A field study of macroinvertebrates within two distinct soil conditions of sun exposures and a laboratory study of specifically Lumbricus terrestris choice under manipulations of soil type and water moisture held under four temperatures by:
Tu Ong
Saint Martin's University
Biology 401
May 9, 2007
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TABLE OF CONTENTS
ABSTRACT ....................................................................................................................................1
INTRODUCTION ..........................................................................................................................1
METHODS .....................................................................................................................................5
Field component...................................................................................................................5
Laboratory component .........................................................................................................7
RESULTS .......................................................................................................................................9
Field component...................................................................................................................9
Laboratory component .........................................................................................................9
DISCUSSION ...............................................................................................................................12
Field component.................................................................................................................12
Laboratory component .......................................................................................................13
ACKNOWLEDGEMENTS ........................................................................................................14
LITERATURE CITED ...............................................................................................................15
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ABSTRACT
Two studies were conducted, a field study and a laboratory study, to investigate the relationship Lumbricus terrestris had among the factors of temperature, soil moisture and soil type. L. terrestris were held under four temperature treatments, each with three replicates of 1% water moisture and three replicates of 3% water moisture. Each replicate, enclosed in a cylindrical container, was composed of autoclaved soil on one side and non-autoclaved (normal) soil on the other. L. terrestris occurrence in either soil varied with each temperature treatment.
The warm (22.1 ºC) treatment had more L. terrestris in the 3% water moisture than the 1% water moisture (F = 5.92; d.f. = 1; p = 0.041). The room (21.7 ºC) treatment had an interaction between the soil type and percent of water moisture (F = 7.38; d.f. = 1; p = 0.026). The control
(21.5 ºC) treatment showed no difference in the number of L. terrestris found in each soil type or percentage of water moisture (F = 2.77; d.f. = 1; p = 0.135). The cold (15.5 ºC) treatment had more L. terrestris in the normal soil than the autoclaved soil (F = 12.8; d.f. = 1; p = 0.007). The laboratory study suggested that L. terrestris were capable of choosing certain soil qualities over others, as reflected by their distribution within the soil held at the four temperature treatments.
INTRODUCTION
Earthworms can be described as “soil engineers” for their work in mixing the organic topsoil with the deeper soil (Gonzalez and Zou, 1999). Primarily, earthworms could be found in neutral or slightly acidic soils because one particular species, Perichaeta indica , was observed to inhabit soils with a pH of 6 to a pH of 7 (Arrhenius, 1921). In contrast, increased metal contaminants within soil altered the distribution of local earthworm populations (Spurgeon and
Hopkin, 1999). Therefore, earthworms could tolerate changes in pH between 6 and 7, but not
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changes in contaminants. Thus, the distribution of earthworm populations within the soil could be reflected within the condition and composition of the soil (Scullion et al.
,1988; Spurgeon and
Hopkin, 1999). My two studies further developed this relationship between earthworm distributions and their soil habitat.
The earthworm-soil relationship is one of balance, so altering one would alter the other.
Changes in soil composition could result from human disturbances such as exposure to metal contamination (Spurgeon and Hopkin, 1999) or coal mining techniques (Scullion et al.
, 1988).
Continually renewing soil with plant litter (Gonzalez and Zou, 1999) can also change its composition over time and could result in changes of soil fauna. The previously mentioned studies showed distributions of earthworms within collected soil as well as within field observed soil. Field collected data could have uncontrollable variables, so laboratory experiments such as those done by Arrhenius (1921) and De Haas et al.
(2006) were also conducted. My two studies consisted of one field study and one laboratory study. The field study was broad to include all macroinvertebrates. The laboratory study focused primarily on the distribution of one earthworm species.
