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Effect of acidified cattle slurry on a soil collembolan community: A mesocosmos study
European Journal of Soil Biology 94 (2019) 103117
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Effect of acidified cattle slurry on a soil collembolan community: A mesocosmos study
T
Alessandra D'Annibalea, Rodrigo Labouriaub, Peter Sørensena, Paul H. Kroghc, ⁎ Bent T. Christensena, Jørgen Eriksena, a
Department of Agroecology, Aarhus University, AU-Foulum, DK-8830, Tjele, Denmark Department of Mathematics, Aarhus University, DK-8000, Aarhus, Denmark c Department of Bioscience, Aarhus University, DK-8600, Silkeborg, Denmark b
ARTICLE INFO
ABSTRACT
Handling editor: Stefan Schrader
The concept of soil health emphasizes the role of soil organisms in sustainable agriculture and relies on bioindicators to assess the impact of management options. This study employed Collembola, a key group of microarthropods with intimate links to soil microbial activity, as bioindicators to reveal the sustainability of using acidified cattle slurry. While environmental and plant production benefits of acidification are well known, effect of acidified slurry on Collembola is undocumented. We added Collembola to sandy soil mixed with untreated or acidified cattle slurry labelled with 15N and applied at two rates (corresponding to 30 and 90 Mg slurry ha−1). The collembolan community included two species for each vertical life-form: epedaphic (Sinella curviseta and Heteromurus nitidus), hemiedaphic (Proisotoma minuta and Hypogastrura assimilis) and euedaphic species (Folsomia fimetaria and Protaphorura fimata). Replicate mesocosmos were incubated for 28 or 56 days at 15 °C, 65% WHC and a 12 h light-dark cycle. The epedaphic species strongly declined at the end of the experiment, whereas P. minuta and F. fimetaria were positively affected by high rates of untreated and acidified slurry, respectively. The hemiedaphic P. minuta showed the highest abundances and accumulation of slurry-15N, especially when high rate of untreated slurry was applied. We conclude that the impact on Collembola of low rates of untreated and acidified slurry applied to agricultural soils is marginal while higher rates of slurry may subsequently form a favourable habitat for euedaphic species.
Keywords: Cattle slurry Acidification Mesofauna Mesocosmos Arable soil 15 N-labelling
1. Introduction The concepts of soil health and sustainable agriculture emphasize the role of soil organisms and recognize that soil physical and chemical properties represent drivers for the structure and function of the decomposer community [1,2]. While addition of crop residues and animal manures impacts the physical soil environment, it also provides nutrient and energy resources supporting the soil decomposer community. To allow for development of sustainable management, the concept of soil health has to be accompanied by bioindicators that provides a quantitative assessment of the impact on soil of a given management option [e.g. [3, 4, 5, 6]. Recognizing that the turnover of organic matter and nutrients rely on microorganisms under the influence of faunal activity, management impacts on numbers and activity of key groups of soil fauna need to be determined. Collembola are microarthropods with intimate links to soil microbial activity [7], but little is known regarding their reaction to intensive
⁎
nitrogen (N) management. In NW European areas with dairy production, grasslands receive frequent inputs of cattle slurry to provide sufficient herbage yields. To improve N use efficiency, ammonia volatilization during application can be reduced by lowering slurry pH through addition of sulphuric acid [8]. While the economic and environmental benefits of slurry acidification are known [9], effects of acidified slurry on soil Collembola remains unknown. The reduced pH is not expected to have direct negative impacts as animals in arable soils are already adapted to a soil pH around 6. However, acidification of slurry may indirectly affect soil Collembola via its effects on the transformation of ammoniacal-N, especially in slurry hot-spots established by slit injection of high slurry rates. Slit injection of slurry in the field may reduce the number of epigeic earthworms but leave enchytraeids unaffected [10]. Earthworm excreta (mucus and urine) and the ammonium content in these substances has been shown to attract Collembola [11,12] while a repulsive behaviour was demonstrated at high concentrations of ammonium [11]. Domene
Corresponding author. Department of Agroecology, Aarhus University, AU-Foulum, Blichers Allé 20, DK-8830, Tjele, Denmark. E-mail address: [email protected] (J. Eriksen).
https://doi.org/10.1016/j.ejsobi.2019.103117 Received 19 November 2018; Received in revised form 6 August 2019; Accepted 12 August 2019 Available online 18 August 2019 1164-5563/ © 2019 Elsevier Masson SAS. All rights reserved.
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et al. [13] demonstrated that ammonium present in sewage sludge was toxic for the Collembola species Folsomia candida, but also that the level of toxicity depends on soil properties. Thus, the impact of cattle slurry on soil Collembola may well relate to application rate and concentrations of ammoniacal-N. Previous studies have addressed the effect of untreated and anaerobically digested animal manure on Collembola [14,15], but effects of acidified slurry on soil Collembola remain elusive. Using a mesocosmos set-up, we examined the effect of untreated and acidified cattle slurry applied at two rates to a sandy soil amended with a collembolan community constructed with equal representation of the three vertical life-forms: epedaphic, hemiedaphic and euedaphic. These life-froms differ typically by having contrasting habitat preferences, feeding ecology and gut microbiome [16,17]. Thus, the population performance could respond to the rate of slurry and its nutrient content and this is hypothesized to be true for euedaphic collembolans already shown to thrive in organic soil amendments [18]. We envisioned that application of slurry at low rate (i.e. conditions after surface application) has a positive effect on Collembola in terms of abundances, whereas slurry applied at high rate (i.e. conditions in injection slits) is detrimental because of high concentrations of ammoniacal-N. We expect a negative effect of acidified slurry on Collembola as acidification conserves ammoniacal-N temporarily in the soil volume affected by the slurry.
Using adult specimens collected from laboratory cultures, a collembolan community was added to each cylinder at day 0. The community included 30 individuals of each of six species belonging to three life-forms [15]: two epedaphic (Heteromurus nitidus, Templeton 1835 and Sinella curviseta, Brook 1882), two hemiedaphic (Hypogastrura assimilis, Krausbauer 1898 and Proisotoma minuta, Tullberg 1871), and two euedaphic (Folsomia fimetaria, Linnaeus 1758 and Protaphorura fimata, Gisin 1952). The Collembola were added to the soil surface and a perforated lid placed on top of the cylinder. During incubation, cylinders were kept at 15 °C and a 12 h light-dark cycle. Each cylinder was weighed once a week and water was added when needed to maintain the initial water content. The experiment involved two sets of cylinders to be destructively harvested for Collembola after 28 and 56 days of incubation. For each harvest time, three replicate cylinders were prepared for each of the treatments: control without slurry (C0), untreated slurry at low rate (UL), untreated slurry at high rate (UH), acidified slurry at low rate (AL), and acidified slurry at high rate (AH). For each treatment, one extra set of replicates without animals was included for determination of initial (day 0) contents of soil water content, pH, and NH4–N and NO3–N extracted with 1 M KCl. In total, 45 cylinders were prepared. 2.2. Analyses at harvest At harvest, four soil subsamples (each 120 g fresh weight) were collected from each cylinder for extraction of Collembola. Another three subsamples were taken for chemical soil analyses. Collembola were extracted over one week with a high-gradient temperature extractor by increasing the temperature stepwise from 25 to 60 °C. For three of the subsamples for enumeration of Collembola, animals were collected in trays with plaster of Paris (calcium sulphate hemihydrate) mixed with activated charcoal (w/w = 8/1) in order to collect live specimens for 15N analyses. For the last subsample, Collembola were collected in benzoic acid [19]. For each species, the number of adults and juveniles was determined visually according to size. The final number of Collembola was based on both plaster of Paris and benzoic acid samples. Soil subsamples were ground for analyses of total-N and 15N abundance. Inorganic N extracted from soil by 2 M KCl was subjected to micro-diffusion for 15N analysis [20]. Collembola extracted from the cylinders and sampled from the laboratory cultures were also subject to isotope measurements. Slurry samples were extracted with 2 M KCl to determine concentrations of NH4–N and NO3–N. The slurry extracts were processed by micro-diffusion for inorganic 15N measurements as described for soil extracts. All samples for N isotope analyses were wrapped in tin capsules (5 × 8 mm) targeting a sample N content of 20–150 μg. Isotope analysis was performed at the Stable Isotope Facility, University of California, Davis, USA, with a PDZ Europa ANCAGSL elemental analyser interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd, Crewe, Cheshire, UK). The 15N atom percent (Atom %) was determined as:
2. Materials and methods 2.1. Experimental setup The mesocosmos experiment relied on a constructed collembolan community exposed to sandy soil mixed with untreated (U) or acidified (A) cattle slurry applied at low (L) or high (H) rate. The soil was retrieved from 0 to 25 cm depth in a field subject to long-term agricultural management. It had 6% clay, 9% silt, 44% fine sand, 39% coarse sand, pH 6.5, 1.6% carbon (C), and 0.15% N. The soil was dried at room temperature, sieved to < 4 mm and then defaunated by successive freeze-thaw cycles (temperature alternating between −18 °C and +20 °C). Table 1 shows the characteristics of the cattle slurry used in the experiment. The slurry was from dairy cows fed a standard diet (mainly silage) and stored for 15 months at 2 °C in a closed container. One batch of slurry was acidified to pH 5.3 with sulphuric acid (8.0 g kg−1 slurry) while another batch remained untreated and had pH 7.5. At the start of the experiment (day 0), the slurries were labelled with 15N using (15NH4)2SO4 with 99.3 atom percent (Atom-%) to reach a final 5% enrichment of the NH4–N pool in the slurry. Each mesocosmos consisted of a 33 cm high Plexiglas cylinder with an inner diameter of 9 cm. The cylinder was closed at the bottom and then filled with 700 g dry soil homogenously mixed with slurry (L, 3 g per 100 g soil; H, 9 g per 100 g soil). These rates correspond to a field application of 30 and 90 Mg slurry ha−1 incorporated into a 0–7 cm soil layer. Demineralized water was then added to reach 65% of the soil water holding capacity (WHC).
