately after shooting or the specimen
was placed on ice and transported to our field laboratory at Fort Howes Ranger
Station, Powder River County, for dissection. Collecting was done in Rosebud
County along Rosebud, Cow and Greenleaf Creeks within 15 km of Colstrip. Some
specimens were collected in Powder River County, usually within 20 km of Fort
Howes.
In the laboratory we employed standard biometric methods and whole car-
cass and organ analysis to evaluate compositional and mensural changes as a
function of age, sex, season, and physiologic state in relation to the physi-
cal and biotic environment, (for details, see Lewis and Morton, 1976; Lewis et
al. , 1976, and below).
Frozen specimens were thawed and analyzed as follows:
1. Molt classifications were determined by detailed observation, the
method being adapted from Morton et al ., (1969). The just-thawed
carcass was then dissected and processed all or in part as follows:
276
-------
2. Liver—wet weight, dry weight, fat content, fat-free dry weight; to
tissue bank.
3. Left kidney—wet weight, dry weight; to tissue bank.
4. Adrenals—wet weight, return to carcass.
5. Thyroid—wet weight, return to carcass.
6. Gonads--wet weight of testes, ovaries and oviducts, return to
carcass.
7. Spleen—wet weight, return to carcass.
8. Gizzard—wet weight, return to carcass.
9. Integument (plumage)--dry weight (does not include beak or leg
scutes), return to carcass.
10. Carcass—dry weight (wet weight determined in field on date of
collection), dried tissue ground and aliquots used to determine fat
content; remainder to tissue bank.
Specific techniques used in making the measurements outlined above:
1. Wet weights of organs were made on a torsion balance as soon as they
were dissected free and blotted.
2. Grinding of carcasses for production of homogeneous aliquots was
done in a Model 4-E Quaker City Laboratory mill.
3. Fat content was determined by placing specimens in thimbles and
extracting them in a soxhlet apparatus with 1,2-dichloro-ethane for
24 hours. Samples were then dried at 75°C in convection ovens and
the fat-free dry weight recorded.
4. Total nitrogen was determined by the standard micro-Kjeldahl tech-
nique .
5. Caloric content was measured by combusting aliquots of dried speci-
mens in a Parr adiabatic calorimeter.
6. Mensural techniques on plumage and appendages were adapted from
Baldwin et al-, (1931).
RESULTS
The western meadowlark is monotypic and considered to be a sibling spe-
cies of the eastern meadowlark, S. magna (Lanyon, 1962). It breeds from the
277
-------
Great Lakes region westward from Mexico to southern Canada. Since the turn of
the century, in association with settlement and deforestation by man, there
has been a significant extension of its range to the northeast (Lanyon, 1956).
This expansion is apparently still progressing (Rohwer, 1973).
Meadowlarks may range into mountain parks and foothills but are better
known as inhabitants of prairies and grassy plains. They winter in parts of
Mexico and in southern U.S. border states but have been recorded as residing
the whole winter as far north as Miles City, Montana (Cameron, 1907). They may
begin arriving in Montana on spring migration in late February or early March
(Weydemeyer, 1973), but an 18-year average first arrival time for Custer County
was 30 March (Gross, 1958). Fall migration usually begins in October but a few
birds are known to have departed Montana and similar latitudes as late as
mid-November.
The time of reproduction is, of course, the single most critical period
in any organism's annual cycle. In the present study primary evaluation of
this period was made by the use of weights of reproductive organs used to
define the normal limits of gonadal development and the temporal limits of the
breeding period.
Gonads
Our collection of meadowlarks began in early April when the first indi-
viduals arrived in the study area. Most of the birds collected throughout
April were adult males, 126 of 145 or 87 percent. Testes of males collected
in April were often one-third to one-half the size of those collected during
May and June (Table 22.1); the size difference was highly significant (P <
0.01, t-test of means). The first males to arrive quickly established and
began defending territories. They were conspicuous because of their frequent
bouts of singing from elevated perches.
Females were not regularly present until May, at which time their ovaries
averaged more than 500 mg, significantly larger (P < 0.01) than those taken in
April (Table 22.2). In both sexes, considerable gonadal growth thus occurred
in many individuals after reaching the study area.
Judging from gonadal and oviduct weights, and follicular diameters,
adults of both sexes typically maintained reproductive function until mid or
late July (Tables 22.1 and 22.2). Gonadal involution in July was rapid,
spanning only a few weeks in the population that we sampled.
A few fledglings were present during the first half of June but they
could not be found with regularity until the last half of the month. There
was no detectable seasonal change in gonad weights of juveniles (Tables 22.1
and 22.3). Note that data on meadowlark juveniles are presented according to
sex. Differences in body size between the sexes were eventually evident to us
(see beyond), and the data were therefore segregated.
In most avian species, the right ovary and oviduct do not persist beyond
early development, and ovarian and oviduct weights referred to here include
only those of the left side. Oviduct size and functional status is primarily
278
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TABLE 22.1. SEASONAL CHANGES IN PAIRED TESTES WEIGHTS (mg) IN WESTERN
MEADOWLARKS COLLECTED NEAR COLSTRIP, MONTANA, 1974-1978
Adults
April
May
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
N
51
33
33
39
35
43
25
18
13
7
8
9
4
Mean
282.7
479.5
539.5
722.6
677.7
673.6
572.3
298.0
16.4
10.5
8.4
8.2
21.0
S.D.
139.2
189.7
152.3
146.8
176.6
183.3
220.0
257.5
7.1
4.4
1.4
3.2
20.2
N
--
--
2
12
8
14
11
29
34
23
6
Juveniles
Mean
--
--
4.8
3.6
3.9
6.7
8.0
5.2
4.3
3.6
4.0
S.D.
--
--
--
1.3
1.3
2.5
2.9
2.5
1.9
1.7
1.5
under the control of ovarian hormones although it does have independent vas-
cular and nerve supplies (see reviews by Lofts and Morton, 1973; Sturkie and
Mueller, 1976). The cycle seen in adult female meadowlarks (Table 22.2),
therefore, reflects seasonal changes in secretion of ovarian hormones (estro-
gens and progesterone). The diminutive size and lack of change in oviducts of
juveniles indicates that their ovaries were nonsecretory from June through
September (Table 22.3).
Body Weights and Body Composition
Body weights afford the most convenient standard for comparisons of body
size and energetics (Baldwin and Kendeigh, 1938). Weights of meadowlarks
tended to decrease during the season and did not swing upward until August
(Table 22.4). Body weights of juveniles increased through time as one would
expect from normal growth. Both sexes of juveniles were approximately equiva-
lent to their adult counterparts in body mass by September (Table 22.4).
279
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TABLE 22.2. SEASONAL CHANGES IN WEIGHTS (mg) OF REPRODUCTIVE ORGANS AND DIAMETER (mm) OF PRE-OVULATORY
FOLLICLES IN ADULT WESTERN MEADOWLARKS COLLECTED NEAR COLSTRIP, MONTANA, 1974-1978.
April 1-15
16-30
May 1-15
16-31
June 1-15
to 16-30
oo
o
July 1-15
16-31
Aug. 1-15
16-31
Sept. 1-15
16-30
Oct. 1-15
N
1
12
35
33
22
15
15
13
6
9
10
5
1
Ovaries
Mean
81.10
107.78
560.00
537.79
414.65
519.35
108.97
42.05
15.05
13.45
20.04
21.36
10.91
S.D.
--
49.76
839.50
900.41
520.63
694.80
218.07
16.61
10.14
4.86
11.54
6.34
— —
N
1
13
35
32
21
17
17
12
5
8
9
3
1
Oviducts
Mean
357.90
275.23
1864.61
1575.46
1102.49
1319.94
356.02
128.14
57.08
40.36
51.55
22.41
23.54
Pre-ovulatory Follicles
S.D.
--
166.55
1824.54
1697.50
1412.55
1503.95
826.28
79.05
29.39
21.57
30.67
13.76
__
N Mean S.D.
__
58 2.22 0.55
141 3.53 2.28
140 3.45 2.53
63 3.68 2.99
63 3.86 2.91
19 3.60 3.21
6 1.08 0.48
—
__
__
__
-------
No large seasonal fluctuations in measured body components were apparent
in adult (Table 22.5) or juvenile (Table 22.6) meadowlarks, but a few trends
occurred. Mean body weight, for example, of adult males decreased during the
last half of the season. This corresponded temporarily with postnuptial molt
(see beyond) but the possible physiological significance of this relationship
may be complex in that a similar trend did not occur in females.
TABLE 22.3. SEASONAL CHANGES IN WEIGHTS (mg) OF REPRODUCTIVE ORGANS IN FEMALE
JUVENILE WESTERN MEADOWLARKS COLLECTED NEAR COLSTRIP, MONTANA,
1974-1978
June
July
Aug.
Sept.
16-20
1-15
16-31
1-15
16-31
1-15
16-30
N
11
8
7
8
16
19
15
Ovaries
Mean
3.11
5.04
5.71
3.90
3.53
5.06
8.06
S.D.
1.78
3.08
2.61
2.77
1.85
2.75
6.70
N
13
8
7
9
11
18
15
Oviducts
Mean
7.00
10.61
10.75
8.32
9.71
10.36
9.95
S.D.
2.85
1.57
1.63
2.47
6.41
5.79
4.28
The nitrogen content of meadowlark carcasses (Table 22.7) and pectoral
muscle (Table 22.8) was remarkably constant.
Water content of adult males tended to be low in early April
(Table 22.5). Comparison of the early and late April means indicates that
they are marginally different (0.10 > P > 0.05). This could be a real effect
because newly arrived migrants are sometimes noticeably dehydrated (Zimmerman,
1965). Rehydration is probably complete within the first day after arrival,
however, and one must be able to determine exact arrival schedules of indivi-
duals to have confidence in the carcass composition data. Unfortunately we do
not have precise information on arrival times of individuals.
Apparently collections ceased before adults began premigratory fattening,
although the three males collected in October were fatter than birds collected
earlier (Table 22.5). October samples of juveniles did not have significantly
more fat than those of September (P < 0.05) when all data on juveniles are
treated together. Note that sex was undetermined in 42 juvenile specimens
but, because of its inherent value, data on their body composition is included
in Table 22.6).
281
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TABLE 22.4. SEASONAL CHANGES IN BODY WEIGHTS (g) IN WESTERN MEADOWLARKS NEAR COLSTRIP, MONTANA
1974-1978
00
NJ
Adult Males
April
May
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
N
76
50
46
46
42
51
28
29
22
12
12
12
3
Mean
118.67
116.61
113.66
114.70
112.93
110.56
110.54
110.89
115.21
118.53
116.79
118.88
131.70
S.D.