Earthworms are not the only macroinvertebrates found within the soil. A study by
Haskell (2000) revealed that numerous other macroinvertebrates, in addition to earthworms, were impacted by soil conditions. Haskell (2000) conducted a study on the impact constructed roads had through the continuous forest of the Cherokee National Forest in Tennessee. He randomly chose sites near constructed roads and gathered soil samples 1, 5, 15, 35, 60, and 100 m from the road’s edge. His results indicated that 14 out of 16 taxa declined as the distance from the roads decreased from 15 to 1 m away from the roads; thus showing that constructed roads
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had an impact on soil fauna. In my field study, I had not examined how roads impacted macroinvertebrate numbers, but instead I focused on how sun exposure impacted their numbers.
I focused primarily on earthworm abundance in my research like Spurgeon and Hopkin did in 1999. They studied soil and earthworms from samples collected in a 10 km vicinity of a primary smelting works, a metal production factory, in Avonmouth, United Kingdom, and established that summer had the lowest soil moisture when compared to spring, autumn, and winter. The largest numbers of earthworms were found during the spring and winter, while lower numbers were found in the autumn and summer. Spurgeon and Hopkin (1999) suggested that lower numbers of earthworms in the summer had arisen from earthworms traveling to deeper soil layers due to lack of moisture. Thus, I measured how soil temperature and moisture influenced the population of the earthworm, Lumbricus terrestris.
These results were observed out in the field, where earthworms were not confined to where they lived. If soil conditions in one area changed, then they were free to move either deeper or further away from the dry areas
(Spurgeon and Hopkin, 1999). Spurgeon and Hopkin (1999) showed moisture, along with soil composition, also influenced the motility of earthworms. I took earthworm motility into consideration and confined my earthworm subjects within cylindrical containers. Each container composed of autoclaved soil on one side and normal soil on the other, which produced two different soil types. While Spurgeon and Hopkin (1999) focused on several species, I focused on one particular species.
My experiment with L. terrestris was similar to the following experiment done by De
Haas et al.
(2006). De Haas et al.
(2006) experimented with chironomid, or small, flying, insect larvae. The experiments involved the chironomid larvae, Chironomes riparius , and sediment gathered from seven floodplains. The researchers combined all possible combinations of two
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using the seven sediments, including combinations of the same sediments, to measure choice and no choice reactions of C. riparius (De Haas et al.
, 2006). The two sediments were placed in containers that were then placed into one larger container. The gap was filled with sand and aerated overnight. I assumed aeration was for the purpose of removing water from the sediments.
The larvae were then put in between the sediments and allowed 3 days to choose. After 10 days the sediment containers were capped and the larvae numbers were recorded. When the sediments were identical, or there was no choice in sediment, the numbers of C. riparius larvae were similar in both sides. When the sediments were different, or there was choice, there was a difference in the number of C. riparius larvae found in the two sediments. I devised a similar experiment to De Haas et al.
(2006) and observed how soil composition influenced the position of initial numbers of L. terrestris when they were exposed two different soil types. I believed that if chironomids could choose between sediments, then L. terrestris might be able to choose between soils as well.
As previously discussed, earthworm, along with other macroinvertebrate, distributions are restricted by certain soil disturbances, additions, compositions, and pH levels. Specifically, earthworms react to soil changes and properties by avoiding them when possible or showing changing distributions within the soil. I measured how soil properties could affect earthworm distributions by conducting two studies, one field study and one laboratory study. My field study investigated the hypothesis that greater numbers of different macroinvertebrates would be found in the soil samples from mostly shaded soil than fully exposed soil. I believed this hypothesis was valid because Haskell (2000) had suggested a difference in macroinvertebrate numbers in soils exposed to the sun and wind as soils were, when next to the roads in Cherokee National
Forest in Tennessee. Intuitively, I believed this hypothesis was also valid because shaded areas
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would have more moisture retention than exposed areas. I studied my field study hypothesis by collecting soil samples from fully sun exposed soil and mostly to full shaded soil and recording the number of different macroinvertebrates found within the samples. My laboratory study investigated the hypothesis that L. terrestris would be found throughout the normal soil and not the autoclaved soil. I believed this hypothesis was valid because I assumed that the normal soil would retain more moisture than the autoclaved soil. I tested my laboratory study hypothesis by using L. terrestris along with similar techniques done by De Haas et al.