Atom %15N =
Characteristics
a
15 N × 100 N + 15N
and the Atom Percent Excess of 15N (15N APE) was calculated as the difference between 15N abundance in the labelled sample and the corresponding unlabelled control sample. For some Collembola species, samples for isotope analysis required pooling of individuals from replicate cylinders to achieve a sufficient amount of N for the analysis. The 15N recovery was calculated for total N, inorganic N (NH4–N + NO3–N) and organic N. The recovery of total N and inorganic N was calculated as:
Table 1 Characteristics of the cattle slurry.
Untreated slurry total N Untreated (labelled) slurry NH4–Na Acidified (labelled) slurry NH4–Na Dry Matter (DM) on fresh weight Untreated slurry pH Acidified slurry pH
14
3.36 g kg−1 1.35 g kg−1 1.63 g kg−1 8.7% 7.5 5.3
Recovery (%) =
Values used for the15N recovery calculations in soil.
APEsoil N × soil × 100 APEslurry Nslurry
where Nsoil is total N or inorganic N. The recovery of 15N in the organic 2
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N pool was calculated as the difference between total N and inorganic N. For the inorganic N pool, the calculation of APE for soil and slurry samples applied the correction of the Atom % given by Sørensen and Jensen [20].
dependency between observations from the same cylinder; and one residual random component taking different values for each observation and allowing the model to represent the over-dispersion present in the data [25,26]. Note that an equivalent model without the random component representing overdispersion does not adjust to the data (see Appendix for details). Both random components were defined in such a way that they have different variances for the observations made with each of the two Collembola collection methods (plaster of Paris or benzoic acid). The models contained a fixed effect composed by a factor representing the combinations of the treatments (C0, UL, UH, AL and AH) and harvest time (day 28 and 56). The parameters associated with the fixed effect defined above are precisely the expected values of the number of animals per g of soil for the corresponding treatments and harvest time (see the appendix A for details); these parameters (and the corresponding confidence intervals) were then multiplied by 1000 for expressing the abundances as number of animals per kg soil.
2.3. Statistical analyses 2.3.1. Modelling the soil parameters For soil, the total N, NO3–N, and the total, inorganic and organic 15 N recoveries were analysed using generalized linear mixed models (GLMM; [21, 22] with fixed effects representing the combinations of the treatments (C0, UL, UH, AL and AH) and harvest time (day 0, 28 and 56). The GLMMs contained a Gaussian random component specially designed to represent the dependency between observations from the same cylinder. All models were defined with the identity link function and the normal distribution, except the model for NO3−-N that was defined with an inverse Gaussian distribution [23]. Tests for the presence of partial interactions, obtained by eliminating the control treatment from the analyses, revealed statistically significant effect modification between acidification and slurry application rate. This rules out the use of additive effects of these two factors. Interactions followed different patterns at different harvest times (p-values < 0.00001) in all cases, except for total-N (p-value = 0.98). Detailed descriptions of the models are given in the Appendix. The NH4–N contents of different combinations of treatments and time were characterized through a Kruskal-Wallis test [24], due to the lack of adherence of Gaussian or other classic parametric models and due to the high variability of the data. The results of this parameter were therefore reported by the medians for each combination of treatment and harvest time, and the corresponding confidence intervals obtained by non-parametric bootstrap (10,000 bootstrap simulations).
2.3.4. Modelling the Collembola population age compositions The age composition of each species of Collembola was characterized by classifying individuals as adults or juveniles. The proportions of adults in each population were estimated using a binomial logistic mixed model with random components and fixed effects defined as in the models for the abundance defined above. Note that these models account for the over-dispersion present in the data, in the same way as the Poisson model describing the abundances defined above. See Appendix for details. 2.3.5. Statistical inference procedures The models described above (except for soil NH4–N) are all instances of GLMM. The inference for these models were performed using the functions lmer (for the Gaussian models), glmer (for the nonGaussian models, as the inverse Gaussian, the Poisson and the binomial models) from the package {lme4} [27] and the function lm for the 15N APE of P. minuta and F. fimetaria, implemented in the R-software [28]. The post-hoc pairwise comparisons for all models were done using the R-package {pairwiseComparisons} [29]. The p-values implicitly used in the post-hoc pairwise comparisons were adjusted for multiple comparisons using the one-step method for the parametric models (implemented in the R package {multcomp} [30], and the fdr (controlled false discovery rate) method for the non-parametric tests [31].
2.3.2. Modelling the 15N accumulation in each species The 15N Atom Percentage Excesses (15N APE) in each species were analysed using Gaussian multiple regression models where the slurry rate entered as a continuous explanatory variable and different slope parameters were estimated for each slurry treatment (untreated or acidified). The slope parameters indicate the expected increases in 15N assimilation when slurry rate increases one unit, i.e. 1 g slurry 100 g−1 soil (within the range 3–9 g slurry per 100 g soil). It was not always possible to obtain enough Collembola biomass to make precise determinations of 15N APE with the sample from one single replicate; in those cases it was then necessary to physically pool samples from replicate cylinders; only cylinders with the same treatment (UL, UH, AL or AH), slurry rate (L or H) and harvest time (da3 to ys 28 and 56) were pooled. The models contained three random components indicating whether each cylinder entered in the pooling of the corresponding observation, allowing in this way to account for the variance heterogeneity generated by the sample pooling process (see Appendix for details). In the case of the species P. minuta and F. fimetaria it was necessary to pool all the samples collected at the same harvest time and subject to the same treatment/slurry application rate; in this case the model did not contain any random component.
3. Results 3.1. Soils Table 2 shows soil pH at day 0 and after 28 and 56 days of incubation. Addition of acidified slurry reduced soil pH compared to corresponding rates of untreated slurry. For all slurry treatments, the soil pH dropped about one unit from day 0 to day 28 and then remained constant. The initial concentration of NH4–N reflected the addition of slurry (Table 3). Soil concentrations of NH4–N peaked at the beginning of the experiment, with higher values in soils with acidified slurry than in soils with corresponding rates of untreated slurry, but concentrations were subsequently reduced to very low levels. However, treatment AH
2.3.3. Modelling the Collembola abundances The abundance (calculated as the number of animals per kg of soil) of each of the Collembola species (except H. assimilis, which did not grow sufficiently during the experiment in any of the treatments) was modelled by a Poisson mixed model with over-dispersion that accounted for differences in the amount of soil sampled, the heterogeneity of dispersion and the dependency of the observations. This model was adjusted, for each species, by using a GLMM defined with a Poisson distribution, a logarithmic link function, an offset with the logarithm of the soil mass corresponding to the observation, and two specially designed Gaussian random components: one taking the same value for the observations arising from the same cylinder, representing the
Table 2 Soil pH measured at the start of incubation (Day 0) and after 28 and 56 days of incubation.
