Survival of Aporrectodea caliginosa and its effects on nutrient availability in biosolids amended soil

Applied Soil Ecology 71 (2013) 1–6

Contents lists available at SciVerse ScienceDirect

Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil

Survival of Aporrectodea caliginosa and its effects on nutrient availability in biosolids amended soil Jacob P. McDaniel a,∗ , Mary E. Stromberger a , Kenneth A. Barbarick a , Whitney Cranshaw b a

Department of Soil and Crop Sciences, 1170 Campus Delivery, Colorado State University, Fort Collins, CO 80523-1170, United States Department of Bioagricultural Sciences and Pest Management, 1177 Campus Delivery, Colorado State University, Fort Collins, CO 80523-1177, United States b

a r t i c l e

i n f o

Article history: Received 4 February 2013 Received in revised form 25 April 2013 Accepted 28 April 2013 Keywords: Earthworm Soil nitrogen availability Colorado Organic matter

a b s t r a c t Few earthworms are present in production agricultural fields in the semi-arid plains of Colorado, where earthworm populations may be constrained by limited water and/or organic matter resources. We conducted a 12-week laboratory incubation study to determine the potential of a non-native endogeic earthworm (Aporrectodea caliginosa) to survive in a low-organic matter Colorado soil (1.4% organic C content), supplemented with or without biosolids, and to determine the effects of A. caliginosa on soil microbial biomass and soil nutrient availability. A factorial design with three main effects of A. caliginosa, biosolids addition, and time was used. Data was collected through destructively sampling at one, two, four, eight, and twelve weeks. During the 12-week study, 97.5% of the worms in the soil survived, and the survival of the earthworms was not significantly affected by the addition of biosolids. The addition of biosolids, however, did significantly reduce the gain in mass of the earthworms (8% mass gain compared to 18% in soil without biosolids). The presence of A. caliginosa significantly increased soil NH4 -N, and NO3 -N concentrations by 31% and 4%, respectively, which was less than the six fold increases in both soil NH4 -N, and NO3 -N concentrations supplied from biosolids. Microbial biomass carbon was not affected by A. caliginosa, but microbial biomass N was affected by an earthworm × biosolids interaction at week 1 and 12. We concluded that A. caliginosa can survive in a low-organic matter Colorado soil under optimal moisture content and that once established, A. caliginosa can provide modest increases in inorganic N availability to crops Colorado agroecosystems. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The effects of earthworms on soil were first documented by Darwin (1886), and for many years, farmers and soil scientist have associated the presence of earthworms as an indication of good soil quality (Doran and Safley, 1997; Roming et al., 1996; Yeates et al., 1998). This is namely due to the effects of earthworms on plant nutrient availability, particularly on increasing the availability of N in the soil. Earthworms can also improve soil quality through their effects on soil physical properties, including mixing and reorganizing soil (Darwin, 1886; Martin and Marinissen, 1993; Oades, 1993; Schrader and Zhang, 1997), creating macropores (Tisdall, 1978, 1985), and changing and improving water and gas flow (Lee and Foster, 1991). Currently there are few earthworms present in production agricultural fields in eastern Colorado, but the transition to notillage (NT) practices could improve conditions for earthworms by

∗ Corresponding author. Tel.: +1 970 491 0636; fax: +1 970 491 5676. E-mail address: [email protected] (J.P. McDaniel). 0929-1393/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsoil.2013.04.010

reducing physical disturbance, increasing water holding capacity, and/or increasing organic matter content (Edwards and Lofty, 1982; House and Parmelee, 1985; VandenBygaart et al., 1999). Besides the conversion from conventional tillage to no-tillage, another way to increase the soil organic matter would be the addition of an organic amendment such as manure or biosolids. The addition of organic matter has been shown to increase earthworm populations in irrigated, forage agroecosystems in Colorado (Hurisso et al., 2011). As earthworms expand into agricultural fields in Colorado, it will become important to understand the effect of earthworms on soil fertility to make correct nutrient management decisions. Earthworms can dramatically affect the concentration of plant available N through the mineralization of soil organic matter and excretion of nitrogenous wastes (Edwards and Lofty, 1977). This process is important when the fertility of the soil is dependent on the mineralization of organic materials such as biosolids or manure (Lubbers et al., 2011). Earthworms aid not only by direct mineralization of organic matter but also by stimulating microbial activity in soil (Curry and Schmidt, 2007). Earthworms primarily affect N availability by increasing the concentration of ammonium-nitrogen (NH4 -N) in the soil due to digestion and excretions of wastes, as