(2006) for conditions that allowed L. terrestris to choose a preferable soil when exposed to two different or similar soil conditions.
METHODS
Field component
I began my experiment with the collection of soil samples from two sites northeast of the
Saint Martin’s University campus: one site completely shaded and the other fully exposed to the sun. I chose those areas because they resembled Haskell’s (2000) samples roads in Cherokee
National Forest, where the soil near the edge of the roads was exposed to sunlight and soil further away from the roads was less exposed to sunlight. In my study, I collected three replicates at each site in a triangular pattern, approximately 1 m from each other, similar to
Haskell’s (2000) sampling of triplets for a better representation of his sampled sites. At each of the three points of the triangle I noted the top layer content and moved all the contents aside and exposed the topsoil layer. Then I noted the weather before I collected my replicates as a survey of the sunlight exposure conditions during sampling times. I collected the soil samples into plastic, cylindrical ice cream containers for later analysis in the laboratory. My replicates were
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3375 cm
3
(15 cm x 15 cm x 15 cm) each which gave the total of 10125 cm
3
of soil to represent each site. I chose 3375 cm 3 as the volume because it was small enough to fit into my containers and large enough to adequately represent my two sites. In the laboratory I sorted through the samples twice and noted the number of different macroinvertebrates found within the samples. I sorted through each replicate the first time noting all the macroinvertebrates in the entire replicate, and then I took a 250 ml volume sample of each replicate for further detailed examination. I differentiated the macroinvertebrates by physical appearance and tallied each macroinvertebrates’ abundance within my collected topsoil samples. I was more concerned with the number of different macroinvertebrates rather than the classification of the macroinvertebrates.
I collected my soil samples on three consecutive Saturdays, February 3, 10, and 17. I sampled in the same northeast vicinity of Saint Martin’s University each Saturday. The shaded areas were close to trails and the exposed areas were littered with wood chippings. Table 1 was an example of my data spreadsheet.
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Table 1. Experimental data spreadsheet for noting the number of macroinvertebrates for the two sampled sites within my study. The sampling sites were located in the northeast vicinity of Saint Martin’s
University. Data was collected for three consecutive weeks in the month of February, 2007.
Collected area
Replicate 1
(A)
Replicate 2
(B)
Replicate 3
(C)
Weather Weather Weather
Shaded soil
Top litter composition
Top litter composition
Top litter composition
Macroinvertebrate Macroinvertebrate Macroinvertebrate
Description Tally Description Tally Description Tally
Weather Weather Weather
Exposed soil
Top litter composition
Top litter composition
Top litter composition
Macroinvertebrate Macroinvertebrate Macroinvertebrate
Description Tally Description Tally Description Tally
Laboratory component
My laboratory study resembled that of De Haas et al.
(2006). In my choice experiment L. terrestris was investigated in two habitable potting soils divided into left and right sections within cylindrical containers (Table 2). I used Sta-Green All Purpose Potting Mix for my soil. I only used left and right divisions because L. terrestris are mostly surface inhabits, so top and bottom divisions would result in irrelevant data. The data was irrelevant because L. terrestris would only be found at the top soil division of the soil whether or not the two soil layers differed in composition. Unlike the study of De Haas et al.
(2006), I counted the number of L. terrestris instead of recording their masses. In the laboratory I conducted four total temperature treatments
– the control was in the same room as the room temperature treatment – each with 1% moisture and 3% moisture, as shown in Table 2. Each temperature treatment, as well as the control, had three replicates. The temperature treatments were conducted in one cool (15 °C) storage room, one room temperature (21 °C) storage room, and one warm (25 °C) storage room. These rooms
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were available in the Old Main building of Saint Martin’s University. I recorded the soil temperatures on the first, second and last day of my laboratory experiment and calculated the means of the temperatures for each treatment. I chose to record the temperatures on the first, second and last day of my laboratory experiment because I assumed the temperatures would not fluctuate as much as they would be shown on those days.