3
Treatment
Day 0
28 days
56 days
C0: Control without slurry UL: Untreated slurry, low rate UH: Untreated slurry, high rate AL: Acidified slurry, low rate AH: Acidified slurry, high rate
6.5 6.9 7.4 6.1 5.8
5.9 5.7 5.5 5.3 4.9
5.9 5.7 5.5 5.4 4.9
European Journal of Soil Biology 94 (2019) 103117
Mean (95% CI)
– – – – –
– 98 (94–103) b,A 101 (97–105) b,A 88 (84–93) a,A 90 (86–94) a,A
Mean (95% CI)
(21–30) (22–30) (16–25) (15–24)
(13–25) a,A (10–22) a,B (14–26) a,A (6–18) a,B
– – – – –
– 26 26 20 19
– 19 16 20 12
– 91 93 91 82
(85–97) (87–99) (84–97) (76–88)
a,A a,B a,A a,B
% %
a,A a,A a,A a,A
Total15N recovery Organic15N recovery
(0.3–0.4) (0.3–0.4) (0.5–0.6) (0.4–0.4) (1.3–1.4)
a,B ab,B c,C b,C d,C
39.1 (35.4–42.8) a,C 91.3 (89.7–93) b,C 208.9 (206.4–211.4) d,C 96.5 (94.9–98.1) c,C 210.1 (207.6–212.6) d,C
1.55 1.59 1.79 1.57 1.81
(1.49–1.6) a,A (1.53–1.64) a,A (1.73–1.84) b,A (1.52–1.63) a,A (1.75–1.86) b,A
– 72 76 71 71
(70–74) (75–78) (69–72) (69–73)
a,A b,A a,B a,A
The five collembolan species subject to statistical analyses (the original community except H. assimilis – see Materials and Methods) showed different population growth patterns (Figs. 1–3). During the first 28 days of incubation, the epedaphic species S. curviseta and H. nitidus showed a three-fold increase in population density regardless of treatment (Fig. 1). The increase was accompanied by a substantial drop in the proportion of adults and a corresponding increase in the proportion of juveniles. After 28 days, these two species had a significantly lower proportion of adults in UH compared to the treatment without slurry addition (C0). From day 28 to day 56, both species showed a significant decline in numbers regardless of soil treatment and at day 56, the number of individuals was smaller than that added initially (day 0) and the proportion of adults had increased significantly, in particular for S. curviseta. The proportion of H. nitidus adults was significantly lower in UH than in C0 after 28 days; this was also true for the treatments UL and UH at day 56. At day 28, the number of the hemiedaphic species Proisotoma minuta did not differ between soil treatments and was only marginally higher than the initial population (Fig. 2). Except for the treatment UH, the number of individuals and the adult proportions did not differ between treatments after 56 days of incubation. At this harvest time, P. minuta showed a significantly higher density in the UH treatment which is the highest population density found in this study. The proportion of adults was similar after 28 and 56 days of incubation. The exception was soil without slurry addition; here the proportion of adults was higher at day 28 than at day 56. The euedaphic species F. fimetaria did not differ significantly between treatments neither in total densities nor in adult proportions after 28 days of incubation (Fig. 3). The data for this harvest are associated with large variability but apparently numbers of individuals were similar for day 0 and day 28. The number of individuals declined significantly from day 28 to day 56 in all the treatments, except for AH. At day 56, this treatment showed the highest number of animals whereas the proportion of adults was similar to that of the other slurry treated soils. At the end of the experiment, the lowest population density was found for C0 (the population was almost extinct) while the proportion of adults was significantly higher compared to the slurry treatments (Fig. 3). The other euedaphic species P. fimata was significantly less abundant in AH after 28 days compared to AL and C0, without differences in adult proportions. For the AH treatment, the population of P. fimata increased significantly from day 28 to day 56. Table 4 shows that the 15N Atom Percent Excess (15N APE) in Collembola was lowest in the two epedaphic species S. curviseta (specifically in UL after 28 days) and H. nitidus (in AL after 28 days). The highest values was found for the hemiedaphic species P. minuta (in UH and AH after 28 days). For S. curviseta the 15N-APE values were higher in treatments with acidified slurry than treatments with untreated slurry. This was also true for H. nitidus and P. minuta and for the
C0: Control without slurry UL: Untreated slurry, low rate UH: Untreated slurry, high rate AL: Acidified slurry, low rate AH: Acidified slurry, high rate 56 days
0.3 0.4 0.5 0.4 1.3
bc,A c,A a,A b,A (71–74) (74–76) (67–69) (69–72) – 73 75 68 71 (1.44–1.55) a,A (1.6–1.71) b,A (1.84–1.95) c,B (1.61–1.72) b,B (1.82–1.92) c,A 1.49 1.66 1.90 1.67 1.87 31.1 (26–36.1) a,B 82.8 (81.1–84.4) b,B 190.3 (188–192.6) d,B 85.7 (84.1–87.4) b,B 168 (166–170) c,B C0: Control without slurry UL: Untreated slurry, low rate UH: Untreated slurry, high rate AL: Acidified slurry, low rate AH: Acidified slurry, high rate 28 days
0.5 (0.1–0.5) a,B 0.6 (0.1–0.6) ab,B 0.8 (0.7–0.9) c,B 0.6 (0.6–0.6) b,B 27.1 (25.8–29.3) d,B
– – – – – (1.44–1.61) a,A (1.5–1.67) a,A (1.68–1.85) b,A (1.48–1.65) a,AB (1.71–1.88) b,A (−0.9-26.8) (−1.3-26.8) (−1.6-26.9) (−1.7-26.7) (−1.8-26.8) 12.9 12.8 12.7 12.5 12.5 C0: Control without slurry UL: Untreated slurry, low rate UH: Untreated slurry, high rate AL: Acidified slurry, low rate AH: Acidified slurry, high rate Day 0
3.5 (3.1–4.3) a,A 45.3 (34.7–47.7) b,A 121.7 (83.9–128.6) d,A 57.1 (56.5–59.1) c,A 156 (140.9–164.6) e,A
Mean (95% CI)
mg kg−1 mg kg−1
Median (95% CI) Treatment Time
NO3−-N
a,A a,A a,A a,A a,A
1.52 1.58 1.76 1.57 1.80
Mean (95% CI) Mean (95% CI)
% g kg−1
NH4++15NO3− recovery 15
Total N
remained higher in NH4–N after 28 and 56 days. Initial (day 0) concentrations of NO3–N did not differ between treatments, but increased significantly during the incubation. After 28 days, the concentration was significantly higher in UH, compared to all the other treatments. After 56 days of incubation, there was no difference in NO3–N concentration between UH and AH, while the concentration was significantly higher in AL compared to UL (Table 3). Total N in soil showed the highest values in soils receiving the high rate of slurry. The recovery of 15N in total N after 28 days of incubation was significantly higher in soil with untreated slurry than in soil with acidified slurry, regardless of addition rate (Table 3), but after 56 days, treatments did not differ. For soils taking high rates of slurry, the recovery of 15 N decreased significantly from day 28 to day 56. The recovery of 15N as inorganic N (15NH4+ 15NO3) ranged from 68 to 76% with significantly higher recovery in soil with the high rate of untreated slurry. 3.2. Collembola
NH4–N
Table 3 Ammonium-N, nitrate-N, total N, and15N recovery rates (inorganic, organic and total) in soil initially and after 28 and 56 days of incubation for each treatment. Different small letters indicate significant (p < 0.05) differences between treatments at a given sampling time, while different capital letters indicate significant differences between 28 and 56 days of incubation.