8.57
6.36
6.47
4.96
5.78
5.93
5.87
6.21
5.02
5.06
5.70
3.98
13.04
Adult Females
N
1
18
41
42
27
29
29
25
18
18
19
9
—
Mean
99.40
92.38
93.91
92.65
89.75
89.22
86.81
85.74
87.58
91.53
89.38
94.48
—
S.D.
6.56
6.76
8.99
6.67
9.61
5.69
5.96
6.77
5.29
4.59
3.61
—
Juvenile Males
N
--
--
2
14
15
33
31
46
44
33
11
Mean
--
--
69.67
87.83
94.28
98.89
104.23
107.33
110.34
115.21
121.366
S.D.
--
--
--
9.87
11.05
7.42
8.45
14.66
6.48
8.47
7.76
Juvenile Females
N
--
--
--
15
9
32
30
32
37
23
4
Mean S.D.
--
--
--
71.37
76.68
79.42
81.47
83.34
90.21
90.80
94.33
--
--
--
9.16
7.31
4.53
8.94
5.51
9.20
7.58
1.77
-------
TABLE 22.5. SEASONAL CHANGES IN BODY COMPOSITION OF ADULT WESTERN
MEADOWLARKS, 1974-1976
Water (% body weight) Lean (% body weight) Fat
MALES
April 1-15
16-30
May
June
July
Aug.
Sept.
Oct.
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
N
48
33
19
27
16
33
13
20
9
3
9
13
3
Mean
64.91
66.24
67.48
67.19
67.55
67.12
70.33
69.15
70.15
71.67
66.53
66.19
61.49
S.D.
6.31
1.87
1.89
2.52
1.96
1.62
3.64
3.13
2.15
2.15
11.68
3.47
2.85
N
46
30
18
25
16
21
12
20
6
3
9
12
3
Mean
29.44
28.66
28.58
27.96
28.09
28.14
24.97
26.17
25.11
24.05
25.92
27.54
27.97
3
2
1
2
1
1
3
2
3
2
4
3
1
S.D.
.17
.11
.52
.81
.99
.34
.16
.98
.10
.04
.88
.18
.27
N
46
30
18
24
16
31
12
20
6
3
9
12
3
(% body weight)
Mean S.D.
6.49
5.14
4.30
5.22
4.42
4.72
5.09
4.70
4.54
4.28
4.34
6.16
10.55
2.23
1.32
1.14
1.38
1.14
1.00
1.77
0.80
1.34
0.75
1.18
1.70
4.03
FEMALES
April
May
June
July
Aug.
Sept.
16-30
1-15
16-30
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
13
28
22
12
21
16
19
7
5
9
7
63.90
64.52
67.86
67.63
68.30
67.20
68.72
70.75
68.96
67.36
68.05
2.18
6.69
3.10
3.99
1.39
5.99
2.53
4.04
1.73
2.61
4.76
13
24
22
11
21
16
17
6
5
9
7
28.26
27.58
26.67
26.55
26.94
27.86
26.79
24.91
26.10
27.67
26.45
1
1
2
1
1
1
2
3
2
.12
.28
.53
.88
.31
.24
.31
.78
.90
2.01
23.48
13
24
22
11
21
16
17
6
5
9
7
8.08
6.78
4.82
5.46
4.82
5.01
4.46
3.94
4.93
5.28
5.73
2.08
2.30
0.93
0.86
0.93
0.84
1.32
0.90
1.40
2.30
2.41
283
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TABLE 22.6. SEASONAL CHANGES IN BODY COMPOSITION OF JUVENILE WESTERN
MEADOWLARKS, 1974-1976
MALES
June
July
Aug.
Sept.
Oct.
f
1-15
16-30
1-15
16-30
1-15
16-31
1-15
16-30
1-15
Water
N
2
5
6
21
17
33
30
27
11
(% body
Mean
69.60
72.30
68.30
70.09
71.62
70.09
69.42
67.76
64.16
weight)
S.D.
1.78
9.48
3.17
2.65
1.92
2.66
2.48
3.35
Lean
N
2
5
6
21
14
29
29
24
11
(% body
Mean
22.72
23.85
27.46
24.92
23.54
25.46
26.81
26.92
27.46
weight)
S
2
9
2
2
1
1
1
1
.D.
.16
.52
.55
.18
.83
.99
.46
.47
Fat
N
2
5
6
21
14
29
29
24
11
(% body
Mean
7.69
3.92
4.24
4.91
4.64
4.31
4.08
4.95
8.38
weight)
S.D.
1.55
1.26
1.43
1.09
0.87
0.95
1.60
4.02
FEMALES
June
July
Aug.
Sept.
Oct.
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
4
13
13
21
25
19
3
70.13
70.44
70.63
70.41
68.64
67.60
62.76
1.49
1.16
3.57
2.43
2.32
1.46
2.81
4
12
12
18
23
18
3
24.80
23.49
24.33
24.78
26.38
27.30
26.71
1
5
3
1
1
1
1
.95
.27
.99
.62
.78
.31
.32
4
12
12
18
24
18
3
5.20
6.03
5.00
4.44
4.44
4.99
10.53
1.07
5.00
1.92
1.03
1.18
1.10
4.14
SEX UNDETERMINED
July
Aug.
Sept.
1-15
16-31
1-15
16-31
1-15
16-30
4
3
9
5
5
13
70.54
70.55
72.79
69.91
70.73
67.42
3.15
4.82
4.83
1.57
1.46
1.37
4
3
9
5
5
13
24.12
24.99
22.83
24.04
25.05
26.51
2
4
4
5
1
.38
.82
.10
.55
.72
.82
4
3
9
5
5
13
5.35
4.46
4.21
4.04
4.10
5.30
2.61
0.41
1.74
1.09
1.02
1.52
Oct.
1-15
64.77 3.06
28.20 1.75
7.03 4.04
284
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TABLE 22.7. SEASONAL CHANGES IN CARCASS NITROGEN (PERCENT DRY WEIGHT) IN WESTERN MEADOWLARKS,
1974-1976
to
00
Adult Males
April
May
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
N
20
13
3
9
6
16
13
18
5
3
8
12
4
Mean
11.6
11.3
10.8
11.6
11.6
11.8
11.8
12.3
11.8
11.7
12.0
11.5
10.0
S
1
1
0
1
0
0
0
1
0
0
1
1
1
.D.
.0
.2
.5
.0
.7
.7
.7
.8
.7
.4
.0
.1
.5
Adult Females
N
3
10
5
8
14
13
9
6
1
9
4
--
Mean
10.6
11.4
11.7
11.5
11.6
11.7
12.1
11.7
11.6
11.5
10.4
—
S.D
1.
1.
1.
0.
1.
0.
0.
0.
--
1.
1.
--
•
1
0
1
9
2
9
7
3
6
6
Juvenile Males
N
--
--
1
4
4
16
6
19
27
21
7
Mean
—
--
11.9
11.7
11.7
12.4
11.5
12.2
12.0
12.1
10.8
S.D.
--
—
--
1.3
0.9
1.5
0.8
1.0
0.9
1.9
1.3
Juvenile Females
N
--
--
--
2
1
10
13
9
21
13
3
Mean
—
—
--
11.8
13.1
10.9
11.8
12.3
11.9
11.4
9.8
S.D.
--
—
--
--
--
1.6
0.8
1.6
0.8
1.3
2.2
-------
TABLE 22.8. SEASONAL CHANGES IN PECTORALIS NITROGEN (PERCENT DRY WEIGHT) IN WESTERN MEADOWLARKS,
1974-1976
00
Adult Males
April
May
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
N
12
11
7
11
9
10
10
7
2
3
5
7
3
Mean
13.4
13.3
13.0
13.1
13.0
13.5
13.0
13.7
13.6
12.7
12.8
13.8
12.5
S.
0.
0.
0.
1.
0.
1.
0.
1.
--
0.
0.
0.
1.
D.
6
6
1
0
8
0
6
8
6
6
8
2
Adult Females
N
--
4
10
12
7
7
9
5
6
--
5
6
—
Mean
--
13.0
13.1
13.8
12.9
12.2
13.0
12.6
12.7
__
12.9
12.9
—
S.D
--
0.
0.
2.
0.
1.
0.
0.
0.
--
0.
0.
—
•
2
6
4
7
3
6
6
4
9
8
Juvenile Males Juvenile Females
N Mean
--
__
__
__
__
2 13.3
2 13.2
8 13.1
11 13.7
16 12.7
17 13.0
13 12.7
11 13.3
S.D. N Mean
__
__
__
__
__
1 20.4
1 12.8
0.5 6 12.8
1.1 8 12.8
0.8 16 12.9
0.8 24 13.6
0.7 10 13.2
0.6 3 12.8
S.D.
--
--
--
--
--
--
--
0.6
1.2
1.6
2.2
0.5
0.4
-------
TABLE 22.9. SEASONAL CHANGES IN CALORIES PER GRAM ASH-FREE DRY WEIGHT (CARCASS AND INTEGUMENT) OF
WESTERN MEADOWLARKS, 1974
July
Aug.
Sept.
Adult Males
N Mean S.D.
16-31 1 4707
1-15
16-31 1 4783
1-15 1 4853
16-30
Adult Females
N
1
1
--
1
2
Mean S.D.
4865
4644
--
5335
4897
Juvenile Males
N
2
5
11
6
7
Mean
4845
4914
4851
4885
5010
S.D.
--
65
114
94
243
Juvenile Females
N
4
4
10
13
5
Mean
4922
4851
4865
5001
5015
S.D.
175
85
72
175
137
(~0
00
-------
TABLE 22.10. SEASONAL CHANGES IN LIVER WET WEIGHT (mg) IN WESTERN MEADOWLARKS, 1974-1976
00
Adult Males
April
May
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
N
44
31
14
18
12
26
9
17
5
5
10
13
3
Mean
2752.5
3051.4
2780.0
2689.0
2626.5
2733.4
2705.2
3218.3
2809.1
3572.9
3284.2
3313.8
3585.0
S.D.
691.4
346.6
458.8
349.0
438.6
343.6
450.7
570.4
436.5
947.4
422.8
536.6
780.3
Adult Females
N
--
12
26
16
9
16
7
16
5
5
10
6
—
Mean
--
2252.6
2681.1
2662.8
2611.1
2604.6
2580.8
2439.1
2361.9
3075.2
2551.4
2682.0
—
S.D.
--
285.7
610.3
519.4
488.1
472.8
366.2
369.8
210.5
386.0
284.9
296.6
—
Juvenile Males
N
--
--
--
--
2
5
4
9
13
30
36
27
12
Mean
--
--
--
--
1954.2
2640.2
3093.7
3022.1
3332.8
3284.7
3316.5
3522.4
3599.8
S.D.