2

J.P. McDaniel et al. / Applied Soil Ecology 71 (2013) 1–6

well as their release of mucus (Whalen et al., 2000). The NH4 -N may be oxidized to nitrate-nitrogen (NO3 -N) by nitrifying bacteria, which are stimulated by earthworm burrowing activities (Parkin and Berry, 1999) that increase the oxygen concentration deeper in the soil profile (Costello and Lamberti, 2008). Biosolids have been studied extensively as a soil amendment (for a review, see Haynes et al., 2009), and in Colorado, a main focus area of research has been to study the accumulation of metals in soil with repeated biosolids application (Ippolito and Barbarick, 2008). Outside of Colorado, studies have been conducted to investigate heavy metal availability due to biosolids and earthworm interactions (Protz et al., 1993; Tomlin et al., 1993), but have not focused on the potential of biosolids to prove organic matter to support earthworm populations. Historically, earthworms have been employed for toxicity tests of biosolids materials (Artuso et al., 2011; Moreira et al., 2008; Natal-da-Luz et al., 2009). Many of these studies utilized anecic or epigeic species, such as Lumbricus terrestris and Eisenia andrei, respectively. Anecic earthworms create permanent vertical burrows and move organic matter from the soil surface deeper into the soil profile, whereas epigeic species live on the surface and feed on organic residues. These earthworms may have a larger effect on nutrient availability due to the potential to redistribute surface-applied biosolids down into the burrows. In contrast, Aporrectodea caliginosa appears to be the most common species in Colorado (Reynolds, 2011) and has the greatest potential for colonization of agricultural fields. A. caliginosa is an endogeic species that primarily creates temporary horizontal burrows and feeds on soil organic matter rather than surface organic matter. Because A. caliginosa feeds on soil organic matter, its expansion in agricultural soils of eastern Colorado may be constrained by low quantities of organic matter, relative to less arid regions of the world. We conducted a 12-week laboratory incubation study to determine the potential of A. caliginosa to survive in a low organic-matter agricultural soil from Colorado. We hypothesized that an amendment of incorporated biosolids would enhance earthworm survival and prevent weight loss by increasing organic matter availability to the earthworms. We also investigated the effects of A. caliginosa, in the presence or absence of biosolids amendments, on plant available nitrogen, and other nutrients, and microbial biomass.

2. Methods and materials The soil selected for this study was a sandy loam (56% sand, 30% silt, 14% clay; 1.4% organic C content, 7.46 pH) Adena (Ustic Paleargid)-Colby (Aridic Ustorthents) complex that represents approximately 900k ha in the western United States (Soil Survey Staff, 2012). The soil was obtained from the top 10 cm of a dryland no-till wheat-corn-fallow research plot near Byers, Colorado (latitude 39.7631921, longitude 103.7973089). The soil was passed through a 1.0-cm sieve and homogenized using a cement mixer. The equivalent of 1 kg of oven-dried soil was added to plastic containers (13.5 cm × 11.0 cm × 9.5 cm), and packed to a bulk density of 1 Mg m−3 (1 g cm−3 ). Prior to adding the soil, a total of 16 holes approximately 0.65 cm in diameter were made in the sides of the containers and covered with fiberglass screen to provide air flow and prevent earthworms from escaping. A plastic lid was then placed on each container. Water was added to the containers of soil to adjust the gravimetric water content to approximately 14% gravimetric water content (approximately 70% of field capacity). The containers were then placed in an incubator at 17 ◦ C for four days prior to the start of the incubation study to allow the microbial communities to adjust to the new environment. The water content was held near constant conditions throughout the study with the use of weekly watering.

Table 1 Chemical properties of anaerobically digested biosolids utilized in this laboratory incubation study. Parameter (units)

−1

Solids (mg kg ) pH EC (dS m−1 ) Organic N (mg kg−1 ) NH4 -N (mg kg−1 ) NO3 -N (mg kg−1 ) Ag (mg kg−1 ) Al (mg kg−1 ) As (mg kg−1 ) Ba (mg kg−1 ) Be (mg kg−1 ) Cd (mg kg−1 ) Cr (mg kg−1 ) Cu (mg kg−1 ) Fe (mg kg−1 ) Hg (mg kg−1 ) K (mg kg−1 ) Mn (mg kg−1 ) Mo (mg kg−1 ) Ni (mg kg−1 ) P (mg kg−1 ) Pb (mg kg−1 ) Se (mg kg−1 ) Zn (mg kg−1 ) a

Biosolids utilized

796,000 6.60 15.7 24,400 13,300 1.51 1.53 75,000 5.36 27.9 0.02 1.45 17.9 708 14,300 1.15 1920 279 11.6 10.6 23,500 15.4 6.48 665