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Fig. 1. Interaction plot of the total densities and adults proportions of the epedaphic species in the experimental treatments at the different experimental time points. The expected values of the treatments at the same observation time are connected with lines as a visual aid for identifying interactions between the observation time and the treatments. Bars indicate 95% CI. Different small letters indicate significant (p < 0.05) differences between treatments within the same sampling, while different capital letters indicate significant differences between 28 and 56 days. Abbreviations: C0: Control without slurry, UL: Untreated slurry, low rate, UH: Untreated slurry, high rate, AL: Acidified slurry, low rate, AH: Acidified slurry, high rate.
euedaphic species at high slurry concentrations after 56 days. At the end of incubation, the euedaphic species P. fimata showed higher slope coefficient for 15N APE in acidified treatments, although the slope was not significantly different from untreated slurry after 56 days. No differences in the slope coefficients were detected for the other species. However, our data suggest a higher increase (i.e. estimated slope) in 15 N accumulation from low to high slurry application rate in P. minuta (ca. 0.12) and F. fimetaria (ca. 0.07–0.11) compared to the other species.
collembolan abundances due to the potential toxicity of elevated ammonium concentrations in the soil. However, most of the ammonium in the soil and that applied with slurry at the start of the incubation was nitrified or immobilized within the first 28 days of incubation; after 56 days of incubation initial concentrations of ammonium were reduced by 99% (Table 3). The change in concentrations of ammoniacal-N during day 0 to day 28 is unknown, but we surmise that any impact of ammonium would be short-term because of its almost complete disappearance after 28 days. The soil receiving a high rate of acidified slurry (AH) was the only treatment with slightly elevated ammonium content at day 28. This treatment was more acid than any other treatment at day 28 and also at day 56 (Table 2). Although the ammonium concentration after 56 days was significantly higher in AH than in the other treatments, the concentration was very low. Detrimental effects of ammonium on Collembola have been claimed in studies involving manure and biosolids applied to soils [13,15,32], but our results lend little support for this claim. As expected, the concentration of nitrate increased during the
4. Discussion Slurry acidification is adopted to reduce ammonia volatilization during surface application and when slits remain open after slurry injection. With high rates of acidified slurry, the effect of acidification may linger on after application whereby elevated levels of ammonium can be retained in the slurry affected soil volume. We envisioned that high rates of slurry could negatively affect 5
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Fig. 2. Interaction plot of the total densities and proportion of adults of the hemiedaphic species P. minuta in the experimental treatments at the different experimental time points. The expected values of the treatments at the same observation time are connected with lines as a visual aid for identifying interactions between the observation time and the treatments. Bars indicate 95% CI. Different small letters indicate significant (p < 0.05) differences between treatments within the same sampling, while different capital letters indicate significant differences between 28 and 56 days. Abbreviations: C0: Control without slurry, UL: Untreated slurry, low rate, UH: Untreated slurry, high rate, AL: Acidified slurry, low rate, AH: Acidified slurry, high rate.
experiment; soils receiving high slurry rates reaching the highest concentrations. The average net immobilization of 15N, that is 15N recovered in organic N pools, accounted for 20% of the ammonium added initially while ammonium plus nitrate held 72% of the 15N label. Immobilization did not differ between treatments neither at day 28 nor at day 56, but the treatments with high slurry rates decreased in organically bound 15N from day 28 to day 56. Thus, our results suggest that ammonium contents and nitrification in the soils were defined by slurry addition rate whereas acidification had little impact. Previous studies based on Collembola extracted from soil in field experiments show a positive effect of organic amendments on numbers of animals. Bolger and Curry [32] observed a beneficial effect of adding 55 and 110 Mg ha−1 of cattle slurry to permanent grassland while an extremely high slurry rate (550 Mg ha−1) significantly depressed collembolan numbers. However, they concluded that in the longer term, the soil fauna would respond positively to the food source provided by the slurry. Similarly, Weil and Kroontje [33] reported a positive effect on numbers of Collembola in soils subjected for 5 years to fresh poultry manure at annual rates ranging from 27 to 110 Mg ha−1. Positive effects on numbers of Collembola have also been reported after soil additions of cattle slurry, green-waste composts, and digestates from biogas [4,14,34]. However, Pommeresche et al. [15] found a negative effect of cattle slurry on collembolan numbers extracted 3 and 48 days after application. After 56 days of incubation, we found that rates of cattle slurry corresponding to 90 Mg ha−1 had a positive effect on the abundance of the species P. minuta (untreated slurry, UH) and F. fimetaria (acidified slurry, AH), whereas other species were unaffected by slurry. The population of F. fimetaria declined from day 28 to day 56 except in soil with high rate of acidified slurry; in this treatment F. fimetaria numbers increased and the adult-to-juvenile ratio differed significantly between day 28 and day 56. This suggests that the application of high rates of acidified slurry sustained the reproduction of this species during the 56 days of incubation. Without slurry addition, F. fimetaria may be negatively affected by competition from other Collembola species. For the two epedaphic species (S. curviseta and H. nitidus), the number of animals increased significantly during the first 28 days of incubation regardless of treatment, while numbers after 56 days fell to below initial levels. These changes were mirrored in the proportion of
adults; the number of juveniles decreased from day 28 to day 56. A clear difference between the epedaphic and the other species in terms of population growth was also reflected in their 15N accumulation. The epedaphic species had the lowest average 15N APE values, while P. minuta showed the highest values, especially at high slurry rates. In a laboratory study, Larsen et al. [35] found P. minuta to have a higher metabolic rate and reproductive investment than P. fimata, suggesting that P. fimata has lower nutritional requirements. This may explain why the population of P. fimata remained rather constant during the entire incubation period. Initially, P. fimata showed lower densities in the AH treatment. However, the population recovered after 56 days and reached final densities that were comparable to the other species. After 56 days of incubation, the highest number of specimens (approximately 67% of the community) was observed for the hemiedaphic species P. minuta in the UH treatment, indicating that a high concentration of untreated slurry constituted a favourable environment for the growth of this species. At the end of the incubation, effects of slurry added at low rate were generally absent while P. minuta and F. fimetaria were positively affected by the high slurry rate. Although the two species differ in lifeforms, they belong to the same family Isotomidae (order Entomobryomorpha). Since this was the only obvious effect of slurry treatments, we conclude that application of slurry at low rate may have but a marginal effect on collembolan community while high rates of slurry may have a positive longer-term effect as suggested by Bolger and Curry [32]. Moreover, the collembolan species involved in the present study were generally unaffected by slurry acidification, notwithstanding that the high rate of acidified slurry favoured the reproduction of F. fimetaria. A shift of the collembolan community towards more abundant euedaphic and hemiedaphic species as a result of slurry application may improve their contribution to slurry decomposition and support agroecosystem functions. The hemiedaphic species H. assimilis did not successfully survive in any treatment and was excluded from analyses as reported previously [36,37]. The selection of Collembola species for mesocosmos studies will depend the objective of a given test and on the stocks of laboratory raised animals at hand. Our experimental set-up relied on addition of the same number of individuals for each species, recognizing that these contribute differently to the collembolan biomass in the soil. Our 6
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Fig. 3. Interaction plot of the total densities and proportion of adults of the euedaphic species in the experimental treatments at the different experimental time points. The expected values of the treatments at the same observation time are connected with lines as a visual aid for identifying interactions between the observation time and the treatments. Bars indicate 95% CI. Different small letters indicate significant (p < 0.05) differences between treatments within the same sampling, while different capital letters indicate significant differences between 28 and 56 days. Abbreviations: C0: Control without slurry, UL: Untreated slurry, low rate, UH: Untreated slurry, high rate, AL: Acidified slurry, low rate, AH: Acidified slurry, high rate.
overall objective was to link the impact of acidified slurry to soil health, using a mesocosmos set-up and Collembola as bioindicators. The limitations of this approach when studying the effect of complex substrates such as slurry is recognized, and one alternative approach is to extract Collembola from field plots subject to different management [e.g. 14, 15]. However, seasonal and annual fluctuations in collembolan numbers and species severely limit the suitability of Collembola as indicator of soil health when based on field samplings [38–40].
their population abundances during the experiment, regardless of the treatment. Acidification of slurry is an important step towards higher N use efficiency and thereby improved sustainability in agricultural crop production. In this study, the low rate of slurry simulates soil conditions following surface application while the high rate was taken to simulate soil conditions in the injection slit. We conclude that the impact on Collembola of low rates of untreated and acidified slurry applied to agricultural soils is marginal while higher rates of slurry may subsequently form a favourable habitat for euedaphic species and preserve their decomposition potential.
5. Conclusions Using a mesocosmos set-up with a constructed population of six species of Collembola incubated for 4 and 8 weeks, we found that application of slurry at a rate corresponding to 30 Mg ha−1 had little impact on the collembolan community while a slurry rate corresponding to 90 Mg ha−1 showed a positive effect. The collembolan species adopted in this study were generally unaffected by slurry acidification. However, two epedaphic species strongly decreased in
Financial support This study was financially supported through a PhD grant from the Graduate School of Science and Technology (GSST) at Aarhus University.