--
--
--
--
--
315.5
388.9
611.9
393.9
620.8
511.7
627.4
948.1
Juvenile Females
N Mean
__
--
__
--
__
5 2222.2
1 2732.1
13 2724.0
11 2650.1
18 2635.3
28 2755.4
19 2789.6
2 2799.2
S.D.
--
--
--
--
--
300.2
--
426.0
377.8
221.8
479.4
266.7
— —
-------
A few data were obtained on caloric values of meadowlark tissue
(Table 22.9). These values were fairly consistent among themselves but, in
general, they fell slightly below those reported previously for birds (Brisbin,
1968), Skar et al., 1972). Constancy in caloric value has usually been found
in animal tissues (Richman, 1958; Richman and Slobodkin, 1960; Golley, 1961).
These data, and those of other species in this study, support the generaliza-
tion that nonfat components of migratory birds are fairly homeostatic consider-
ing the great seasonal swings that occur in their energy expenditures (see
Odum et al., 1965).
Liver
Weights and chemical composition of livers were measured in nearly 600
meadowlarks (Tables 22.10- 22.14). Livers of adult males tended to enlarge
TABLE 22.11.
SEASONAL CHANGES IN LIVER COMPOSITION OF ADULT WESTERN
MEADOWLARKS, 1974-1976
Water % liver weight)
Males
April
May
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-30
" -15
1 31
1-15
16-31
1-15
16-30
1-15
N
42
26
14
17
12
26
9
17
6
5
10
12
3
Mean
70.40
71.59
70.08
70.51
69.69
70.78
70.26
71.86
71.72
71.59
71.93
70.31
68.63
S
2
4
1
1
1
2
1
1
1
1
0
1
0
.D.
.42
.40
.59
.58
.07
.06
.98
.44
.74
.49
.88
.49
.57
Lean
N
42
25
14
17
12
26
9
14
4
4
8
11
3
(% liver weight)
Mean
24.67
24.31
25.86
25.53
25.29
24.32
24.57
24.47
23.69
24.67
24.46
24.58
25.55
S.D.
2.61
4.06
1.24
1.22
2.83
1.35
2.22
1.33
1.75
2.32
0.85
1.07
2.52
Fat (% liver
' N
42
25
14
17
12
26
9
14
4
4
8
11
3
Mean
4.95
4.14
4.06
3.96
4.40
4.90
5.17
3.59
4.21
3.88
3.83
5.07
5.83
weight)
S.D.
2.05
1.41
1.10
0.69
1.05
1.53
0.69
0.72
1.05
0.68
1.01
1.48
2.80
Females
April
May
June
July
Aug.
Sept.
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
11
22
16
9
15
9
16
5
5
10
8
70.35
70.28
70.40
71.52
71.51
70.85
72.07
71.94
71.70
71.06
69.82
1
2
1
2
2
2
2
1
1
1
1
.49
.20
.72
.55
.17
.26
.13
.82
.72
.40
.74
11
21
16
9
13
9
16
4
5
9
8
25.32
25.03
24.85
23.42
23.02
24.45
23.56
24.03
24.14
24.88
25.12
1.39
1.36
2.67
2.45
2.87
1.69
2.21
1.66
1.13
1.00
1.43
11
21
16
9
13
9
16
4
5
9
8
4.34
5.09
4.96
5.06
5.28
4.81
4.37
4.10
4.16
4.31
5.46
0.45
1.39
1.31
0.68
1.25
1.30
1.10
1.31
1.68
0.84
1.69
289
-------
TABLE 22.12.
SEASONAL CHANGES IN LIVER COMPOSITION OF JUVENILE WESTERN
MEADOWLARKS, 1974-1976
Males
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
Water
N
2
5
4
17
13
30
36
27
11
% liver
Mean
73.27
71.34
72.27
72.69
72.71
72.06
71.56
69.79
68.53
weight)
S.D.
— _
1.26
0.19
1.31
2.03
1.29
1.66
2.87
2.19
Lean
N
2
5
4
16
11
28
34
25
11
(% liver
Mean
23.27
23.88
23.62
23.72
24.11
24.39
24.36
25.32
25.12
weight)
S.D.
1.73
1.22
1.36
1.62
1.53
1.39
1.58
1.79
Fat (%
N
2
5
4
16
11
28
34
25
11
liver
Mean
3.46
4.78
4.12
3.64
3.73
3.56
4.03
5.06
6.16
weight)
S.D.
—
0.58
1.05
0.80
0.81
0.98
0.86
2.21
3.36
Females
June
July
Aug.
Sept.
Oct.
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
5
1
14
11
18
28
19
3
70.12
73.54
72.36
71.85
71.44
71.06
70.22
70.72
0.79
—
1.15
1.92
1.46
1.67
2.08
0.58
5
--
13
9
17
28
18
2
23.23
21.46
23.40
24.85
25.23
24.84
25.13
25.18
3.11
--
1.03
1.46
1.30
1.44
1.47
— —
5
1
13
9
17
28
18
2
6.64
5.04
4.26
3.69
3.31
4.11
4.71
4.43
2.94
--
1.00
1.09
0.58
0.96
1.68
— —
during the last two months of the collection period (Table 22.8). These were
matched in size by those of juvenile males. This change in liver size of
males was unmatched by seasonal alterations in liver composition; the reported
components of the liver remained remarkably constant in terms of relative
proportions.
Because of its central role in intermediary metabolism, especially as a
storage site for nutrients, the liver may vary considerably in mass according
to diet, feeding habits, and energy expenditures (Oakeson, 1953; Hanson, 1962;
Pendergast and Boag, 1973; Ankney, 1977).
Of pertinence to the present study, seasonal changes in liver mass have
been noted in migrants in that they decrease sharply during spring migration
(Oakeson, 1953). Liver weight may also decrease during incubation especially
in species, such as geese, that exhibit reduced rates of feeding at that time
(Ankney, 1977).
290
-------
TABLE 22.13. SEASONAL CHANGES IN LIVER DRY LIPID INDEX (FAT AS PERCENT DRY WEIGHT) IN WESTERN
MEADOWLARKS, 1974-1976
K3
Adult Males
April
May
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
N
48
27
17
26
13
31
12
15
5
4
8
11
3
Mean
20.32
19.19
15.57
14.60
16.91
19.61
20.97
15.17
17.90
16.03
15.75
20.63
23.71
S.D.
12.56
8.18
4.58
3.15
4.70
6.36
4.52
4.12
4.13
4.08
4.60
6.45
14.04
Adult Females
N
--
11
23
18
3
15
10
20
6
5
9
7
__
Mean
--
17.44
20.45
18.65
21.68
27.91
22.17
18.78
18.46
17.34
17.38
22.09
—
S.D.
--
1.83
6.03
5.11
6.13
16.61
7.54
6.64
5.12
7.11
3.58
7.63
—
Juvenile Males
N
--
--
--
--
2
5
5
19
14
29
34
24
10
Mean
--
--
--
--
15.22
20.27
18.00
16.93
16.32
14.69
16.43
20.10
25.32
S.D.
--
--
--
--
--
4.07
4.84
5.14
3.90
4.59
3.79
8.17
18.10
Juvenile Females
N Mean
__
__
__
__
__
5 30.66
8 18.73
8 18.21
12 14.35
21 14.68
25 17.07
12 17.98
3 23.16
S.D.
--
--
--
—
—
19.59
5.35
5.50
2.75
4.03
6.05
5.24
9.64
-------
TABLE 22.14. SEASONAL CHANGES IN LIVER NITROGEN (PERCENT DRY WEIGHT) IN WESTERN MEADOWLARKS, 1974-1976
NO
Adult Males
April
May
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
N
12
18
5
4
1
6
6
5
8
3
5
7
1
Mean
13.0
12.9
12.7
12.7
12.6
12.8
12.7
12.5
13.0
12.5
13.0
12.9
11.0
S.D.
0.6
0.6
0.5
0.4
--
0.4
0.5
0.6
0.8
0.3
0.6
0.7
—
Adult
N
--
6
8
3
2
5
7
9
3
4
5
4
—
Females
Mean
--
13
13
12
12
12
12
12
12
13
12
13
_
.0
.1
.7
.6
.9
.9
.9
.6
.1
.5
.1
_
S
-
0
0
0
-
0
0
0
0
1
0
0
_
.D.
-
.8
.3
.4
-
.4
.2
.7
.5
.1
.3
.5
_
Juvenile Males
N Mean S.D.
__
--
--
__
--
1 11.8
1 13.4
11 12.7 1.0
7 12.7 0.4
13 13.0 0.6
25 13.1 0.7
10 13.0 0.6
5 12.5 0.7
Juvenile Females
N Mean S.D.
__
__
__
—
__
1 12.9
1 12.7
4 13.5 0.7
4 12.5 0.2
7 13.3 0.5
10 13.0 0.7
5 13.0 0.3
2 11.8
-------
Other Organs
Other organs of meadowlarks that were weighed routinely included heart
(Table 22.15), lungs (Table 22.16), spleen (Table 22.17), adrenal glands
(Table 22.18), thyroid glands (Table 22.19), and left kidney (Table 22.20).
One kidney was weighed because there is no weight asymmetry between left and
right kidneys in birds (Johnson, 1968). Kidneys were also examined for nitro-
gen content (Table 22.21). Spleens tended to become much larger as the season
progressed. Spleens in September were, for example, nearly twice as large as
those of April in both adult sexes, a difference that was significant in both
cases (P < 0.05). This is at least partly related to increased production of
blood (and perhaps immune bodies) during molt.
The highly practical aspects of understanding cardiac function has led to
its intensive study in a wide range of higher animals. In birds a rather
complete review of heart anatomy and physiology has been completed by Sturkie
(1976) . Heart weights have been reported for many avian species (Hartman,
1955; Brush, 1966), There seems to be a predictable, but allometric relation-
ship between heart size and body size. Hearts are relatively larger in
smaller birds. In birds of the approximate size utilized in the present study
the heart would be expected to comprise 1 percent or more of body weight.
Environmental factors, such as high altitude, are known to affect heart size
in birds (see review by Carey and Morton, 1976), but regular seasonal oscilla-
tions are unreported. An apparent decrease of heart weight throughout the
breeding season in adult female meadowlarks is not readily explanable by us.
Splenic function has been poorly studied in birds, especially wild birds.
However, it is known to receive about 3-4 percent of cardiac output in chickens
(Sapirstein and Hartman, 1959). And it may vary seasonally in mass in both
sedentary (Riddle, 1928b) and migratory (Oakeson, 1953) birds. We find no
reported data on breeding birds that can be used for comparative purposes with
our data.
Size of adrenal glands varies with species, sex, age, health and a host
of factors related to physical stress such as temperature, disease, vitamin
deficiency, and exercise (see review by Ringer, 1976a). Daily and seasonal
rhythmicities in function are known to occur, particularly in relation to
photocycle. Measurement of these functions cannot be determined reliably by
whole gland weight; plasma or urinary corticosteroid titers are required (see
review by Assenmacher, 1973). Cytologic changes occur seasonally in adrenals
of migratory birds (Burger, 1938; Fromme-Bouman, 1962; Lorenzen and Farner,
1964).