The National Sewage Sludge Surveya

Median

Mean

Minimum

Maximum

13.6 11,200 4.96 426 0.278 1.76 32.7 463 15,700 0.827

31.6 13,600 7.09 567 0.391 2.67 81.5 558 27,700 1.24

1.94 1400 1.18 76.9 0.04 0.208 6.74 115 1580 0.19

856 57,300 49.2 2120 2.34 11.8 1160 1720 195,000 7.5

420 11.5 23.5 19,300 48.2 6.25 784

1220 16.3 48.9 22,400 76.6 7.12 994

34.8 2.51 7.61 5720 5.81 1.1 216

14,900 86.4 77,100 350 24.2 8550

Robert Brobst, USEPA, personal communication.

Adult A. caliginosa were used for this study, which were collected by hand sorting in September 2009 from the edge of an irrigated alfalfa field at Colorado State University (CSU) Agricultural Research Development and Education Center (ARDEC). Earthworms were taken back to the laboratory and placed in a large plastic container of soil from the ARDEC site. The worms were allowed to equilibrate in field moist, ARDEC soil for four days. After four days, two worms were placed in each of 80 petri dishes on wet filter paper and placed back in the incubator overnight for gut evacuation of ARDEC soil. A. caliginosa were rinsed with deionized water and blotted dry before an evacuated, fresh weight was obtained on each pair of earthworms. Two earthworms were added to each container, which approximated 400 earthworms m−2 . While this density was higher than what has been reported in one Colorado study (Hurisso et al., 2011), it is lower than the density used in another laboratory incubation study which examined the effects of earthworms on nitrogen mineralization (Willems et al., 1996). Moreover, Hurisso et al. (2011) reported that the dry mass of earthworms was 2.3–13 g m−2 in the field, which was near the dry mass in the containers, determined at the end of this study to be 16.2 g m−2 . We believe that the earthworm density in this study is not an unreasonable representation of what could occur in Colorado field soil. A factorial design was used for this study with the main effects of biosolids application, earthworms, and time. At each sampling time, there were four replicates of each treatment: the control, with biosolids, with earthworms, and with earthworms and biosolids. It was assumed that the size of the worms would be related to the worms’ activity; therefore, the experiment was blocked by the starting mass of the earthworms. There were five time points that were designated for destructive sampling (one, two, four, eight, and 12 weeks). Dry, anaerobically digested biosolids were obtained from the Littleton/Englewood Wastewater Treatment Plant in Englewood, CO. Biosolids chemical properties are shown in Table 1. Biosolids were mixed with soil for each pot individually before filling the pots at rate equivalent to 11.2 Mg ha−1 (based on 20 cm soil depth).

J.P. McDaniel et al. / Applied Soil Ecology 71 (2013) 1–6

3

Table 2 Summary of statically significant effects of incubation time (0, 1, 2, 4, 8, and 12 weeks), addition of incorporated biosolids (0 Mg ha−1 , and 11.2 Mg ha−1 ), addition of two adult earthworms (Aporrectodea caliginosa), and their interactions during a laboratory incubation study in a sandy loam soil. (NS = P-value > 0.1). Parameter

Week

Biosolids

Week × biosolids

Earthworms

Week × earthworms

Biosolids × earthworms

Week × biosolids × earthworms

Earthworms × block

Block

Nitrate Ammonium Microbial biomass C Microbial biomass N EC AB-DTPA extractable Ca Cu Fe K Mg Mn P Zn