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Table 4 15 N Atom % Excess (15N-APE) of Collembola exposed to untreated or acidified cattle slurry applied at low or high rate. Means ± SEM are given with number of samples in brackets. The slope coefficient Est. is the effect of a unit increase slurry concentration (in the range from 3 to 9 g per 100 g). 95% confidences intervals (CI) are reported in brackets and different letters indicate significant (p < 0.05) differences between harvest time and treatment combinations. “na” indicates “not available”. Time
Treatment
Low rate
High rate
Est. (95% CI)
Untreated Acidified Untreated Acidified
S. curviseta 0.034 ± 0.012 (5) 0.042 ± 0.004 (4) 0.038 ± 0.007 (5) 0.061 ± 0.01 (4)
0.108 0.116 0.095 0.124
0.01 0.01 0.01 0.01
Treatment
Low rate
High rate
Est. (95% CI)
Untreated Acidified Untreated Acidified
P. minuta 0.558 ± 0.106 (2) 0.600 (1) 0.629 (1) 0.648 (1)
1.039 1.007 0.832 0.969
0.12 0.12 0.10 0.12
Treatment
Low rate
High rate
Epedaphic species 28 days 56 days Time
Hemiedaphic species 28 days 56 days Time
Euedaphic species 28 days 56 days
Untreated Acidified Untreated Acidified
P. fimata 0.169 ± 0.221 ± 0.399 ± 0.336 ±
0.035 0.011 0.034 0.035
(2) (3) (5) (4)
0.435 0.445 0.542 0.656
(1) ± 0.012 (2) ± 0.016 (5) ± 0.02 (6)
± 0.059 (2) ± 0.086 (2) ± 0.019 (2) (1)
± ± ± ±
(0.01–0.02) (0.01–0.02) (0.01–0.01) (0.01–0.02)
(0.09–0.16) (0.08–0.15) (0.07–0.13) (0.07–0.17)
a a a a
(2) (2) (5) (2)
0.03 0.03 0.04 0.05
(0.01–0.04) (0.01–0.05) (0.03–0.06) (0.04–0.07)
Acknowledgement [13]
We gratefully acknowledge the technical assistan[1–3]ce of Mette Nielsen.
[14]
Appendix A. Supplementary data [15]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ejsobi.2019.103117.
[16]
References
[17] [18]
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8
High rate
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H. nitidus 0.077 ± 0.021 (3) 0.03 ± 0.028 (2) 0.112 (1) 0.120 (1)
0.235 0.278 0.199 0.212
0.03 0.03 0.02 0.03
Low rate
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Est. (95% CI)
F. fimetaria 0.266 (1) 0.340 (1) na 0.396 (1)
0.626 0.622 0.730 0.933
0.07 0.07 0.08 0.11
(1) ± 0.102 (2) (1) (1)
(0.01–0.04) (0.02–0.04) (0.01–0.04) (0.01–0.04)
a a a a
a a a a
Est. (95% CI)
0.011 0.003 0.034 0.139
Low rate
b b ab a
(1) (1) (1) ± 0.044 (2)
(0.05–0.09) (0.06–0.09) (0.06–0.10) (0.09–0.12)
a a a a
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Contents lists available at ScienceDirect
European Journal of Soil Biology journal homepage: www.elsevier.com/locate/ejsobi
Effect of acidified cattle slurry on a soil collembolan community: A mesocosmos study
T
Alessandra D'Annibalea, Rodrigo Labouriaub, Peter Sørensena, Paul H. Kroghc, ⁎ Bent T. Christensena, Jørgen Eriksena, a
Department of Agroecology, Aarhus University, AU-Foulum, DK-8830, Tjele, Denmark Department of Mathematics, Aarhus University, DK-8000, Aarhus, Denmark c Department of Bioscience, Aarhus University, DK-8600, Silkeborg, Denmark b
ARTICLE INFO
ABSTRACT
Handling editor: Stefan Schrader
The concept of soil health emphasizes the role of soil organisms in sustainable agriculture and relies on bioindicators to assess the impact of management options. This study employed Collembola, a key group of microarthropods with intimate links to soil microbial activity, as bioindicators to reveal the sustainability of using acidified cattle slurry. While environmental and plant production benefits of acidification are well known, effect of acidified slurry on Collembola is undocumented. We added Collembola to sandy soil mixed with untreated or acidified cattle slurry labelled with 15N and applied at two rates (corresponding to 30 and 90 Mg slurry ha−1). The collembolan community included two species for each vertical life-form: epedaphic (Sinella curviseta and Heteromurus nitidus), hemiedaphic (Proisotoma minuta and Hypogastrura assimilis) and euedaphic species (Folsomia fimetaria and Protaphorura fimata). Replicate mesocosmos were incubated for 28 or 56 days at 15 °C, 65% WHC and a 12 h light-dark cycle. The epedaphic species strongly declined at the end of the experiment, whereas P. minuta and F. fimetaria were positively affected by high rates of untreated and acidified slurry, respectively. The hemiedaphic P. minuta showed the highest abundances and accumulation of slurry-15N, especially when high rate of untreated slurry was applied. We conclude that the impact on Collembola of low rates of untreated and acidified slurry applied to agricultural soils is marginal while higher rates of slurry may subsequently form a favourable habitat for euedaphic species.
Keywords: Cattle slurry Acidification Mesofauna Mesocosmos Arable soil 15 N-labelling
1. Introduction The concepts of soil health and sustainable agriculture emphasize the role of soil organisms and recognize that soil physical and chemical properties represent drivers for the structure and function of the decomposer community [1,2]. While addition of crop residues and animal manures impacts the physical soil environment, it also provides nutrient and energy resources supporting the soil decomposer community. To allow for development of sustainable management, the concept of soil health has to be accompanied by bioindicators that provides a quantitative assessment of the impact on soil of a given management option [e.g. [3, 4, 5, 6]. Recognizing that the turnover of organic matter and nutrients rely on microorganisms under the influence of faunal activity, management impacts on numbers and activity of key groups of soil fauna need to be determined. Collembola are microarthropods with intimate links to soil microbial activity [7], but little is known regarding their reaction to intensive
⁎
nitrogen (N) management. In NW European areas with dairy production, grasslands receive frequent inputs of cattle slurry to provide sufficient herbage yields. To improve N use efficiency, ammonia volatilization during application can be reduced by lowering slurry pH through addition of sulphuric acid [8]. While the economic and environmental benefits of slurry acidification are known [9], effects of acidified slurry on soil Collembola remains unknown. The reduced pH is not expected to have direct negative impacts as animals in arable soils are already adapted to a soil pH around 6. However, acidification of slurry may indirectly affect soil Collembola via its effects on the transformation of ammoniacal-N, especially in slurry hot-spots established by slit injection of high slurry rates. Slit injection of slurry in the field may reduce the number of epigeic earthworms but leave enchytraeids unaffected [10]. Earthworm excreta (mucus and urine) and the ammonium content in these substances has been shown to attract Collembola [11,12] while a repulsive behaviour was demonstrated at high concentrations of ammonium [11]. Domene
Corresponding author. Department of Agroecology, Aarhus University, AU-Foulum, Blichers Allé 20, DK-8830, Tjele, Denmark. E-mail address: [email protected] (J. Eriksen).
https://doi.org/10.1016/j.ejsobi.2019.103117 Received 19 November 2018; Received in revised form 6 August 2019; Accepted 12 August 2019 Available online 18 August 2019 1164-5563/ © 2019 Elsevier Masson SAS. All rights reserved.
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et al. [13] demonstrated that ammonium present in sewage sludge was toxic for the Collembola species Folsomia candida, but also that the level of toxicity depends on soil properties. Thus, the impact of cattle slurry on soil Collembola may well relate to application rate and concentrations of ammoniacal-N. Previous studies have addressed the effect of untreated and anaerobically digested animal manure on Collembola [14,15], but effects of acidified slurry on soil Collembola remain elusive. Using a mesocosmos set-up, we examined the effect of untreated and acidified cattle slurry applied at two rates to a sandy soil amended with a collembolan community constructed with equal representation of the three vertical life-forms: epedaphic, hemiedaphic and euedaphic. These life-froms differ typically by having contrasting habitat preferences, feeding ecology and gut microbiome [16,17]. Thus, the population performance could respond to the rate of slurry and its nutrient content and this is hypothesized to be true for euedaphic collembolans already shown to thrive in organic soil amendments [18]. We envisioned that application of slurry at low rate (i.e. conditions after surface application) has a positive effect on Collembola in terms of abundances, whereas slurry applied at high rate (i.e. conditions in injection slits) is detrimental because of high concentrations of ammoniacal-N. We expect a negative effect of acidified slurry on Collembola as acidification conserves ammoniacal-N temporarily in the soil volume affected by the slurry.