The avian thyroid is paired and lobes are located in the neck next to the
great blood vessels. As in mammals, the gland is involved in a wide range of
functions such as growth, heat production, carbohydrate metabolism, and sexual
maturation. It also affects migratory behavior and onset of molt in some
birds and growth of young feathers (Assenmacher, 1973).
293
-------
TABLE 22.15. SEASONAL CHANGES IN HEART WET WEIGHT (mg) IN WESTERN MEADOWLARKS, 1974-1976
Adult Males
April
May
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
N
50
26
13
20
14
29
11
18
7
3
10
13
3
Mean
1560.03
1604.48
1575.75
1537.21
1393.93
1422.66
1397.69
1381.65
1311.71
1262.97
1372.38
1408.00
1793.30
S.D.
287.80
180.23
204.36
137.63
138.43
189.06
197.66
224.40
157.49
82.15
197.86
346.78
320.03
Adult Females
N
--
12
25
17
8
14
12
19
5
5
10
8
—
Mean
--
1257.81
1198.23
1132.81
1020.63
1030.04
1035.21
977.65
976.44
999.94
975.02
1067.71
—
S.D.
--
103.50
163.03
147.89
80.08
194.29
91.50
315.30
118.04
158.75
110.40
127.30
—
Juvenile Males
N
—
--
--
--
2
6
4
20
17
32
34
27
10
Mean
--
—
--
—
749.20
786.10
1176.94
1138.90
1169.20
1131.30
1224.40
1290.20
1383.10
S.D.
--
--
--
—
--
176.90
390.60
298.40
153.40
141.00
171.90
151.30
77.70
Juvenile Females
N Mean
__
--
__
__
__
5 773.40
1 723.00
13 957.20
13 877.10
18 898.50
24 972.43
19 1017.30
2 1053.90
S.D.
--
--
--
--
--
150.50
--
146.90
168.30
131.10
148.80
117.30
—
-------
TABLE 22.16. SEASONAL CHANGES IN WET WEIGHT (mg) OF BOTH LUNGS IN WESTERN MEADOWLARKS, 1974-1976
Adult Males
April
May
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1=15
16-30
1-15
N
31
19
6
13
8
19
7
13
4
4
8
12
3
Mean
1678.7
1663.4
1801.0
1601.8
1516.7
1584.9
1670.2
1786.5
1551.4
1689.4
1585.3
1501.6
1621.4
S.D.
224.0
227.1
325.3
180.2
246.9
214.1
289.5
316.1
249.9
165.9
105.3
261.1
286.6
Adult Females
N
8
20
14
8
11
11
13
4
4
7
5
—
Mean
1351.7
1227.8
1265.3
1198.7
1154.1
1131.0
1254.9
1122.8
1245.4
1157.5
1158.4
__
S.D.
142
143
172
205
238
182
191
64
222
119
118
—
.1
.0
.6
.0
.8
.7
.2
.0
.7
.3
.6
Juvenile Males
N
--
--
1
3
2
17
12
27
29
24
8
Mean
--
--
928.4
1096.8
1555.9
1415.0
1381.8
1435.0
1419.5
1453.9
1453.2
S.D.
--
--
--
313.1
--
196.0
179.0
190.5
196.2
225.3
330.4
Juvenile
N
--
--
--
3
1
11
11
18
21
17
3
Females
Mean
--
--
--
1013
937
1298
1046
1147
1127
1074
1064
.4
.9
.7
.6
.6
.2
.0
.7
S.D.
--
--
--
145.0
--
617.1
151.7
453.8
215.1
156.2
30.4
-------
TABLE 22.17. SEASONAL CHANGES IN SPLEEN WET WEIGHT (mg) IN WESTERN MEADOWLARKS, 1974-1976
Adult Males
April 1-15
16-30
May 1-15
16-31
June 1-15
16-30
NJ
S July 1-15
16-31
Aug. 1-15
16-31
Sept. 1-15
16-30
Oct. 1-15
N
47
29
36
32
30
41
20
21
16
6
7
11
1
Mean
141.7
121.0
130.3
174.7
120.8
180.8
180.0
243.4
268.8
446.1
235.7
287.5
164.9
S.D.
111.1
94.3
86.2
209.6
100.8
131.0
167.0
121.1
238.5
467.9
130.2
165.3
—
Adult Females
N
1
14
28
30
22
13
17
18
9
9
10
8
0
Mean
109.4
137.1
152.1
136.9
117.2
141.5
141.8
152.8
134.5
173.1
191.5
221.7
—
S.D.
--
104
128
98
74
81
105
101
86
103
67
100
—
.0
.0
.7
.7
.1
.0
.3
.8
.4
.6
.0
Juvenile Males
N Mean
__
__
__
__
__
13 129.5
15 70.3
25 130.3
18 218.2
25 182.8
28 178.6
24 140.7
11 201.7
S.D.
--
--
—
--
--
126.6
31.2
104.2
208.3
199.3
175.8
85.5
115.5
Juvenile Females
N Mean
__
__
__
--
__
16 77.9
8 70.0
31 178.9
11 193.7
14 156.8
25 138. 5
19 178.7
4 162.6
S.D.
—
--
--
--
--
34.8
29.2
254.1
198.0
158.6
71.6
138.9
102.3
-------
TABLE 22.18. SEASONAL CHANGES IN WET WEIGHTS (mg) OF PAIRED ADRENAL GLANDS IN WESTERN MEADOWLARKS,
1974-1978
I-O
VO
Adult
April
May
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
N
34
23
31
28
34
43
20
19
10
5
8
10
1
Males
Mean
7
7
6
6
7
6
7
6
6
6
6
7
7
.2
.6
.2
.3
.1
.7
,0
.8
.9
.0
.3
.4
.9
S.D.
2.8
3.4
2.1
2.4
3.1
2.8
2.7
2.1
1.6
1.6
3.0
1.7
—
Adult
N
9
27
24
21
13
19
18
10
6
6
8
—
Females
Mean
5
6
6
6
7
6
5
6
5
7
8
_
.9
.4
.0
.5
.1
.4
.1
.9
.3
.0
.9
_
S
2
3
2
2
3
2
2
2
1
2
3
.D.
.9
.3
.6
.7
.4
.0
.0
.9
.3
.4
.5
__
Juvenile Males
N
--
1
11
12
27
15
26
32
21
8
Mean
-
7
7
6
6
6
5
7
8
7
-
.2
.1
.2
.9
.9
.9
.3
.0
.8
S
-
-
2
3
2
2
2
3
2
2
.D.
-
-
.1
.2
.2
.8
.3
.0
.4
.1
Juvenile Females
N
--
--
16
8
31
14
15
19
15
3
Mean
-
-
5
4
6
5
5
7
6
9
-
-
.7
.3
.0
.7
.6
.3
.2
.3
S.D.
—
--
2.4
2.0
2.9
1.9
1.5
3.1
2.1
2.9
-------
TABLE 22.19. SEASONAL CHANGES IN WET WEIGHTS (rag) OF PAIRED THYROID GLANDS IN WESTERN MEADOWLARKS,
1974-1978
00
Adult Males
April
May
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
N
39
23
33
29
30
40
17
19
14
5
5
5
—
Mean
6.4
6.1
6.3
8.5
7.4
9.7
8.7
8.8
8.4
8.2
7.0
8.5
—
S.D.
2.6
2.0
2.7
3.6
3.1
4.8
3.8
3.1
2.9
3.4
3.4
3.5
—
Adult Females
N
1
7
23
16
18
16
18
18
8
10
4
2
—
Mean
5.0
7.3
8.1
8.5
8.0
8.5
6.6
7.3
7.5
5.7
7.7
3.3
—
S.D.
2.3
6.2
4.3
4.8
4.3
3.9
3.1
2.2
2.2
2.7
--
—
Juvenile Males
N
--
2
12
13
23
10
14
21
11
—
Mean
--
11.7
8.4
5.2
7.6
6.9
7.7
8.2
8.3
S.D.
--
--
4.4
1.4
2.9
2.2
2.3
2.0
2.2
Juvenile Females
N
--
--
15
7
25
6
5
8
5
1
Mean
--
--
7.3
5.2
5.3
7.3
5.8
7.7
8.7
11.6
S.D.
--
--
4.8
1.3
2.1
2.7
1.2
3.2
2.2
_ _
-------
TABLE 22.20. SEASONAL CHANGES IN WET WEIGHTS (rag) OF LEFT KIDNEY IN WESTERN MEADOWLARKS, 1974-1976
Adult Males
April
May
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
N
29
17
9
16
11
26
7
15
5
3
9
7
3
Mean
385.5
502.9
478.4
471.6
464.3
487.5
479-2
569.8
546.8
595.9
592.6
517.6
617.8
S.D.
127.7
58.3
45.6
43.2
61.6
54.9
68.9
99.0
50.4
77.3
63.5
69.0
90.7
Adult Females
N
--
10
22
18
9
13
7
13
4
4
8
7
—
Mean
--
434.7
460.6
443.9
424.4
449.9
458.0
437.2
409.2
541.6
463.0
468.1
—
S.D.
--
43.5
85.2
55.8
70.0
45.9
46.4
51.2
52.7
101.5
49.3
60.6
—
Juvenile Males
N
--
--
--
--
1
5
5
15
10
28
33
25
10
Mean
--
--
--
--
343.3
409.1
385.2
500.2
471.4
478.8
542.9
512.2
603.8
S.D.
--
--
--
--
--
66.4
40.8
79.7
109.9
133.6
86.3
86.9
47.3
Juvenile Females
N Mean
--
__
__
__
__
2 377.0
1 282.6
11 423.8
11 415.8
18 419.7
22 449 . 1
17 464.3
2 464.7
S.D.
--
--
--
--
--
--
--
57.8
63-6
80.8
57.8
41.5
-------
TABLE 22.21. SEASONAL CHANGES IN KIDNEY NITROGEN (PERCENT DRY WEIGHT) IN WESTERN MEADOWLARKS, 1976
o
o
Adult Males
April
May
June
July
Aug.
Sept.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
N
13
6
4
9
7
17
4
11
5
1
3
2
Mean
12.3
12.5
12.3
12.3
12.0
12.3
12.4
12.4
12.5
13-9
13.6
15.6
S.
9.
1.
2.
4.
2.
5.
0.
4.
7.
--
4.
--
D.
3
9
2
2
4
5
8
8
7
2
Adult Females
N
--
4
8
4
5
9
4
8
1
4
3
_-
Mean
--
12.4
12.3
12.4
12.2
12.0
12.1
12.7
11.8
13.9
13.1
--
S
-
5
10
6
5
1
1
14
--
6
1
-
.D.