<0.001 <0.001 <0.001 <0.001 <0.001

<0.001 <0.001 0.020 NS <0.001

<0.001 <0.001 NS <0.001 <0.001

0.082 0.084 NS NS NS

NS NS NS 0.099 NS

NS NS NS NS NS

NS NS NS NS NS

NS NS NS NS NS

NS NS 0.001 0.020 NS

<0.001 <0.001 <0.001 <0.001 NS <0.001 <0.001 <0.001

NS NS NS NS NS NS NS 0.001

NS NS NS NS NS NS NS NS

NS NS NS NS NS NS NS NS

NS NS NS NS NS NS NS NS

NS NS NS NS NS NS NS NS

NS NS NS NS NS NS NS NS

NS NS NS NS NS NS NS NS

0.002 NS <0.001 NS NS 0.008 0.001 NS

This rate is typical of what would be applied for irrigated maize production in Colorado, and was selected as a means to increase the organic matter content of the soil. Four replications of each treatment were destructively sampled at the end of each time period and individually homogenized in a large beaker. Two subsamples from each container were taken and either air-dried (for chemical analyses) or refrigerated at 4 ◦ C (for NH4 -N and NO3 -N and microbial biomass determinations). If the container included earthworms, the earthworms were removed before the soil samples were collected and placed in petri dishes with wet filter paper. The worms were placed in the incubator overnight and an evacuated, fresh weight was obtained for each pair of worms. Soil NO3 -N and NH4 -N samples were extracted two days after sampling, following the Mulvaney (1996) procedure, on soil that had been stored in the refrigerator. The samples were then frozen until analysis of NO3 -N and NH4 -N on an Alpkem Flow Solution IV Automated wet chemistry system (O.I. Analytical, College Station, TX). Plant available nutrients (Ca, Cu, Fe, K, Mg, Mn, P, and Zn) were determined by an AB-DTPA soil extraction (Barbarick and Workman, 1987). The samples were refrigerated until analyzed using inductively coupled plasma-atomic emission spectrometry (ICP-AES) (IRIS ADVANTAGE Radial ICP, TJA Solutions, West Palm Beach, FL). Soil pH (Thomas, 1996) and electrical conductivity (EC) (Rhoades, 1996) analyses were performed on air-dried previously screened (2-mm sieve) soil using the saturated paste method (Rhoades, 1996). Total soil C and N were determined by sample combustion using a LECO CHN-100 Autoanalyser® (St. Joseph, Michigan). Inorganic C was analyzed by the pressure transducer method (Sherrod et al., 2002). Microbial biomass C and N were analyzed using a by the chloroform fumigation extraction method (Brookes et al., 1985; Vance et al., 1987) two days after destructive sampling on soil that had been stored in the refrigerator. Extract samples were frozen prior to analysis on a total organic carbon analyzer (Shimadzu Total Organic Carbon Analyzer (model TOC-Vcpn+TNM-1, Columbia, MD)) which simultaneously measured both C and N; a 10-fold dilution was performed on the samples prior to analysis. Data analysis was performed with SAS version 9.2 (SAS Institute, 2008) using Proc Glimmix. An alpha value of 0.1 was used for determining statistical significance. The data were checked for normality and homogeneity of variance, and when necessary a log transformation was used. The data presented in the results are the non-transformed data. When interactions were significant, only the interaction was further considered rather than individual main effects. When effects were significant, means were separated by the least significance difference (LSD) method.

There were two containers in the A. caliginosa treatment of which only one of two worms was found at the time of sampling (one replicate from the week one no biosolids treatment, and one replicate from the week 4 with biosolids treatment). Soil data from these containers were not included in the statistical analysis and were treated as missing data points instead. This was done because the effect of earthworms would not be accurately represented in these samples as the exact time of the earthworm loss was unknown. In addition, there would be less interaction of the earthworms with the soil and biosolids, resulting in a bias in the data. 3. Results and discussion Below we present the results of earthworm survival, and report the effects of earthworms on soil nutrients and biomass. In many cases soil nutrients and microbial biomass were significantly affected by biosolids or the biosolids × time interaction (Table 2), but of these results, only those that are relevant to explaining earthworm effects are discussed below. 3.1. Earthworm survival and mass The organic C content of the study’s soil was low (1.4%) and typical of agricultural soils of eastern Colorado. Addition of biosolids increased the organic C content from 1.4% to 1.5%. In order to determine if there was enough organic matter to support earthworms, the change in mass was studied, as well as the survival of the earthworms. Survival of earthworms was not affected by biosolids. This is in agreement with Artuso et al. (2011), who found that A. caliginosa is not affected by moderate additions of biosolids. By the end of the study, the average number of earthworms was 1.95 in both the control and biosolids-amended containers. A total of two containers had less than two earthworms at the end of study (one from a no-biosolids control container and one in a biosolids-amended container). Regardless of whether biosolids were added, the mass of the earthworms increased over incubation time. The change in the mass of A. caliginosa on a percent of starting mass basis was affected by the addition of biosolids (P = 0.038). There was a 17.7% increase without biosolids and a 7.65% increase with biosolids. There was not a significant time effect (P = 0.185), indicating biosolids did not have an increasing negative effect on earthworms over time. The gain in mass of the earthworms in the control soil (no biosolids) was about twice the gain of earthworm biomass in biosolids amended soil. This is perhaps due to the increase in soil EC in response to biosolids addition. There was a significant time × biosolids interaction (P ≤ 0.001) on soil EC that resulted in an increase from 0.59 dS m−1 at that start of the study to 2.74 dS m−1