Using adult specimens collected from laboratory cultures, a collembolan community was added to each cylinder at day 0. The community included 30 individuals of each of six species belonging to three life-forms [15]: two epedaphic (Heteromurus nitidus, Templeton 1835 and Sinella curviseta, Brook 1882), two hemiedaphic (Hypogastrura assimilis, Krausbauer 1898 and Proisotoma minuta, Tullberg 1871), and two euedaphic (Folsomia fimetaria, Linnaeus 1758 and Protaphorura fimata, Gisin 1952). The Collembola were added to the soil surface and a perforated lid placed on top of the cylinder. During incubation, cylinders were kept at 15 °C and a 12 h light-dark cycle. Each cylinder was weighed once a week and water was added when needed to maintain the initial water content. The experiment involved two sets of cylinders to be destructively harvested for Collembola after 28 and 56 days of incubation. For each harvest time, three replicate cylinders were prepared for each of the treatments: control without slurry (C0), untreated slurry at low rate (UL), untreated slurry at high rate (UH), acidified slurry at low rate (AL), and acidified slurry at high rate (AH). For each treatment, one extra set of replicates without animals was included for determination of initial (day 0) contents of soil water content, pH, and NH4–N and NO3–N extracted with 1 M KCl. In total, 45 cylinders were prepared. 2.2. Analyses at harvest At harvest, four soil subsamples (each 120 g fresh weight) were collected from each cylinder for extraction of Collembola. Another three subsamples were taken for chemical soil analyses. Collembola were extracted over one week with a high-gradient temperature extractor by increasing the temperature stepwise from 25 to 60 °C. For three of the subsamples for enumeration of Collembola, animals were collected in trays with plaster of Paris (calcium sulphate hemihydrate) mixed with activated charcoal (w/w = 8/1) in order to collect live specimens for 15N analyses. For the last subsample, Collembola were collected in benzoic acid [19]. For each species, the number of adults and juveniles was determined visually according to size. The final number of Collembola was based on both plaster of Paris and benzoic acid samples. Soil subsamples were ground for analyses of total-N and 15N abundance. Inorganic N extracted from soil by 2 M KCl was subjected to micro-diffusion for 15N analysis [20]. Collembola extracted from the cylinders and sampled from the laboratory cultures were also subject to isotope measurements. Slurry samples were extracted with 2 M KCl to determine concentrations of NH4–N and NO3–N. The slurry extracts were processed by micro-diffusion for inorganic 15N measurements as described for soil extracts. All samples for N isotope analyses were wrapped in tin capsules (5 × 8 mm) targeting a sample N content of 20–150 μg. Isotope analysis was performed at the Stable Isotope Facility, University of California, Davis, USA, with a PDZ Europa ANCAGSL elemental analyser interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd, Crewe, Cheshire, UK). The 15N atom percent (Atom %) was determined as:
2. Materials and methods 2.1. Experimental setup The mesocosmos experiment relied on a constructed collembolan community exposed to sandy soil mixed with untreated (U) or acidified (A) cattle slurry applied at low (L) or high (H) rate. The soil was retrieved from 0 to 25 cm depth in a field subject to long-term agricultural management. It had 6% clay, 9% silt, 44% fine sand, 39% coarse sand, pH 6.5, 1.6% carbon (C), and 0.15% N. The soil was dried at room temperature, sieved to < 4 mm and then defaunated by successive freeze-thaw cycles (temperature alternating between −18 °C and +20 °C). Table 1 shows the characteristics of the cattle slurry used in the experiment. The slurry was from dairy cows fed a standard diet (mainly silage) and stored for 15 months at 2 °C in a closed container. One batch of slurry was acidified to pH 5.3 with sulphuric acid (8.0 g kg−1 slurry) while another batch remained untreated and had pH 7.5. At the start of the experiment (day 0), the slurries were labelled with 15N using (15NH4)2SO4 with 99.3 atom percent (Atom-%) to reach a final 5% enrichment of the NH4–N pool in the slurry. Each mesocosmos consisted of a 33 cm high Plexiglas cylinder with an inner diameter of 9 cm. The cylinder was closed at the bottom and then filled with 700 g dry soil homogenously mixed with slurry (L, 3 g per 100 g soil; H, 9 g per 100 g soil). These rates correspond to a field application of 30 and 90 Mg slurry ha−1 incorporated into a 0–7 cm soil layer. Demineralized water was then added to reach 65% of the soil water holding capacity (WHC).
Atom %15N =
Characteristics
a
15 N × 100 N + 15N
and the Atom Percent Excess of 15N (15N APE) was calculated as the difference between 15N abundance in the labelled sample and the corresponding unlabelled control sample. For some Collembola species, samples for isotope analysis required pooling of individuals from replicate cylinders to achieve a sufficient amount of N for the analysis. The 15N recovery was calculated for total N, inorganic N (NH4–N + NO3–N) and organic N. The recovery of total N and inorganic N was calculated as:
Table 1 Characteristics of the cattle slurry.
Untreated slurry total N Untreated (labelled) slurry NH4–Na Acidified (labelled) slurry NH4–Na Dry Matter (DM) on fresh weight Untreated slurry pH Acidified slurry pH
14
3.36 g kg−1 1.35 g kg−1 1.63 g kg−1 8.7% 7.5 5.3
Recovery (%) =
Values used for the15N recovery calculations in soil.
APEsoil N × soil × 100 APEslurry Nslurry
where Nsoil is total N or inorganic N. The recovery of 15N in the organic 2
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N pool was calculated as the difference between total N and inorganic N. For the inorganic N pool, the calculation of APE for soil and slurry samples applied the correction of the Atom % given by Sørensen and Jensen [20].
dependency between observations from the same cylinder; and one residual random component taking different values for each observation and allowing the model to represent the over-dispersion present in the data [25,26]. Note that an equivalent model without the random component representing overdispersion does not adjust to the data (see Appendix for details). Both random components were defined in such a way that they have different variances for the observations made with each of the two Collembola collection methods (plaster of Paris or benzoic acid). The models contained a fixed effect composed by a factor representing the combinations of the treatments (C0, UL, UH, AL and AH) and harvest time (day 28 and 56). The parameters associated with the fixed effect defined above are precisely the expected values of the number of animals per g of soil for the corresponding treatments and harvest time (see the appendix A for details); these parameters (and the corresponding confidence intervals) were then multiplied by 1000 for expressing the abundances as number of animals per kg soil.
2.3. Statistical analyses 2.3.1. Modelling the soil parameters For soil, the total N, NO3–N, and the total, inorganic and organic 15 N recoveries were analysed using generalized linear mixed models (GLMM; [21, 22] with fixed effects representing the combinations of the treatments (C0, UL, UH, AL and AH) and harvest time (day 0, 28 and 56). The GLMMs contained a Gaussian random component specially designed to represent the dependency between observations from the same cylinder. All models were defined with the identity link function and the normal distribution, except the model for NO3−-N that was defined with an inverse Gaussian distribution [23]. Tests for the presence of partial interactions, obtained by eliminating the control treatment from the analyses, revealed statistically significant effect modification between acidification and slurry application rate. This rules out the use of additive effects of these two factors. Interactions followed different patterns at different harvest times (p-values < 0.00001) in all cases, except for total-N (p-value = 0.98). Detailed descriptions of the models are given in the Appendix. The NH4–N contents of different combinations of treatments and time were characterized through a Kruskal-Wallis test [24], due to the lack of adherence of Gaussian or other classic parametric models and due to the high variability of the data. The results of this parameter were therefore reported by the medians for each combination of treatment and harvest time, and the corresponding confidence intervals obtained by non-parametric bootstrap (10,000 bootstrap simulations).
2.3.4. Modelling the Collembola population age compositions The age composition of each species of Collembola was characterized by classifying individuals as adults or juveniles. The proportions of adults in each population were estimated using a binomial logistic mixed model with random components and fixed effects defined as in the models for the abundance defined above. Note that these models account for the over-dispersion present in the data, in the same way as the Poisson model describing the abundances defined above. See Appendix for details. 2.3.5. Statistical inference procedures The models described above (except for soil NH4–N) are all instances of GLMM. The inference for these models were performed using the functions lmer (for the Gaussian models), glmer (for the nonGaussian models, as the inverse Gaussian, the Poisson and the binomial models) from the package {lme4} [27] and the function lm for the 15N APE of P. minuta and F. fimetaria, implemented in the R-software [28]. The post-hoc pairwise comparisons for all models were done using the R-package {pairwiseComparisons} [29]. The p-values implicitly used in the post-hoc pairwise comparisons were adjusted for multiple comparisons using the one-step method for the parametric models (implemented in the R package {multcomp} [30], and the fdr (controlled false discovery rate) method for the non-parametric tests [31].