-
.2
.4
.0
.1
.7
.0
.5
.5
.5
-
Juvenile Males Juvenile Females
N
--
--
--
--
1
3
3
11
3
13
4
6
Mean S.D. N Mean S.D.
--
__
__
__
12.0
11.8 0.6 2 12.5
12.0 0.6
12.4 3.9 5 12.3 4.3
12.4 2.5 2 13.9
13.9 13.1 2 13.3
14. 2 15.3 3 14.1 18.2
15.1 16.3 4 16.1 21.75
-------
TABLE 22.22. SEASONAL CHANGES IN DIAMETER (mm) OF LARGEST THYMUS LOBE IN WESTERN MEADOWLARKS, 1974-1975
u>
o
Adult Males
April
May
June
July
Aug.
Sept.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
N
22
15
7
11
9
8
5
6
1
3
4
5
Mean
0.4
2.5
3.0
3.6
3.6
3.9
5.3
4.4
4.4
6.7
5.9
5.7
S
0
1
0
0
1
1
1
1
-
1
2
1
.D.
.8
.2
.7
.7
.1
.0
.6
.3
-
.4
.8
.1
Adult
N
--
6
14
14
7
8
6
5
4
--
4
4
Females
Mean
--
2.
3.
3.
4.
3.
4.
4.
4.
--
5.
5.
8
5
8
1
6
1
1
8
1
6
S.
--
0.
0.
0.
1.
1.
1.
1.
1.
--
0.
1.
D.
9
9
6
2
4
6
1
7
5
2
Juvenile Males Juvenile Females
N Mean S.D. N Mean S.D.
__
__
__
—
__
1 6.8 — I 4.2
2 5.5 -- I 4.6
6 5.5 1.2 6 6.1 1.3
9 5.7 1.4 5 6.0 1.5
15 5.6 1.1 14 5.9 1.1
14 5.3 0.7 18 5.0 1.2
12 5.2 1.2 10 5.0 0.8
-------
Weight of thyroid glands varies seasonally, being largest in winter in
most, (see Miller, 1939; Wilson and Parner, 1960), but not all, species.
Increased size can be a function of both cell size or number (see reviews by
Hohn, 1950 and Ringer, 1976b). Thyroid (and adrenal) weights sometimes vary
inexplicably in wild birds (Hartman, 1946). Some of this variance may be
related to shifts in diet(Riddle and Fisher, 1926) as well as in temperature.
There is, however, a general positive correlation of both glands with body
size (Hartman and Brownell, 1961). And within a given species, seasonal
changes in weight of thyroids may serve as a useful index to secretory
activity (Kendeigh and Wallin, 1966). The perspective provided by histological
examination is necessary to understand functional condition of thyroids at a
given time; weights alone are unsatisfactory for this purpose.
TABLE 22.23. SEASONAL CHANGES IN WET WEIGHT (mg) OF BURSA OF FABRICIUS IN
JUVENILE WESTERN MEADOWLARKS, 1974-1976
Males
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
N
1
16
14
30
21
33
34
24
10
Mean
51
99
98
170
173
186
196
192
160
.5
.5
.8
.1
.4
.1
.2
.2
.6
S.D
--
34
34
71
63
54
72
60
47
•
.1
.8
.0
.9
.7
.2
.3
.4
N
--
14
7
29
14
18
24
19
2
Females
Mean
--
83
121
152
156
171
170
158
169
.8
.7
.6
.1
.2
.8
.6
.7
S.D
--
36
41
66
76
29
60
39
__
.1
.0
.0
.7
.5
.5
.5
The kidneys of birds are symmetrically paired structures located in bony
depressions of the fused pelvis. They usually comprise about 1 percent of
the body weight in birds. Their relationship to body weight is allometric,
however, and they are relatively larger in small birds and in some desert and
marine types. The kidneys through osmoregulatory controls, maintain water and
electrolyte balance (see review by Shoemaker, 1972).
Data were also gathered on the diameter of the largest thymus lobe
(Table 22.22), weight of the bursa of Fabricius (Table 22.23), and gizzard
weight (Table 22.24). The bursa of Fabricius is a dorsal diverticulum of the
302
-------
TABLE 22.24. SEASONAL CHANGES IN EMPTY GIZZARD WET WEIGHT (mg) IN WESTERN MEADOWLARK, 1974-1976
o
Co
Adult Males
April
May
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
N
47
29
17
24
16
34
15
21
8
5
10
11
3
Mean
2945.6
2403.3
2145.5
1957.9
1803.1
1867.8
1845.9
2021.7
2353.8
2716.1
2720.2
3135.8
3453.5
S.D.
390.5
471.9
222.1
306.9
330.9
382.8
220.6
406.8
243.4
285.2
221.4
369.9
216.4
Adult Females
N
--
11
26
20
14
21
11
17
8
5
10
7
—
Mean
--
1010.9
1811.6
1817.5
1707.5
1548.6
1520.6
1608.8
1839.7
2265.8
2283.4
2411.4
—
S.D.
--
207.1
238.0
237.4
218.0
179.4
154.6
203.9
354.0
326.8
146.1
192.2
—
Juvenile Males
N
--
--
--
--
1
5
5
20
16
32
35
27
11
Mean S.D.
__
__
__
__
1955.6
1726.8 273.1.
1746.0 379.5
1984.8 212.6
2512.6 316.2
2700.6 386.0
2908.3 459.8
3060.6 303.7
3140.3 266.4
Juvenile Females
N
--
--
--
--
--
5
1
13
12
18
26
18
1
Mean
--
—
--
—
--
1459.4
1465.2
1828.1
1969.6
2200.0
2424.8
2548.1
2774.7
S.D.
—
--
—
--
--
127.9
--
178.0
292.0
194.8
319.0
279.9
-------
proctodeal region of the cloaca. Its major functions include the regulation
of humoral antibody production (Click, 1964; Chang et al,, 1957; Warner and
Szenberg, 1964; Cooper et al., 1967). The secretion of a diffusible factor
which acts on lymphoid tissue (Click, 1960a; Jankovich and Leskovitz, 1965; St.
Pierre and Ackermann 1965), and the synthesis of immunoglobulins (Grossi et al.j
1968; Thornbecke et al., 1968; Zaccheo et al., 1968; Click, 1977). Basically
it contributes to immunological competence.
The bursa is restricted to birds, being largest in young birds and tend-
ing to involute with advancing age. Maximum size is reached during the first
few weeks of life, but the exact time varies among species, being about 69
days in the pigeon (Riddle, 1928a), 110 days in;he pheasant (Kirkpatrick 1944)
and 56 days in the mallard (Johnson 1961). After the bursa obtains maximum
size it involutes and eventually disappears (Jolly, 1913; Schauder, 1923, Click,
1960b; Ward and Middleton, 1971).
Both the thymus and the bursa of the western meadowlark persist for an as
yet undefined period following the assumption of the winter plumage. This
allows us to easily distinguish adult birds and birds of the year at least
throughout the fall.
The bursa is necessary for the development of antibody-mediated
responses. It normally involutes when a bird reaches sexual maturity. How-
ever, it will involute earlier if the younger bird is subjected to stress. It
will, for example, regress in the presence of glucocorticoids (see Lewis et
al. , 1976). Consequently, it should be very useful for identifying stressors
of young birds. Sudden regressive changes in the structure of the bursa are
more likely to be translated quickly into histological changes than into
reductions in the weight of the gland.
The thymus gland is an elongate structure, usually with seven lobes, that
lies laterally in the mid- and lower neck. The avian thymus, like that of
mammals, is involved in immunological processes, including lymphocyte forma-
tion. Along with the bursa it is thought to comprise a dual immunologic
system in birds. Both organs direct the maturation of immunologically com-
petent cells capable of reacting to antigens (Assenmacher, 1973).
In all vertebrates the thymus typically increases in size until sexual
maturity and then regresses markedly. In birds, however, the process can be
reversed and the gland may enlarge again following reproduction (Hohn, 1956).
This was observed in meadowlarks in that juveniles had relatively large glands
whereas those of adults were small during the first half of the summer (the
period of reproduction) and tended to enlarge thereafter (Table 22.22).
The gizzard or stomach of seed-eating birds characteristically is highly
muscular in keeping with its function as the site where food is pulverized
before passage to the main digestive and absorptive portions of the gastro-
intestinal tract. In this capacity it is, of course, the functional analog of
teeth in birds. It may also serve as a chamber for food storage and for acid
proteolysis (Ziswiler and Farner, 1972).
Seasonal changes in gizzard size are to be anticipated as a result of
dietary changes; both due to food choice or to quantity of intake. Reduced
304
-------
TABLE 22.25. PERCENT OF WESTERN MEADOWLARKS SHOWING MOLT, 1974-1977
o
Ln
Adult Males
Number
N in molt
April
May
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
76
49
47
46
42
51
27
28
14
7
12
14
3
0
0
0
0
0
1
11
23
13
5
12
13
1
Percent
molting
--
--
--
1.96
40.74
82.14
92.86
71.43
100.00
92.85
33.33
Adult
Nunber
N in molt
1
15
45
36
26
29
28
24
12
13
17
8
—
0
0
0
0
0
0
5
10
9
13
16
7
--
Females
Juvenile Males
Percent Number
molting N in molt
--
2
2
8
17.86 19
41.67 30
75.00 33
100.00 45
94.12 52
87.50 28
11
--
0
0
1
7
23
28
44
52
25
9
Percent
molting
--
0
0
12.50
36.84
76.67
84.85
97.78
100.00
89.29
81.82
Juvenile Female
Number
N in molt
--
--
1
8
7
33
25
44
38
22
3
--
--
0
2
5
17
23
31
36
22
2
Percent
molting
—
--
0
25.00
71.43
51.52
92.00
70.45
94.74
100.00
66.67
-------
TABLE 22.26. SEASONAL CHANGES OF INTEGUMENT DRY WEIGHT (rag) IN WESTERN MEADOWLARKS, 1974-1977
CT\
April
May
June
July
Aug.
Sept.
Oct.
1-15
16-30
1-15
16-31
1-15
16-30
1-15
16-31
1-15
16-31
1-15
16-30
1-15
N
49
35
19
27
17
34
16
22
8
4
10
12
3
Adult
Males
Mean
10001
9397
8822
8844
8548
8037
7788
7228
7181
8187
9043
10119
11913
.0
.0
.2
.4
.3
.0
.4
.2
.1
.1
.4
.0
.7
S.D.