J.P. McDaniel et al. / Applied Soil Ecology 71 (2013) 1–6 30 With Biosolids Without Biosolids

A 25

20 -1

at week 12 in the biosolids treated soil. This increase of approximately 2 dS m−1 may have impacted the ability of earthworms to gain mass. A soil EC of 1.58 dS m−1 has been shown to negatively affect earthworms mass after more than 30 days of exposure (Jun et al., 2012). Also, the soil was not allowed to freely drain in the present study. Consequently, salts were unable to move deeper into the profile, thus allowing for an increase in the EC of the soil. In contrast another study showed that the addition of biosolids is beneficial to earthworm populations (Baker et al., 2002). This study indicated that biosolids amendment added significant organic matter to their soil, which aided in the survival of earthworms. Baker et al. (2002) showed that the benefits of biosolids were related to the application rate, with no additional benefit at rates higher than 30 Mg ha−1 . The application rate in our study was 11.2 Mg ha−1 , which is higher than the typical application rate for winter wheat production but typical for irrigated maize in Colorado. Barbarick and Ippolito (2000) have recommend a rate of 4.4 Mg ha−1 of dry biosolids for the production of winter wheat in Colorado. A larger increase in earthworm mass might have been observed at this lower application rate because of reduced negative impacts of biosolids constituents, such as salts.

NH4-N (mg kg )

4

A 15

10

B B

5 CD 0

DE

F

DE

EF

2

4

C

C

-5 0

6

8

10

12

14

Week Fig. 1. Changes in soil NH4 -N concentrations over time, in a sandy loam soil incubated in the presence or absence of biosolids (0 Mg ha−1 , and 11.2 Mg ha−1 ) averaged across the presence or absence of Aporrectodea caliginosa. Biosolids and time had a significant interactive effect on the log transformed soil NH4 -N concentrations. Values labeled with the same letter are not significantly different at an alpha value of 0.10.

3.2. Soil properties mineralization and subsequently nitrification. The effect of earthworms on the increase in NO3 -N was significant (P = 0.082), although the actual increase in NO3 -N was only very slight (4%). Earthworms presumably enhanced nitrification by supplying NH4 N and oxygen through burrowing activity, as shown by others (Blair et al., 1995). During the incubation period, the concentration of NO3 -N in soil without biosolids increased threefold, but with the addition of biosolids, there was a six-fold increase. The factorial design of the experiment should have allowed for separation of earthworm and biosolids effects but due to the magnitude of the biosolids, earthworm effects were less influential. Based on these results, A. caliginosa has the potential to increase N availability to crops in Colorado agricultural soils, mainly by increasing NH4 -N concentrations. In contrast, NO3 -N concentrations would be affected mainly by biosolids land application, rather than the presence of earthworms. Additional studies are necessary, however, to confirm if these trends would occur under field conditions.

140 A

A 120

100

-1

NO3-N (mg kg )

A log transformation was performed on the soil NO3 -N and NH4 -N data to correct for heterogeneity of variance. The addition of the earthworms influenced both NO3 -N (P = 0.082) and NH4 -N (P = 0.084), when averaged across biosolids treatments. The NO3 -N levels increased 4% with the addition of earthworms to 53.7 mg kg−1 NO3 -N compared to 51.3 mg kg−1 NO3 -N without earthworms. In comparison, NH4 -N levels increased by 31% in the presence of earthworms, from 1.91 mg kg−1 NH4 -N in containers without earthworms to 2.51 mg kg−1 NH4 -N in containers with earthworms. This net increase in inorganic N due to A. caliginosa is equivalent to ∼6 kg of additional plant available N per hectare furrow slice. Earthworms produce a mucous coat to reduce the loss of moisture, facilitate respiration, and to act as a lubricant as the earthworm moves through the soil (Whalen et al., 2000). The mucus contains N-rich mucoproteins and mucous excretions account for approximately one-half of the N loss from earthworms each day (Needham, 1957). The remainder of the N loss from earthworms is from urine, which contain primarily NH4 and urea (Edwards and Bohlen, 1996), and from casts that contains most of the NH4 -N that is released from earthworms (Tillinghast, 1967). The increase in the NH4 -N concentration that was measured is in large part a result of the direct release of N by the earthworms. There was also an interaction between sampling time and biosolids (P ≤ 0.001), where NH4 -N concentrations increased over time but at different rates between control and biosolids-amended soil (Fig. 1). In the absence of biosolids (averaged over the presence or absence of earthworms), there was a slight increase in the NH4 -N concentration from approximately 0 mg kg−1 at time zero to 1.4 mg kg−1 over the 12-week period. This presumably was due to mineralization of soil organic matter by microorganisms and earthworms (when present). When biosolids were present, there was a large increase in NH4 -N during the first week of the study. This initial peak in mineralization activity was likely due to a pulse of microbial activity in the soil immediately after the addition of biosolids, which then slowed when the most available food sources were depleted. The application of biosolids also added a small amount of NH4 -N (Table 1). Soil NO3 -N concentrations increased over the incubation period, and the increase in NO3 -N was largely influenced by an interaction between time and biosolids application (Fig. 2). Biosolids contain a large amount of organic N that can undergo