2.3.2. Modelling the 15N accumulation in each species The 15N Atom Percentage Excesses (15N APE) in each species were analysed using Gaussian multiple regression models where the slurry rate entered as a continuous explanatory variable and different slope parameters were estimated for each slurry treatment (untreated or acidified). The slope parameters indicate the expected increases in 15N assimilation when slurry rate increases one unit, i.e. 1 g slurry 100 g−1 soil (within the range 3–9 g slurry per 100 g soil). It was not always possible to obtain enough Collembola biomass to make precise determinations of 15N APE with the sample from one single replicate; in those cases it was then necessary to physically pool samples from replicate cylinders; only cylinders with the same treatment (UL, UH, AL or AH), slurry rate (L or H) and harvest time (da3 to ys 28 and 56) were pooled. The models contained three random components indicating whether each cylinder entered in the pooling of the corresponding observation, allowing in this way to account for the variance heterogeneity generated by the sample pooling process (see Appendix for details). In the case of the species P. minuta and F. fimetaria it was necessary to pool all the samples collected at the same harvest time and subject to the same treatment/slurry application rate; in this case the model did not contain any random component.
3. Results 3.1. Soils Table 2 shows soil pH at day 0 and after 28 and 56 days of incubation. Addition of acidified slurry reduced soil pH compared to corresponding rates of untreated slurry. For all slurry treatments, the soil pH dropped about one unit from day 0 to day 28 and then remained constant. The initial concentration of NH4–N reflected the addition of slurry (Table 3). Soil concentrations of NH4–N peaked at the beginning of the experiment, with higher values in soils with acidified slurry than in soils with corresponding rates of untreated slurry, but concentrations were subsequently reduced to very low levels. However, treatment AH
2.3.3. Modelling the Collembola abundances The abundance (calculated as the number of animals per kg of soil) of each of the Collembola species (except H. assimilis, which did not grow sufficiently during the experiment in any of the treatments) was modelled by a Poisson mixed model with over-dispersion that accounted for differences in the amount of soil sampled, the heterogeneity of dispersion and the dependency of the observations. This model was adjusted, for each species, by using a GLMM defined with a Poisson distribution, a logarithmic link function, an offset with the logarithm of the soil mass corresponding to the observation, and two specially designed Gaussian random components: one taking the same value for the observations arising from the same cylinder, representing the
Table 2 Soil pH measured at the start of incubation (Day 0) and after 28 and 56 days of incubation.
3
Treatment
Day 0
28 days
56 days
C0: Control without slurry UL: Untreated slurry, low rate UH: Untreated slurry, high rate AL: Acidified slurry, low rate AH: Acidified slurry, high rate
6.5 6.9 7.4 6.1 5.8
5.9 5.7 5.5 5.3 4.9
5.9 5.7 5.5 5.4 4.9
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Mean (95% CI)
– – – – –
– 98 (94–103) b,A 101 (97–105) b,A 88 (84–93) a,A 90 (86–94) a,A
Mean (95% CI)
(21–30) (22–30) (16–25) (15–24)
(13–25) a,A (10–22) a,B (14–26) a,A (6–18) a,B
– – – – –
– 26 26 20 19
– 19 16 20 12
– 91 93 91 82
(85–97) (87–99) (84–97) (76–88)
a,A a,B a,A a,B
% %
a,A a,A a,A a,A
Total15N recovery Organic15N recovery
(0.3–0.4) (0.3–0.4) (0.5–0.6) (0.4–0.4) (1.3–1.4)
a,B ab,B c,C b,C d,C
39.1 (35.4–42.8) a,C 91.3 (89.7–93) b,C 208.9 (206.4–211.4) d,C 96.5 (94.9–98.1) c,C 210.1 (207.6–212.6) d,C
1.55 1.59 1.79 1.57 1.81
(1.49–1.6) a,A (1.53–1.64) a,A (1.73–1.84) b,A (1.52–1.63) a,A (1.75–1.86) b,A
– 72 76 71 71
(70–74) (75–78) (69–72) (69–73)
a,A b,A a,B a,A
The five collembolan species subject to statistical analyses (the original community except H. assimilis – see Materials and Methods) showed different population growth patterns (Figs. 1–3). During the first 28 days of incubation, the epedaphic species S. curviseta and H. nitidus showed a three-fold increase in population density regardless of treatment (Fig. 1). The increase was accompanied by a substantial drop in the proportion of adults and a corresponding increase in the proportion of juveniles. After 28 days, these two species had a significantly lower proportion of adults in UH compared to the treatment without slurry addition (C0). From day 28 to day 56, both species showed a significant decline in numbers regardless of soil treatment and at day 56, the number of individuals was smaller than that added initially (day 0) and the proportion of adults had increased significantly, in particular for S. curviseta. The proportion of H. nitidus adults was significantly lower in UH than in C0 after 28 days; this was also true for the treatments UL and UH at day 56. At day 28, the number of the hemiedaphic species Proisotoma minuta did not differ between soil treatments and was only marginally higher than the initial population (Fig. 2). Except for the treatment UH, the number of individuals and the adult proportions did not differ between treatments after 56 days of incubation. At this harvest time, P. minuta showed a significantly higher density in the UH treatment which is the highest population density found in this study. The proportion of adults was similar after 28 and 56 days of incubation. The exception was soil without slurry addition; here the proportion of adults was higher at day 28 than at day 56. The euedaphic species F. fimetaria did not differ significantly between treatments neither in total densities nor in adult proportions after 28 days of incubation (Fig. 3). The data for this harvest are associated with large variability but apparently numbers of individuals were similar for day 0 and day 28. The number of individuals declined significantly from day 28 to day 56 in all the treatments, except for AH. At day 56, this treatment showed the highest number of animals whereas the proportion of adults was similar to that of the other slurry treated soils. At the end of the experiment, the lowest population density was found for C0 (the population was almost extinct) while the proportion of adults was significantly higher compared to the slurry treatments (Fig. 3). The other euedaphic species P. fimata was significantly less abundant in AH after 28 days compared to AL and C0, without differences in adult proportions. For the AH treatment, the population of P. fimata increased significantly from day 28 to day 56. Table 4 shows that the 15N Atom Percent Excess (15N APE) in Collembola was lowest in the two epedaphic species S. curviseta (specifically in UL after 28 days) and H. nitidus (in AL after 28 days). The highest values was found for the hemiedaphic species P. minuta (in UH and AH after 28 days). For S. curviseta the 15N-APE values were higher in treatments with acidified slurry than treatments with untreated slurry. This was also true for H. nitidus and P. minuta and for the
C0: Control without slurry UL: Untreated slurry, low rate UH: Untreated slurry, high rate AL: Acidified slurry, low rate AH: Acidified slurry, high rate 56 days
0.3 0.4 0.5 0.4 1.3
bc,A c,A a,A b,A (71–74) (74–76) (67–69) (69–72) – 73 75 68 71 (1.44–1.55) a,A (1.6–1.71) b,A (1.84–1.95) c,B (1.61–1.72) b,B (1.82–1.92) c,A 1.49 1.66 1.90 1.67 1.87 31.1 (26–36.1) a,B 82.8 (81.1–84.4) b,B 190.3 (188–192.6) d,B 85.7 (84.1–87.4) b,B 168 (166–170) c,B C0: Control without slurry UL: Untreated slurry, low rate UH: Untreated slurry, high rate AL: Acidified slurry, low rate AH: Acidified slurry, high rate 28 days
0.5 (0.1–0.5) a,B 0.6 (0.1–0.6) ab,B 0.8 (0.7–0.9) c,B 0.6 (0.6–0.6) b,B 27.1 (25.8–29.3) d,B
– – – – – (1.44–1.61) a,A (1.5–1.67) a,A (1.68–1.85) b,A (1.48–1.65) a,AB (1.71–1.88) b,A (−0.9-26.8) (−1.3-26.8) (−1.6-26.9) (−1.7-26.7) (−1.8-26.8) 12.9 12.8 12.7 12.5 12.5 C0: Control without slurry UL: Untreated slurry, low rate UH: Untreated slurry, high rate AL: Acidified slurry, low rate AH: Acidified slurry, high rate Day 0
3.5 (3.1–4.3) a,A 45.3 (34.7–47.7) b,A 121.7 (83.9–128.6) d,A 57.1 (56.5–59.1) c,A 156 (140.9–164.6) e,A
Mean (95% CI)
mg kg−1 mg kg−1
Median (95% CI) Treatment Time
NO3−-N
a,A a,A a,A a,A a,A
1.52 1.58 1.76 1.57 1.80
Mean (95% CI) Mean (95% CI)
% g kg−1
NH4++15NO3− recovery 15
Total N
remained higher in NH4–N after 28 and 56 days. Initial (day 0) concentrations of NO3–N did not differ between treatments, but increased significantly during the incubation. After 28 days, the concentration was significantly higher in UH, compared to all the other treatments. After 56 days of incubation, there was no difference in NO3–N concentration between UH and AH, while the concentration was significantly higher in AL compared to UL (Table 3). Total N in soil showed the highest values in soils receiving the high rate of slurry. The recovery of 15N in total N after 28 days of incubation was significantly higher in soil with untreated slurry than in soil with acidified slurry, regardless of addition rate (Table 3), but after 56 days, treatments did not differ. For soils taking high rates of slurry, the recovery of 15 N decreased significantly from day 28 to day 56. The recovery of 15N as inorganic N (15NH4+ 15NO3) ranged from 68 to 76% with significantly higher recovery in soil with the high rate of untreated slurry. 3.2. Collembola
NH4–N
Table 3 Ammonium-N, nitrate-N, total N, and15N recovery rates (inorganic, organic and total) in soil initially and after 28 and 56 days of incubation for each treatment. Different small letters indicate significant (p < 0.05) differences between treatments at a given sampling time, while different capital letters indicate significant differences between 28 and 56 days of incubation.