Table 2. The left and right sectional division treatments of the laboratory component. This schematic is an aerial view of my containers. In actuality my containers were circular.
Control*
(approximately 21 °C)
Cool storage room
(approximately 15 °C)
Room storage room
(approximately 21 °C)
Warm storage room
(approximately 25 °C)
N** N A*** N A N A N
3 replicates at 1% moisture
Control
(approximately 21 °C
N N
3 replicates at 1% moisture
Cool storage room
(approximately 15 °C)
A N
3 replicates at 1% moisture
Room storage room
(approximately 21 °C)
A N
3 replicates at 1% moisture
Warm storage room
(approximately 22 °C)
A N
3 replicates at 3% moisture
3 replicates at 3% moisture
*The control was situated with the Room temperature treatment
3 replicates at 3% moisture
3 replicates at 3% moisture
**N stands for non-autoclaved (normal) topsoil
***A stands for autoclaved topsoil
The two habitable potting soils were actually one brand but one potting soil was not manipulated (normal) and the other was autoclaved. These two potting soils were placed in cylindrical containers separated by netting situated from the bottom of the container to 2/3 the height of the soil for easy soil separation during the counting of L. terrestris within each section at the end of this experiment. Half of the potting soil was autoclaved, or sterilized, under 15 psi at 121.6 ºC for 15 minutes and the other half was maintained in its original state. This difference in soil was factored into my experiment as an observation of Haskell’s (2000) experiment deductions on human influences that might or might not impact the macroinvertebrate
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populations within the soil. The autoclaved potting soil represented soil that differed from the unaltered potting soil. I used sixteen L. terrestris for each replicate. The moisture content was kept in the treatments by adding water volume equivalent to 3% or 1% of the soil volume. L. terrestris was placed between the two soils vertically divided. After five days the locations of L. terrestris were recorded for the warm treatment, two days later data was recorded for the control treatment, another two days later data was recorded for the room treatment and the another two days later data was recorded for the cold treatment. L. terrestris distribution data that I gathered from my trials were subjected to an Analysis of Variance (ANOVA) test. Then as I found significant differences then I subjected the data to a Tukey test for multiple comparisons(Minitab,
2005).
RESULTS
Field component
The field component examined the relationship between the numbers of different macroinvertebrates present to the location of the gathered soil samples, similar to when Haskell
(2000) surveyed the Cherokee National Forest in Tennessee. There was an observed difference in the number of different macroinvertebrates found within the two different sites within the northeast vicinity of Saint Martin’s University (F = 14.56; d.f. = 1; p = 0.002). The mean number of macroinvertebrates was higher within the shaded soil samples (mean = 2.89) than the exposed soil samples (mean = 0.89).
Laboratory component
The laboratory component investigated the effect temperature, water moisture, and soil type played on the numbers of L. terrestris present within each treatment. The mean soil
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temperatures for temperature treatments ranged from 15.5 ºC within the cold treatment, 21.5 ºC within the control treatment, 21.7 ºC within the room treatment, and 22.1 ºC within the warm treatment. A two way ANOVA (Minitab, 2005) was performed on the number of L. terrestris found within the autoclaved and non-autoclaved (normal) potting soils for each temperature treatment. There was no interaction between the soil type and water moisture for the warm (22.7
ºC) treatment (F = 0.24; d.f. = 1; p = 0.640), the control treatment (F = 2.77; d.f. = 1; p = 0.135), and the cold treatment (F = 2.45; d.f. = 1; p = 0.156) but there was an interaction between the soil type and water moisture for the room (21.7 ºC) treatment (F = 7.38; d.f. = 1; p = 0.026). In all aspects the control (21.5 ºC) treatment showed no difference in the number of L. terrestris found in each soil type held at the different water moistures.