1211
809
582
799
681
774
855
648
766
562
1202
780
1613
.6
.6
.9
.9
.6
.1
.6
.2
.3
.4
.2
.5
.9
Adult Females
N
13
23
22
15
22
15
20
9
5
9
7
-_
Mean
7800
7639
7145
6671
6235
6317
5778
5946
6680
7201
7634
—
.0
.9
.9
.3
.2
.4
.0
.0
.8
.4
.7
S.D.
795.8
1116.4
785.2
752.3
538.1
693.9
753.5
1192.7
1708.8
1254.8
864.7
—
Juvenile
N
--
--
2
5
6
21
19
32
38
28
11
Mean
--
--
6336.
4366.
4815.
4903.
5259.
6044.
7858.
8866.
9955.
Males
2
9
7
4
8
8
5
6
1
S.D
--
--
--
741
450
369
715
1552
1456
1530
841
.6
.6
.9
.7
.8
.8
.8
.8
Juvenile
N
--
--
--
1
1
14
12
20
30
19
4
Females
Mean
--
--
--
4461
3591
4117
4624
4909
6611
7455
7964
.6
.5
.3
.8
.6
.8
.0
.7
S.D
--
—
--
--
--
562
1082
751
1665
1228
717
.9
.6
.4
.2
.7
.8
-------
feeding results in decreased gizzard size and increased feeding, as in pre-
migratory hyperphagia, results in hypertrophy (Breitenbach et al., 1963;
Anderson, 1972; Moss, 1974; Ankney, 1977).
Gizzard weight decreased significantly from April to May in adult male
meadowlarks (P < 0.05). It then increased substantially during the last two
months of the season (P < 0.01). Contents of the gizzards of our specimens
were also weighed and categorized at 3-hr intervals throughout the day.
No diurnal feeding pattern was apparent. Uncertainties regarding rate of food
passage in the gut make these data difficult to evaluate.
Molt
Postnuptial molt in the population lasted from July through the end of
collecting and tended to begin earlier in males than in females (Table 22.25).
A complete molt involving flight feathers occurred also in juveniles and their
molt had approximately the same tempo as that of adults (Table 22.25).
Weights of the dry integument increased toward the end of the season as new,
unworn feathers replaced the old (Table 22.26).
Molt and reproduction in most temperate zone migrants do not overlap in
time, a relationship that seems to hold for all species in the present study
(Lewis, Morton and Kern; unpublished data).
Lean body mass was relatively stable during molt in our meadowlarks, a
phenomenon also observed in chaffinches (Fx>i,ngi>11a ooelebs ) by Gavrilov and
Dolnik (1974), in European tree sparrows (Passer1 m.. montanus ) by Myrcha and
Pinowski (1970) , and white-crowned sparrows ( ZonotT-iah-ia leucophpys gambel-i-i^
by Chilgren (1977). Such stability suggests that feather growth was not
achieved at the expense of body protein.
We analyzed data on body lipid as a function of molt stage to better
appreciate the relationship of molt to onset of premigratory — fattening.
Results show that as the molt comes to a close, meadowlarks begin to fatten
(Table 22.27). Birds having completed or nearly completed molt (category 0-1)
had significantly more body lipid than those still growing two or more pairs
of remiges (P < 0.01). Thus molt within the population was not perfectly
synchronous.
Slight increases in body weight during postnuptial molt have been docu-
mented for a number of migratory species, usually this was attributed to the
new plumage. In at least one case, however, a major increase in weight was
due to fattening (Morton and Welton, 1973).
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307
-------
TABLE 22.27. BODY L1PID AS PERCENT OF BODY WEIGHT IN WESTERN MEADOWLARKS DURING AND AT END OF MOLT
u>
o
oo
Adult Males
N
Mean
S.D.
6+
6
3.91
0.92
2-5
10
5.65
0.94
0-1
5
9.92
3.08
Adult Females
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4.13
0.75
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9 2
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1.65
Juvenile Males
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36
4.31
0.96
2-5
13
5.73
2.61
0-1
8
9.49
3.49
Juvenile Females
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22
4.65
1.58
2-5
13
5.18
0.87
0-1
3
12.69
0.50
All Birds
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75
4.36
1.15
2-5
45
5.67
1.72
0-1
18
10.03
3.10
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-------
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314
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SECTION 23
PARTICULATES IN THE LUNGS OF WESTERN MEADOWLARKS
(STURNELLA NEGLECTA) IN SOUTHEASTERN MONTANA
M. D. Kern, R. A. Lewis and M. B. Berlin
ABSTRACT
The lungs of western meadowlarks (Sturnella
negleota), collected within 100 km of the coal-fired
power plant and coal mines at Colstrip, Montana, were
examined histologically for particulates and associated
pulmonary damage. Birds were collected during 1975
(when the power plant was not operating), 1976 (when it
operated intermittently), and 1977-78 (when it was in
full operation).
Three major categories of particulates occurred in
the meadowlark's lung: (1) crystals of variable size,
(2) very small ("50.5 ym) round black flecks, and
(3) larger black particles of variable size and shape.
Most larger particles were confined to the air ducts,
but smaller ones were widespread in the lungs. Crystals
were the only particulates that irritated pulmonary
tissue, sometimes eliciting mild fibrosis within the
parabronchial wall.
Particulates, especially the black forms,
increased in the lining of the parabronchi and the
lumen of the air capillaries between 1975 and 1977.
The number of particulate-containing macrophages in
the lining of the parabronchi also increased between
1975 and 1976, but then declined. However, in 1978,
the particulate content of the lung was at 1975 levels
and there were fewer particulate-containing macrophages
in the parabronchi than in 1975. These declines are
probably related to the fact that most of the meadow-
larks collected in 1978 were obtained at greater
distances from Colstrip than birds collected in 1976-77.
Juvenile birds had smaller particulate burdens
than adults. Among juveniles collected in 1977 and
315
-------
1978, the concentrations of participates were
similar in the parabronchi, but significantly
different in the air capillaries (smaller in
1978). Particulate burdens of male and female
birds were similar in all but one region of the
lung. The number of particulate-containing
macrophages was negatively correlated with the
distance from Colstrip at which a bird was
collected during 1975-77.
INTRODUCTION
Birds have the potential to serve as biological monitors of
particulate air pollution from coal-fired power plants. Small birds have
high metabolic rates and are active at levels where exposure to gaseous and
other respirable pollutants may be severe (Lewis and Lewis, 1979). Their
lungs are a major site of detoxification and contain macrophages that remove
particulate matter from inhaled air. Their pulmonary tissue is easily
irritated by inhaled substances to which it exhibits inflammatory responses
of greater or lesser severity. In this respect, birds appear to be an order
of magnitude more sensitive to aerial pollutants than are humans (Takemoto
et al., 1974). Since little respired material is filtered out in the nasal
passageways (Takemoto et at., 1974) and air flow through the lung is only in
one direction (Bretz and Schmidt-Nielsen, 1971), the avian lung is analogous
to a high volume sampler of air-borne materials.
The few published reports concerning the effects of air pollutants on
wild birds generally pertain to sedentary urban species and support their use
as biomonitors (Lewis and Lewis, 1979). The most striking case of historic
importance is the use of the canary (Serinus eanari-d) in sensing mine gases
and anoxia (Neal and Olstruin, 1971). Levels of lead in the organs of pigeons
(Columbia livid}, both in Japan and the United States, have been related to
aerosol levels of this element at sampling sites (Tansy and Roth, 1970; Ohi
et al. , 1974). Similar relationships have been discovered between the dust
content and associated damage in the lungs of doves and pollutant levels at
sampling sites in Japan (Takemoto et al., 1974); and between the presence of
particulate-laden pulmonary macrophages in the lungs of house sparrows
(Passer domesticus) and pollution levels in areas of California (McArn et al.,
1974). In addition, Tashiro et al. (1974) have demonstrated that birds are
especially sensitive to air-borne pollutants during their breeding season.
Takemoto et al. (1974) found a direct relationship between the age of doves
in polluted regions of Japan and the degree of lung damage.
Western meadowlarks (Sturnella negleota) are the most widely distributed
and abundant passerine in the Colstrip area (Lewis et al., 1976; Preston and
Thompson, 1979). They are neither sedentary nor urban, but presumably return
to nest in the same general vicinity year after year (Lewis, unpublished
banding returns). Consequently, individuals may be exposed to aerial
emissions from the Colstrip power plant for five months each year at a time
when they are reproductively active and hence potentially highly sensitive
316
-------
to such insults on the respiratory system. Furthermore, unless they can
completely clear particulates from their lungs while absent from Montana
(which does not seem to be the case), there may be accumulation of
particulates in the respiratory system with successive years of exposure.
Under these conditions, western meadowlarks may be particularly useful
biomonitors of stack emissions in remote grassland areas surh as Colstrip.
MATERIALS AND METHODS
This study deals with samples of lungs from western meadowlarks
(S. neglecta} collected within 100 km of Colstrip, Montana, between 1975 and
1978. Most birds were taken SSE and SEE of Colstrip, i.e., from sites that
are predominantly downwind of the power source (Miller et al., 1976; Crecelius
et al., 1978). Birds were shot and dissected in the field as described by
Lewis et al. (1976). The caudal tip of the left lung was removed from each
bird and fixed in Bouin's solution or 10 percent neutral buffered formalin.
Fixed samples were later dehydrated, embedded in paraffin, and sectioned at
7.0 urn. Representative sections were stained with haematoxylin and eosin by
American Histolabs (Silver Spring, Md.) and examined by us.
Our histological survey of the lung is modeled after methods of McArn
et at. (1974). Initially, we scanned the sections in order to determine the
types of particulates that were present. Then, we examined 20 transverse
sections of parabronchi and five regions of air capillaries selected at
random within each section more closely. "We determined the particulate
concentrations in (1) macrophages and smooth muscle cells within the lining
of each parabronchus, (2) the lumen of each parabronchus, and (3) the lining
and lumen of the air capillaries, in each case using a scale of 0 (none
present) to 5 (very high concentrations present). In addition, we counted
the number of particulate-containing macrophages in the lining of each of
the 20 parabronchi.
RESULTS AND DISCUSSION
Histological Structure of the Meadowlark's Lung
We begin with a brief statement concerning the microscopic anatomy of
the meadowlark's lung because it has not been described previously. The
meadowlark's lung is structurally similar to that of the domestic fowl
(Hodges, 1974). However, pulmonary lobules, consisting of a parabronchus and
the air capillaries that arise from it, are not as clearly defined as in
chickens. There is no conspicuous connective tissue partition between
adjacent lobules. In the terminology of Hodges (1974), atria, infundibulae,
and air capillaries are all represented in the lobule.
Nature of the Particulates in the Meadowlark's Lung
The lungs of our specimens contained three basic types of particulates
(Figure 23.1):
1. Transparent, tetragonal crystals, ca. 1.5 urn in length and 0.5 ym
in width, scattered individually or in clusters and chains.