B

80

B

C

C

C

60

40

20

D

D

D With Biosolids Without Biosolids

E

0 0

2

4

6

8

10

12

14

Week Fig. 2. Changes in soil NO3 -N concentrations over time, in a sandy loam soil incubated in the presence or absence of biosolids (0 Mg ha−1 , and 11.2 Mg ha−1 ) averaged across the presence or absence of Aporrectodea caliginosa. Biosolids and time had a significant interactive effect on the log transformed soil NO3 -N concentrations. Values labeled with the same letter are not significantly different at an alpha value of 0.10.

J.P. McDaniel et al. / Applied Soil Ecology 71 (2013) 1–6

productivity in eastern Colorado agricultural systems. While growers have alternative options for increasing plant N availability (e.g., biosolids amendment), earthworms may provide additional benefits to growers of semi-arid regions by improving soil structure, macroporosity, and soil water holding capacity.

140 120 -1

Microbial Biomass N (mg kg )

With Aporrectodea caliginosa Without Aporrectodea caliginosa

A

5

100 80 C

B

Acknowledgements

60

CD DE

DEF

Funding for this research was provided by the Littleton/Englewood Wastewater Treatment Plant and the Colorado Agricultural Experiment Station.

40 EFG 20

DEF DEFG

0

FG

G

References

-20 0

2

4

6

8

10

12

14

Week Fig. 3. Changes in microbial biomass N over time, in a sandy loam soil incubated in the presence or absence of Aporrectodea caliginosa averaged across the presence or absence of biosolids. A. caliginosa and time had a significant interactive effect on soil microbial biomass N, and values labeled with the same letter are not significantly different at an alpha value of 0.10.

In this study, A. caliginosa had no significant effect on soil pH, EC, plant nutrients other than N, total organic C, or microbial biomass C. However, there was a significant earthworm × time interaction effect on microbial biomass N (P = 0.099) (Fig. 3). Microbial biomass N increased during the first week, and was greater in soil with earthworms than without. The initial increase may have been due to improved environmental conditions of the soil at the beginning of the study compared to the initial field conditions of the soil. When the soil was collected, it had low moisture content, and it also had been subjected to higher temperature as it was collected at the end of the summer. The transition to a cooler, moister environment would be more favorable for microbial activity, which was also enhanced in the presence of earthworms at week one. Perhaps if the soil had been placed in the incubator for longer than 4 days to adjust to the new conditions prior to the study, the effects seen may have been reduced. Afterwards, earthworms had no effect until week 12, where there was a decrease in microbial biomass N. Other studies have also shown a decrease in microbial biomass N (Hendrix et al., 1998; Zhang and Hendrix, 1995) and it has been suggested this is due to earthworms increasing the rate of turnover of soil microorganisms (Hendrix et al., 1998), although it is unknown why turnover would be higher at week 12 compared to earlier in the incubation. 4. Conclusions The results of this study confirmed that A. caliginosa can survive up to 12 weeks in a low organic-matter soil from eastern Colorado, under ideal moisture and temperature conditions and without additions of exogenous organic matter. However, our hypothesis, that the addition of biosolids would increase the survival of A. caliginosa, was not supported. The biosolids addition did not result in the earthworms losing mass, but there was a reduction in the mass gained on a percent of starting mass basis possibly due to increased soil EC. The addition of biosolids may lead to secondary toxicity effects that reduce the ability of the earthworms to reproduce, gain weight, or develop. A longer-term study conducted under field conditions would need to be performed to determine if the overall effect is negative. As predicted, the addition of earthworms increased the availability of NH4 -N (and NO3 -N slightly), but did not affect any other measured nutrients, soil organic C, or microbial biomass C. Thus, colonization of agricultural soils in Colorado by A. caliginosa could modestly improve plant fertility and