The 22.1 ºC, 21.5 ºC, and 15.5 ºC treatments showed no interaction between the soil type and water moisture so each condition, separately, affected the number of L. terrestris found in each soil type held at each percent water moisture (Figure 1).
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A B
0
-2
4
2
8
6
14
12
10
Autoclaved
Normal
14
12
10
8
6
4
2
0
Autoclaved
Normal
1% 3%
Percentage of water moisture
1% 3%
Percentage of water moisture
C D
14
12
10
8
6
4
2
0
Normal
Normal
14
12
10
8
6
4
2
0
Autoclaved
Normal
1% 3% 1% 3%
Percentage of water moisture Percentage of water moisture
Figure 1: The mean numbers of L. terrestris found within the autoclaved and non-autoclaved (normal) potting soils in the four temperature treatments. Each water moisture condition was done in replicates of three. The percentage of water moisture was based on the water volume to the soil volume within each container. All standard error bars represent one standard deviation from the mean. (A) The warm treatment, held in an incubator at 25.0 ºC, had a mean soil temperature of 22.1 ºC. The mean numbers of
L. terrestris had a range of 1 to 4 individuals within the normal soil and 2 to 4 individuals within the autoclaved soil. (B) The room treatment, held in the storage room on the fourth floor of Old Main, had a mean soil temperature of 21.7 ºC. There was more variation within the autoclaved potting soil than the normal potting soil as observed by the length of the error bars. (C) The control treatment, held in the same storage room as the room treatment, had a mean soil temperature of 21.5 ºC. Unlike all the other treatments the control was done using two normal potting soils. (D) The cold treatment, held in the storage room within the chemistry laboratory on the first floor of Old Main, had a mean temperature of 15.5 ºC. There was more variation in the containers held at 3% water moisture than the containers held at 1% water moisture as shown with the error bars.
The soil type had no effect on the number of L. terrestris in the 22.1 ºC treatment (F = 0.03; d.f.
= 1; p = 0.875). Instead the 22.1 ºC treatment had a substantially greater number of L. terrestris found within the 3% than the 1% water moisture revealed by a Tukey test (F = 5.92; d.f. = 1; p =
0.041) (Minitab, 2005). The cold (15.5 ºC) treatment had a substantial number of L. terrestris found in the normal than the autoclaved potting soil (F = 12.8; d.f. = 1; p = 0.007).
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DISCUSSION
Field component
My results supported my hypothesis. I had hypothesized the number of different macroinvertebrates found in the mostly shaded soil samples would be higher than that of the fully sun exposed soil samples found at the northeast vicinity of Saint Martin’s University because my results showed a significant difference between the mostly shaded soil compared to the fully sun exposed soil (F = 14.56; d.f. = 1; p = 0.002). The difference in locations did lead to the differences in the numbers of macroinvertebrates as shown by Haskell (2002), whom found differences in the number of taxa from the different locations of his sampled sites within the
Cherokee National Forest in Tennessee.
There might have been other factors besides sun exposure that might have affected the number of macroinvertebrates within the soil. The fully exposed soil had wood chip debris on its surface while the mostly shaded soil had leaf litter on its surface. The wood chip debris might have a longer decomposition time than the leaf litter so the leaf litter might have contributed more to the nutrient supply for the macroinvertebrates found within the mostly shaded soil. I had noted the difference in the superficial layer but had not included it into my study. In the future, I recommend further investigations into the number of macroinvertebrates as influenced by the superficial layer on top of the soil. I also think future studies could be more valuable if the two sites were studied for a longer duration, such as a year and the studies expanded to incorporate other areas along with the northeast vicinity of Saint Martin’s University. For future considerations on my field component I encourage a longer study time to include seasonal variations. I would also encourage the recording of soil temperatures prior to collection so it could also be a factor. I also encourage genus identification so the number of individual
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macroinvertebrate abundance could be included with the numbers of different macroinvertebrates.