317
-------
/
Figure 23.1.
Particulates (arrows) in the lungs of western
meadowlarks collected near Colstrip, Montana.
A and B. Crystalline particulates of small and
large size. C. Spherical black particles within
macrophages on the border of a parabronchus.
D. Large black particulates with distinct
projections in an area of air capillaries.
Magnification: A - C, 3750X; D, 7500X.
318
-------
2. Small round black flecks with a diameter ^0.5 ym.
3. Larger black particles; these were round, oval, triangular,
polygonal, splinterlike, or irregular in shape and varied in
size with minimal dimensions between 0.9 and 12.0 ym and maximal
dimensions between 1.4 and 21.6 ym. They were sometimes con-
glomerates of the small flecks.
Black filamentous material and large irregular crystals were also
occasionally present. The latter commonly measured between 2.8 and 32.9 ym
on a side. Most of the larger particulates were confined to the air ducts,
whereas smaller particulates were widespread in both the air ducts and air
capillaries.
The parabronchial wall was occasionally thickened around small groups of
crystalline particulates. This suggests, as does their size, that they are
silicates since (1) the latter are known to elicit fibrotic responses from
pulmonary tissue (Bowden, 1976), and (2) one of the major particulates in
aerosols near Colstip is an aluminum silicate of a size (0.6 to 1.0 ym)
similar to the crystals in the meadowlark lung (Van Valin et al., 1979).
On the basis of their spherical shape and tiny size, the small flecks
in the meadowlark's lung may be sulfur- or chlorine-containing particulates
emitted by the power plant (Van Valin et al., 1979). These particles and the
larger black ones rarely elicited inflammatory responses from the birds'
pulmonary tissue aside from occasional small infiltrations of lymphocytes
around major blood vessels. Extravasation frequently occurred around heavy
concentrations of black particulates, but may be an artifact of the dissection
procedure because (1) other heavy concentrations of particulates in the same
sections were not enveloped by erythrocytes, and (2) parabronchi were commonly
filled with erythrocytes.
Particulate Burdens of the Meadowlark's Lung
The distribution and density of particulates in the meadowlark's lung
appear in Table 23.1. To our knowledge, this is the first time that changes
in particulate burdens have been examined for several months of each of
several years, and with regard to age and sex, in an avian species.
Particulate burdens in the meadowlark's lung increased progressively
between 1975 (when the power plant was not operating), 1976 (when it operated
intermittently), and 1977 (when it was in full operation). This trend is
especially well shown by the particulate content of macrophages in the lining
of the parabronchi. However, it is also reflected by changes in particulate
density at other sites in the parabronchus, and in the lumen of the air
capillaries, although not in the lining of the latter.
All types of particulates increased, but the change in the black forms
was especially noticeable (Figure 23.2). Lungs of many adult birds collected
in 1975 were completely free of particulates. This was not the case in
subsequent years. The change in particulate burdens was especially
319
-------
TABLE 23.1. PARTICULATE CONTENT OF LUNG TISSUE FROM WESTERN MEADOWLARKS (5 turnella neglecta)
COLLECTED AT COLSTRIP, MONTANA, BETWEEN 1975 AND 1978*1"
Particulates associated with parabronchi
Number of
Groups N containing
macrophages per Macrophages
parabronchus (rated 0-5)
Adults:
1975 29 24. 81 ±3. 99 a 1.38 ±0.21
1976 41 30.43±4.26 b 2.19±0.24
U> 1977 130-131 22.64 + 1.06 a 2. 45 ±0.18
NJ
O
1978 27 18.29 + 1.31 c 1.67±0.26
Juveniles:
1977 15 31.05±5.40 a 1.91±0.38
1978 15 27. 43 ±2. 74 a 1.46 + 0.43
Adults:
April 1976-1977 43 27.20±3.76 a 2.07±0.27
May 1975-1977 65 24. 27 ±2. 21 ab 2. 11 ±0.23
Particulate density in
Particulates associated with
air capillaries
Smooth muscle Lumen of Lining of air Lumen of air
cells parabronchus capillaries capillaries
(rated 0-5) (rated 0-5) (rated 0-5) (rated 0-5)
a 1.15 + 0.18 a 0.88 + 0
b 1.31 ± 0.13 a 0.73 ± 0
b 1.60± 0.12 b 1.16 ± 0
a 1.08 + 0.16 a 0.90+0
a 1.34 ± 0.29 a 0.98 ± 0
a 0.98 ± 0.35 a 0.89 ± 0
a 1.26 + 0.19 a 0.88±0
a 1.39±0.14 a 1.09±0
[1975,
0.62 + 0
[1976]
.16 a 1.93 + 0.27
.11 a 1.90±0.18
.09 b 1.95±0.09
.12 a 1.64 ±0.11
.23 a 1.73±0.32
.26 a 0.88 ± 0.36
.14 ab 1.90 ±0.21
.15(49) be 1.88 + 0.13
1977]
.ll(16)a
a 0.40+0.11 a
a 0.40 ± 0.10 a
a 0.63 ±0.07 b
a 0.47 + 0.11 ab
a 0.44 ±0.09 a
b 0.24 + 0.09 b
a 0.60 ± 0.12 ab
a 0.47 + 0.10 a
(continued)
-------
TABLE 23.1. (Continued)
Co
Particulates associated with parabronchi
Number of
Groups N containing
macrophages per Macrophages
parabronchus (rated 0-5)
Particulate density in
Particulates associated with
air capillaries
Smooth muscle Lumen of Lining of air Lumen of air
cells parabronchus capillaries capillaries
(rated 0-5) (rated 0-5) (rated 0-5) (rated 0-5)
June 1975-1977 68 21 . 87 ± 1 . 51(63) b 1.28 + 0.321 9) a 1 . 1 5 ± 0. 16(14) a ]. 22 ±0.12
[1975,1977] [1975] [1975,1976]
33.48±1800( 5)a 2. 51 ± 0. 22(59) b 1 .88 ± 0.14(54) b
[1976] [1976,1977] [l97?]
July 1975, 28 22. 64 ±2. 49 ab 1. 89±0.45
1977-1978
August 1975,1978 22 20. 77 ±3. 61 b 1.57*0.30
Males 171 24.72±1.36 a 2. 19 t 0.18
Females 89 24. 53 ±2. 19 a 1. 94 ± 0.18
Adults 227 23. 61 ±1.19 a 2. 17 ±0.13
Juveniles 33 31. 85 ±3. 20 a 1.67 ±0.27
a 0.85 ± 0.24(13)a 0.97 + 0.20
[1975,1978]
1.79± 0.47(15)ab
[1977]
a 1 .07 ± 0.20 a 0.87 i 0.13
a 1.43 ± 0.10 a 1 .06 ± 0.08
a 1.31 ± 0.12 a 0.90 ± 0.10
a 1.43 ± 0.08 a 1.02 ± 0.07
b 1.13 ± 0.21 b 0.93 ± 0.15
c 2.33±0.37( 9) a 0. 36 i 0. 18(14) a
[1975] [1975,1976]
1.76±0.69( 5)a 0. 65 ± 0. 09(54) b
[1976J [1977]
1.96± 0.10(54)a
[1977]
abc 1.93 ±0.27 a 0.51 ±0.16 ab
abc 1.68 ±0.14 a 0.46+0.12 ab
a ].86±0.09 a 0.43 ±0.08 a
b L,76±0.13 a 0.45 + 0.07 a
a 1.90 ±0.07 a 0.54 ±0.05 a
a 1.30+0.26 b 0.34±0.07 b
*Values In the table are means t C195(N). For the monthly changes in the particulate content of the adult lung, statistically
significant differences existed between years of the study; data were lumped whenever possible, but in some cases were neces-
sarily present.ed separately.
"Within t.he column in each division of the table, values not. followed by the same letter differ significantly at the 0.05 level
(Student, t-tests and Student-Neuman-Keuls tests for data concerning the number of macrophages; Mann-Whitney or Student-Neuman-
Keuls tests for rated data).
-------
•
•
•*.' »•;
•••*••<-•
»_j-*
r
'f • -
. -"v'*1^,
-
. .;
''.'•
Figure 23.2. Changes in the particulate content of lungs from
western meadowlarks collected between 1975 and 1977.
A and B. During 1975, the lining of the parabronchus
(P) and air capillaries (C) was frequently free of
particulates. C and D. During 1977, particulates
occurred in the lining of both parabronchi and air
capillaries (arrows). Magnifications: A and C, 750X;
B and D, 3750X.
3Z2
-------
pronounced in macrophages within the lining of the parabronchi. Curiously,
the number of these macrophages declined in 1977, although the actual
particulate content of the parabronchi increased.
In 1978, the particulate content of the lung was similar to that seen
in 1975. Furthermore, significantly fewer particulate-containing macrophages
occurred in the parabronchi than in 1975. There are several possible
explanations for these declines. Most important in this regard is the fact
that birds shot in 1978 were intentionally collected at distances in excess
of 69 km from the power plant in order to supplement the pre-operational data
base for the Colstrip Project. Collections in earlier years were frequently
made much closer to the power plant. However, since birds with dirty lungs
were collected at these great distances in earlier years, other factors may
also be responsible for the decreases in 1978. Perhaps meadowlarks are now
actively avoiding heavily impacted areas near the power plant, as suggested
by the data of Preston and Thompson (1979), and therefore are exposed to fewer
air-borne aerosols than in earlier years. However, we cannot discount the
possibility that mechanisms for removing particulates from the lungs have
improved (i,.e.} adapted to the increased particulate burdens) during the 4
years of study. Bowden (1976) has shown, for example, that the number of
alveolar macrophages in the mammalian lung is directly related to the number
of small particles that reach the alveoli (also see Stuart, 1976). It is also
possible that the age structure of the adult segment of the population in
1978 was weighted toward young adults. This may be significant in light of
the finding of Takemoto et a.1. (1974) that the trauma produced by aerosols
in avian lungs is directly related to the age of birds. We suspect, however,
that the declines in 1978 are due largely to the collection procedures
followed that year.
Significant differences in the particulate burdens of adult lungs
existed between the months when the birds were collected. However, we are
unable to discern general trends in the data because of differences in some
months (e.g. June) over the 4 years of study and because monthly changes
in one region of the lung are not in the same direction as changes elsewhere
in the same lung (Table 23.1). If a small sample of five birds collected in
June, 1976, is ignored, it is possible to conclude that the number of
particulate-containing macrophages in the parabronchi was higher during
April than between May and August.
Significant differences in particulate burdens as a function of age
were observed; juvenile birds had cleaner lungs (Table 23.1). Among the
juveniles collected during 1977 and 1978, the concentrations of particulates
in the parabronchi were similar, but concentrations in the air capillaries
differed significantly (lower in 1978) . The particulate burdens of male and
female meadowlarks were similar in all but one region of the lung that were
examined.