Artuso, N., Kennedy, T.F., Connery, J., Grant, J., Schmidt, O., 2011. Assessment of biosolids in earthworm choice tests with different species and soils. Glob. Nest. J. 13, 255–265. Baker, G., Michalk, D., Whitby, W., O‘Grady, S., 2002. Influence of sewage waste on the abundance of earthworms in pastures in south-eastern Australia. Eur. J. Soil Biol. 38, 233–237. Barbarick, K.A., Ippolito, J.A., 2000. Nitrogen fertilizer equivalency of sewage biosolids applied to dryland winter wheat. J. Environ. Qual. 29, 1345–1351. Barbarick, K.A., Workman, S.M., 1987. Ammonium bicarbonate-DTPA and DTPA extractions of sludge-amended soils. J. Environ. Qual. 16, 125–130. Blair, J.M., Parmelee, R.W., Lavelle, P., 1995. Influences of earthworms on biogeochemistry. In: Hendrix, P.F. (Ed.), Earthworm Ecology and Biogeography in North America. Lewis Publishers, Boca Raton, FL, pp. 127–158. Brookes, P., Landman, A., Pruden, G., Jenkinson, D., 1985. Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol. Biochem. 17, 837–842. Costello, D., Lamberti, G., 2008. Non-native earthworms in riparian soils increase nitrogen flux into adjacent aquatic ecosystems. Oecologia 158, 499–510. Curry, J.P., Schmidt, O., 2007. The feeding ecology of earthworms – a review. Pedobiologia 50, 463–477. Darwin, C., 1886. The Formation of Vegetable Mould, Through the Action of Worms, With Observations on Their Habits. Appleton, New York, NY. Doran, J.W., Safley, M., 1997. Defining and assessing soil health and sustainable productivity. In: Pankhurst, C., Doube, B., Gupta, V. (Eds.), Biological Indicators of Soil Health. CAB International, Wallingford, Oxon, UK, pp. 1–28. Edwards, C.A., Bohlen, P.J., 1996. Biology and Ecology of Earthworms, third edition. Chapman and Hall, London, England. Edwards, C.A., Lofty, J.R., 1977. Biology of Earthworms, second edition. Wiley, New York, NY. Edwards, C.A., Lofty, J.R., 1982. The effect of direct drilling and minimal cultivation on earthworm populations. J. Appl. Ecol. 19, 723–734. Haynes, R.J., Murtaza, G., Naidu, R., 2009. Inorganic and organic constituents and contaminants of biosolids: implications for land application. In: Sparks, D.L. (Ed.), Advances in Agronomy, vol. 104. Elsevier Academic Press Inc., San Diego, pp. 165–267. Hendrix, P.F., Peterson, A.C., Beare, M.H., Coleman, D.C., 1998. Long-term effects of earthworms on microbial biomass nitrogen in coarse and fine textured soils. Appl. Soil Ecol. 9, 375–380. House, G.J., Parmelee, R.W., 1985. Comparison of soil arthropods and earthworms from conventional and no-tillage agroecosystems. Soil Tillage Res. 5, 351–360. Hurisso, T.T., Davis, J.G., Brummer, J.E., Stromberger, M.E., Stonaker, F.H., Kondratieff, B.C., Booher, M.R., Goldhamer, D.A., 2011. Earthworm abundance and species composition in organic forage production systems of northern Colorado receiving different soil amendments. Appl. Soil Ecol. 48, 219–226. Ippolito, J.A., Barbarick, K.A., 2008. Fate of biosolids trace metals in a dryland wheat agroecosystem. J. Environ. Qual. 37, 2135–2144. Jun, T., Wei, G., Griffiths, B., Liu, X.J., Xu, Y.J., Hua, Z., 2012. Maize residue application reduces negative effects of soil salinity on the growth and reproduction of the earthworm Aporrectodea trapezoides, in a soil mesocosm experiment. Soil Biol. Biochem. 49, 46–51. Lee, K.E., Foster, R.C., 1991. Soil fauna and soil structure. Aust. J. Soil Res. 29, 745–775. Lubbers, I.M., Brussaard, L., Otten, W., van Groenigen, J.W., 2011. Earthworminduced N mineralization in fertilized grassland increases both N2 O emission and crop-N uptake. Eur. J. Soil Sci. 62, 152–161. Martin, A., Marinissen, J.C.Y., 1993. Biological and physicochemical processes in excrements of soil animals. Geoderma 56, 331–347. Moreira, R., Sousa, J.P., Canhoto, C., 2008. Biological testing of a digested sewage sludge and derived composts. Bioresour. Technol. 99, 8382–8389. Mulvaney, R.L., 1996. Nitrogen-inorganic forms. In: Sparks, D.L. (Ed.), Methods of Soil Analysis. SSSA Book Series. Soil Science Society of America, Madison, WI, pp. 1123–1151. Natal-da-Luz, T., Tidona, S., Jesus, B., Morais, P.V., Sousa, J.P., 2009. The use of sewage sludge as soil amendment. The need for an ecotoxicological evaluation. J. Soils Sediments 9, 246–260. Needham, A.E., 1957. Components of nitrogenous excreta in the earthworms Lumbricus terrestris L. and Eisenia foetida (savigny). J. Exp. Biol. 34, 425–446. Oades, J.M., 1993. The role of biology in the formation, stabilization and degradation of soil structure. Geoderma 56, 377–400.