Laboratory component
I rejected my hypothesis. I had hypothesized that L. terrestris would be found in the normal, untreated potting soils throughout all the treatments, but my data showed differences in the different temperature treatments. Under the control temperature treatment, where both the potting soils were normal, the number of L. terrestris on either side showed no preference for one side or the other, as expected because both sides were the same. These control results made the soil preference in the other treatments more apparent when differences did occur due to soil type or water moisture. The significant difference in the number of L. terrestris found between the two water moistures, within the warm treatment, could have been due to more benefits of having more moisture as apparent in the 3% water moisture than the 1% water moisture (F =
5.92; d.f. = 1; p = 0.041). On the other hand, contrast to the warm treatment, the cold treatment had a significant difference between the autoclaved and normal soils (F = 12.8; d.f. = 1; p =
0.007). When I parted the two soils separately, I had noticed that the autoclaved soil was dryer than the normal soil for both of the 1% and 3% water moistures. This difference in dryness could have resulted in the greater number of L. terrestris found within the normal soil. The autoclaved soil, which was dryer, could have been abrasive to L. terrestris so they were found in the normal soil because they might have had greater motility in it. Contrary to both the 22.1 ºC and 15.5 ºC temperature treatments, the 21.7 ºC treatment showed an interaction between the soil type and the water moisture content so I was unable to examine the soil type and water moisture as independent factors.
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In the future, I recommend utilizing another control, in addition to the normal potting soil control, composed of both autoclaved potting soils. Also I would recommend staggering the individual temperatures treatments so experimental duration could be consistent. I would also increase the number of earthworms in each replicate because some replicates had returned with no evident earthworms. My study could also be expanded to other macroinvertebrates that are also mobile within the soil. Further investigations of macroinvertebrate-soil relationships in addition to those previously done by Arrhenius (1921), Scullion et al.
(1988), Gonzalez and Zou,
(1999), Spurgeon and Hopkin (1999), Haskell (2000), and De Haas et al.
(2006), can help to expand our understanding about how macroinvertebrates are influenced by outside manipulations of soil that might go along with our growing residential areas. In conclusion, future studies could broaden the soil and earthworm relationship by exposing several species under the same soil conditions.
ACKNOWLEDGEMENTS
I would like to acknowledge my family for their support and creative input into my project. Dr. Mary Jo Hartman and Dr. Margaret Olney helped in the growth of my research project. Cheryl Guglielmo had provided me with access to the storage rooms. Si’i Vulangi helped me gather my field samples. Wendy Matson suggested presentation tactics. I would also like to thank my Senior Biology Class of 2007 for their friendship and encouragement throughout my research project.
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LITERATURE CITED
Arrhenius, O. 1921. Influence of soil reaction on earthworms. Ecology . 2: 255–257.
De Haas, E.M., Wagner, C., Koelmans, A.A., Kraak, M.H., Admiraal, W. 2006. Habitat selection by chironomid larvae: fast growth requires fast food. The Journal of Animal
Ecology . 75: 148–155.
Gonzalez, G., Zou, X. 1999. Plant and litter influences on earthworm abundance and community structure in a tropical wet forest. Biotropica . 31: 486–493.
Haskell, D.G. 2000. Effects of forest roads on macroinvertebrate soil fauna of the southern
Appalachian Mountains. Conservation Biology . 14: 57–63.
Minitab Inc. 2005. Minitab Release 14.20.
Scullion, J., Mohammed, A.R.A., Richardson, H. 1988. Effect of storage and reinstatement procedures on earthworm populations in soils affected by opencast coal mining. The
Journal of Applied Ecology . 25: 233–240.
Spurgeon, D.J., Hopkin, S.P. 1999. Seasonal variation in the abundance, biomass and biodiversity of earthworms in soils contaminated with metal emissions from a primary smelting works. The Journal of Applied Ecology . 36: 173–183.
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