Pollution Gradient Analysis
Our study appears to be the first to provide a pollution gradient
analysis based on the avian respiratory system. The number of particulate-
containing macrophages per parabronchus declined as the distance between the
323
-------
site of collection and the power plant increased during 1975, 1976, and 1977,
but not during 1978 (Table 23.2). Given the great minimal distance from
Colstrip (and the smaller dispersion about the mean) at which meadowlarks
were shot during 1978 (69 km), the absence of a relationship during that year
is probably not surprising. The inverse relationship between the two
variables in each of the earlier years is both linear and significant,
although weak (Table 23.2).
Whereas significant relationships existed between the mmbev of
particulate-containing macrophages and collection distance during 1975-77,
only one significant relationship occurred between par't'Lculate density in the
parabronchus or air capillaries and collection distance (Table 23.3). These
findings, and the large amount of particulate matter accumulated by pulmonary
macrophages (Figure 23.2), suggest that the number of particulate-laden
macrophages is the most sensitive indicator of pollution impact on the birds.
Data concerning the number of such cells are relatively easy to obtain and
may be a sensitive bioindicator of the power plant's impact on birds until
and unless the air ducts become saturated with particulates because of
repeated exposure to aerosols. Clearly, further information on power plant
emissions and air quality in the vicinity of Colstrip is needed and birds
may prove useful in its acquisition.
In this regard, it is worth adding that our data are consonant with
data obtained with air quality monitoring equipment at Colstrip (Crecelius
et at., 1978). Using the latter, it has been demonstrated that 69 percent (by
weight) of the stack dust at Colstrip is less than 0.3 ym in diameter and
hence can remain air-borne for days or weeks and be widely dispersed. A
continuously recording air quality monitoring station situated 12 km
downwind (SE) from Colstrip has also recorded plume strikes several times
each week when the power plant was in operation.
It is possible, but remains to be demonstrated, that the avian lung is
a more sensitive and reliable sampler of low levels of air-borne particulates
than state-of-the-art monitoring equipment. In contrast to the data from
meadowlark lungs (Table 23.1), the equipment did not record significant
changes in the concentration of elements in Colstrip air after Unit 1 of the
power plant began operating.
Recommendations
Since the particulates in the lungs of western meadowlarks at Colstrip
may originate from several sources (fugitive dust from traffic on secondary
roads or that associated with farming and mining operations; stack emissions
of the power plant), it is important to distinguish the impact of the power
plant from other potential impacts. Consequently, we recommend that samples
of lung be set aside in the future (1) for trace metal analysis, and (2) for
examination with the electron microscope, as well as for histological
examination.
Atomic absorption analyses of trace metals should indicate if emissions
from the power plant are accumulating in the birds' lungs. The stack dust at
Colstrip is relatively rich in Ca, Se and V. Concentrations of these elements
324
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TABLE 23.2. DENSITY GRADIENT ANALYSIS: NUMBER OF PARTICULATE-CONTAINING MACROPHAGES
PER PARABRONCHUS VS. DISTANCE OF MEADOWLARK FROM COLSTRIP AT CAPTURE
OJ
Year N
Mean distance from
Colstrip at capture
[km(range)]
Linear relationship
between number of
macrophages (Y) and
distance from
Colstrip in km (X)*
Correlation
coefficient
for the
equation (r)
Significance
of
correlation
coefficient
1975
1976
1977
1978
22
44
146
42
74.7
60.3
62.4
74.3
(38.94 -
(20.92 -
(18.10 -
(68.80 -
90.12)
96.56)
96.96)
89.31)
Y =
Y =
Y =
Y =
57.30 -
48.16 -
27.92 -
17.12 +
0.40
0.27
0.07
0.06
X
X
X
X
-0
-0
-0
+0
.48
.39
.25
.07
0.02 0.50
*The slopes (regression coefficients) of these four equations are not statistically
different (P>0.05; analysis of covariance)
-------
TABLE 23.3. THE RELATIONSHIP BETWEEN PARTICULATES IN THE
LINING OF THE PARABRONCHI AND AIR CAPILLARIES
VS. DISTANCE OF THE MEADOWLARK FROM COLSTRIP
AT CAPTURE, AS SHOWN BY THE CORRELATION
COEFFICIENTS, IS NOT USUALLY LINEAR
Correlation coefficients (r)
Year N
1975 22
1976 44
1977 146
1978 42
Lining of
parabronchus
-0.11
-0.23
+0.21*
+0.18
Lining of air
capillaries
+0.15
+0.08
-0.04
+0.07
"0.01
-------
(and several others) have been measured in dust at three air monitoring sites
near Colstrip (Crecelius et al., 1978). Assays of these elements in samples
of lung may more closely establish the contribution of the power plant to the
observed particulate burdens in the tissue.
However, atomic absorption analyses will not indicate the distribution
of these and other particulates in the lungs nor the damage they cause to
pulmonary tissue. Hence, routine histological surveys should be continued.
They should be complemented with ultrastructural studies, similar to those
done by McArn et al. (1974), in order to clearly identify particulates of
very small size in the lung.
One limitation of the study to-date involves the method used to fix the
lungs. Extravasation is commonly widespread in the organ and fixation is not
uniformly good. These shortcomings confound histological evaluation and can
be readily avoided if in the future the air duct system of the bird is
perfused with fixative before the lung is removed and fixed. This method of
fixation will also preserve the larger ducts in the respiratory tree. It
would be useful to study the effects of particulates on the mucociliary
apparatus of these larger airways since they are known to be affected by
exposure to such things as SC>2 in other species of birds (Wakabayashi et al,,,
1977) . In addition, a considerable fund of information concerning the
structure of these larger airways of birds, both at the light and electron
microscopic levels, is already available in the literature and therefore
readily available for comparison (Bienenstock et al., 1973; Hodges, 1974;
Walsh and McLelland, 1974 a,b; Jeffery, 1978).
CONCLUSIONS
The meadowlark's lung is structurally similar to that of the domestic
fowl.
Three major categories of particulates occurred in the meadowlark's lung
during 1975-78: (a) crystals of variable size, (b) small C< 0.5 urn) round
black flecks, and (c) larger black particles of variable size and shape. The
crystals may be silicates. The small flecks may be sulfur- or chlorine- con-
taining particles emitted by the power plant. Larger particles were generally
confined to the air ducts, but smaller ones were widespread in the lungs.
Only the crystals caused perceptible irritation to the lung.
All categories of particulates, but especially the black forms, increased
in the lining of the parabronchi and the lumen of the air capillaries between
1975 and 1977. The number of particulate-containing macrophages in the lining
of the parabronchi also increased between 1975 and 1976, but then declined.
In 1978, the particulate content of the lung was at 1975 levels and there were
fewer particulate-containing macrophages in the parabronchi than in 1975.
These declines are probably related to the fact that most of the meadowlarks
collected in 1978 were obtained at greater distances from Colstrip than birds
collected in 1976-77.
Significant monthly variations in particulate burdens occurred at the
various sites of the lung that were examined, but overall monthly trends are
327
-------
difficult to discern. However, the number of particulate-containing macro-
phages was, with one exception, higher during April than between May and
August.
Juvenile birds had smaller particulate burdens than adults.
Among the juveniles collected in 1977 and 1978, the concentrations of
particulates were similar in the parabronchi, but significantly different in
the air capillaries (smaller in 1978).
Particulate burdens of male and female birds were similar in all but one
region of the lung that were examined.
The number of particulate-containing macrophages was significantly and
negatively correlated with the distance from Colstrip at which a bird was col-
lected during 1975, 1976, and 1977-
Only one significant relationship (weak and positive) occurred between
particulate density in the lung and the distance from Colstrip at which a
meadowlark was collected.
Of the histological measurements, the number of particulate-laden macro-
phages in the lining of the parabronchi appears to be the most sensitive in-
dicator of pollution impact on meadowlarks. It appears to be a useful bio-
monitor of the particulate output of the coal-fired power plant at Colstrip.
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Walsh, C., and J. McLelland. 1974a. The Ultrastructure of the Avian
Extrapulmonary Respiratory Epithelium. Acta Anat., 89:412-422.
Walsh, C., and J. McLelland. 1974b. Granular "Endocrine" Cells in Avian
Respiratory Epithelia. Cell Tissue Res., 153(2):269-276.
330
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing/
1. REPORT NO.
4. TITLE AND SUBTITLE
2.
5. REPORT DATE
The Bioenvironmental Impact of a Coal-Fired Power Plant;
Sixth Interim Report, Colstrip, Montana. August, 1980."
6. PERFORMING ORGANIZATION CODE
3. RECIPIENT'S ACCESSION NO.
1. AUTHOR(S) " ~~~
Edited by Eric M. Preston, David W. O'Guinn, and
Ruth A. Wilson
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
U. S. EPA
Corvallis Environmental Research Laboratory
200 S.W. 35th St.
Corvallis, OR 97330
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
SAME
13. TYPE OF REPORT AND PERIOD COVERED
Interim 12/77—12/80
14. SPONSORING AGENCY CODE
EPA/600/02
15. SUPPLEMENTARY NOTES In this series the EPA numbers are: the 1st Interim Rept. (EPA-
600/3-76-002), the 2nd Interim Rept. (EPA-600/3-76-013), the 3rd Interim Rept. (EPA-
600/3-78-021), the 4th Interim Rept. (EPA-6QO/3-79-Q44), and the 5th Interim Rept. (EPA-
16. ABSTRACT -- 600/3~80-052).
The EPA has recognized the need for a rational approach to the incorporation of
ecological impact information into power facility siting decisions in the northern
great plains. Research funded by the Colstrip, Coal-Fired Power Plant project is a
first attempt to generate methods to predict the bioenvironmental effects of air
pollution before damage is sustained. Pre-construction documentation of the environ-
mental characteristics of the grassland ecosystem in the vicinity of Colstrip, Montana
began in the summer of 1974. Since then, key characteristics of the ecosystem have
been monitored regularly to detect possible pollution impacts upon plant and animal
community structure.
In the summer of 1975, field stressing experiments were begun to provide the data
necessary to develop dose-response models for S02 stress on a grassland ecosystem.
These experiments involve continuous stressing of one acre grassland plots with _
measured doses of S02 during the growing season (usually April through October).
Results of the 1979 field season's investigations are summarized in this
publication. This is the last interim report of the six year project. Final reports
will be published in 1981.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
plant and animal response to pollution
coal-fired power plant
air pollutants
grassland ecosystems
coal-fired power plant
emissions
air quality monitoring
51
18. DISTRIBUTION STATEMENT
release to public
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
330
20. SECURITY CLASS (This page)
Unclassif ied
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
331
it US GOVERNMENT PRINTING OFFICE. 1981 -757-064/0252
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