6

J.P. McDaniel et al. / Applied Soil Ecology 71 (2013) 1–6

Parkin, T.B., Berry, E.C., 1999. Microbial nitrogen transformations in earthworm burrows. Soil Biol. Biochem. 31, 1765–1771. Protz, R., Teesdale, W.J., Maxwell, J.A., Campbell, J.L., Duke, C., 1993. Earthworm transport of heavy-metals from sewage-sludge – a micro-pixe application in soil science. Nucl. Instrum. Methods Phys. Res. B: Beam Interact. Mater. Atoms 77, 509–516. Reynolds, J.W., 2011. More earthworms (Oligochaeta: Lumbricidae and Sparganophilidae) from Colorado, USA. Megadrilogica 14, 159–172. Rhoades, J.D., 1996. Salinity: electrical conductivity and total dissolved solids. In: Sparks, D.L. (Ed.), Methods of Soil Analysis, Part 3 – Chemical Methods. Soil Sciences Society of America, Madison, WI, pp. 417–435. Roming, D.E., Farkynd, M.J., Harris, R.F., 1996. Methods for Assessing Soil Quality. Soil Science Society of America, Madison, WI. SAS Institute, 2008. Statistical Analysis System, Version 9.2. SAS Inst., Cary, NC, USA. Schrader, S., Zhang, H.Q., 1997. Earthworm casting: stabilization or destabilization of soil structure? Soil Biol. Biochem. 29, 469–475. Sherrod, L.A., Dunn, G., Peterson, G.A., Kolberg, R.L., 2002. Inorganic carbon analysis by modified pressure-calcimeter method. Soil Sci. Soc. Am. J. 66, 299–305. Soil Survey Staff, N.R.C.S., United States Department of Agriculture, 2012. Official Soil Series Descriptions. Available online at http://soils.usda.gov/ technical/classification/osd/index.html (accessed 16.08.12). Thomas, G.W., 1996. Soil pH and soil acidity. In: Sparks, D.L. (Ed.), Methods of Soil Analysis, Part 3 – Chemical Methods. Soil Sciences Society of America, Madison, WI, pp. 475–490.

Tillinghast, E.K., 1967. Excretory pathways of ammonia and urea in earthworm Lumbricus terrestris L. J. Exp. Zool. 166, 295–300. Tisdall, J.M., 1978. Modification of Soil Structure. New York, NY. Tisdall, J.M., 1985. Earthworm activity in irrigated red-brown earths used for annual crops in Victoria. Aust. J. Soil Res. 23, 291–299. Tomlin, A.D., Protz, R., Martin, R.R., McCabe, D.C., Lagace, R.J., 1993. Relationships amongst organic-matter content, heavy-metal concentrations, earthworm activity, and soil microfabric on a sewage-sludge disposal site. Geoderma 57, 89–103. Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass-C. Soil Biol. Biochem. 19, 703–707. VandenBygaart, A.J., Protz, R., Tomlin, A.D., 1999. Changes in pore structure in a no-till chronosequence of silt loam soils, southern Ontario. Can. J. Soil Sci. 79, 149–160. Whalen, J.K., Parmelee, R.W., Subler, S., 2000. Quantification of nitrogen excretion rates for three lumbricid earthworms using N-15. Biol. Fertil. Soils 32, 347–352. Willems, J., Marinissen, J.C.Y., Blair, J., 1996. Effects of earthworms on nitrogen mineralization. Biol. Fertil. Soils 23, 57–63. Yeates, G.W., Shepherd, T.G., Francis, G.S., 1998. Contrasting response to cropping of populations of earthworms and predacious nematodes in four soils. Soil Tillage Res. 48, 255–264. Zhang, Q.L., Hendrix, P.F., 1995. Earthworm (Lumbricus rubellus and Aporrectodea caliginosa) effects on carbon flux in soil. Soil Sci. Soc. Am. J. 59, 816–823.