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Fig. 1. Interaction plot of the total densities and adults proportions of the epedaphic species in the experimental treatments at the different experimental time points. The expected values of the treatments at the same observation time are connected with lines as a visual aid for identifying interactions between the observation time and the treatments. Bars indicate 95% CI. Different small letters indicate significant (p < 0.05) differences between treatments within the same sampling, while different capital letters indicate significant differences between 28 and 56 days. Abbreviations: C0: Control without slurry, UL: Untreated slurry, low rate, UH: Untreated slurry, high rate, AL: Acidified slurry, low rate, AH: Acidified slurry, high rate.
euedaphic species at high slurry concentrations after 56 days. At the end of incubation, the euedaphic species P. fimata showed higher slope coefficient for 15N APE in acidified treatments, although the slope was not significantly different from untreated slurry after 56 days. No differences in the slope coefficients were detected for the other species. However, our data suggest a higher increase (i.e. estimated slope) in 15 N accumulation from low to high slurry application rate in P. minuta (ca. 0.12) and F. fimetaria (ca. 0.07–0.11) compared to the other species.
collembolan abundances due to the potential toxicity of elevated ammonium concentrations in the soil. However, most of the ammonium in the soil and that applied with slurry at the start of the incubation was nitrified or immobilized within the first 28 days of incubation; after 56 days of incubation initial concentrations of ammonium were reduced by 99% (Table 3). The change in concentrations of ammoniacal-N during day 0 to day 28 is unknown, but we surmise that any impact of ammonium would be short-term because of its almost complete disappearance after 28 days. The soil receiving a high rate of acidified slurry (AH) was the only treatment with slightly elevated ammonium content at day 28. This treatment was more acid than any other treatment at day 28 and also at day 56 (Table 2). Although the ammonium concentration after 56 days was significantly higher in AH than in the other treatments, the concentration was very low. Detrimental effects of ammonium on Collembola have been claimed in studies involving manure and biosolids applied to soils [13,15,32], but our results lend little support for this claim. As expected, the concentration of nitrate increased during the
4. Discussion Slurry acidification is adopted to reduce ammonia volatilization during surface application and when slits remain open after slurry injection. With high rates of acidified slurry, the effect of acidification may linger on after application whereby elevated levels of ammonium can be retained in the slurry affected soil volume. We envisioned that high rates of slurry could negatively affect 5
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Fig. 2. Interaction plot of the total densities and proportion of adults of the hemiedaphic species P. minuta in the experimental treatments at the different experimental time points. The expected values of the treatments at the same observation time are connected with lines as a visual aid for identifying interactions between the observation time and the treatments. Bars indicate 95% CI. Different small letters indicate significant (p < 0.05) differences between treatments within the same sampling, while different capital letters indicate significant differences between 28 and 56 days. Abbreviations: C0: Control without slurry, UL: Untreated slurry, low rate, UH: Untreated slurry, high rate, AL: Acidified slurry, low rate, AH: Acidified slurry, high rate.
experiment; soils receiving high slurry rates reaching the highest concentrations. The average net immobilization of 15N, that is 15N recovered in organic N pools, accounted for 20% of the ammonium added initially while ammonium plus nitrate held 72% of the 15N label. Immobilization did not differ between treatments neither at day 28 nor at day 56, but the treatments with high slurry rates decreased in organically bound 15N from day 28 to day 56. Thus, our results suggest that ammonium contents and nitrification in the soils were defined by slurry addition rate whereas acidification had little impact. Previous studies based on Collembola extracted from soil in field experiments show a positive effect of organic amendments on numbers of animals. Bolger and Curry [32] observed a beneficial effect of adding 55 and 110 Mg ha−1 of cattle slurry to permanent grassland while an extremely high slurry rate (550 Mg ha−1) significantly depressed collembolan numbers. However, they concluded that in the longer term, the soil fauna would respond positively to the food source provided by the slurry. Similarly, Weil and Kroontje [33] reported a positive effect on numbers of Collembola in soils subjected for 5 years to fresh poultry manure at annual rates ranging from 27 to 110 Mg ha−1. Positive effects on numbers of Collembola have also been reported after soil additions of cattle slurry, green-waste composts, and digestates from biogas [4,14,34]. However, Pommeresche et al. [15] found a negative effect of cattle slurry on collembolan numbers extracted 3 and 48 days after application. After 56 days of incubation, we found that rates of cattle slurry corresponding to 90 Mg ha−1 had a positive effect on the abundance of the species P. minuta (untreated slurry, UH) and F. fimetaria (acidified slurry, AH), whereas other species were unaffected by slurry. The population of F. fimetaria declined from day 28 to day 56 except in soil with high rate of acidified slurry; in this treatment F. fimetaria numbers increased and the adult-to-juvenile ratio differed significantly between day 28 and day 56. This suggests that the application of high rates of acidified slurry sustained the reproduction of this species during the 56 days of incubation. Without slurry addition, F. fimetaria may be negatively affected by competition from other Collembola species. For the two epedaphic species (S. curviseta and H. nitidus), the number of animals increased significantly during the first 28 days of incubation regardless of treatment, while numbers after 56 days fell to below initial levels. These changes were mirrored in the proportion of
adults; the number of juveniles decreased from day 28 to day 56. A clear difference between the epedaphic and the other species in terms of population growth was also reflected in their 15N accumulation. The epedaphic species had the lowest average 15N APE values, while P. minuta showed the highest values, especially at high slurry rates. In a laboratory study, Larsen et al. [35] found P. minuta to have a higher metabolic rate and reproductive investment than P. fimata, suggesting that P. fimata has lower nutritional requirements. This may explain why the population of P. fimata remained rather constant during the entire incubation period. Initially, P. fimata showed lower densities in the AH treatment. However, the population recovered after 56 days and reached final densities that were comparable to the other species. After 56 days of incubation, the highest number of specimens (approximately 67% of the community) was observed for the hemiedaphic species P. minuta in the UH treatment, indicating that a high concentration of untreated slurry constituted a favourable environment for the growth of this species. At the end of the incubation, effects of slurry added at low rate were generally absent while P. minuta and F. fimetaria were positively affected by the high slurry rate. Although the two species differ in lifeforms, they belong to the same family Isotomidae (order Entomobryomorpha). Since this was the only obvious effect of slurry treatments, we conclude that application of slurry at low rate may have but a marginal effect on collembolan community while high rates of slurry may have a positive longer-term effect as suggested by Bolger and Curry [32]. Moreover, the collembolan species involved in the present study were generally unaffected by slurry acidification, notwithstanding that the high rate of acidified slurry favoured the reproduction of F. fimetaria. A shift of the collembolan community towards more abundant euedaphic and hemiedaphic species as a result of slurry application may improve their contribution to slurry decomposition and support agroecosystem functions. The hemiedaphic species H. assimilis did not successfully survive in any treatment and was excluded from analyses as reported previously [36,37]. The selection of Collembola species for mesocosmos studies will depend the objective of a given test and on the stocks of laboratory raised animals at hand. Our experimental set-up relied on addition of the same number of individuals for each species, recognizing that these contribute differently to the collembolan biomass in the soil. Our 6
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Fig. 3. Interaction plot of the total densities and proportion of adults of the euedaphic species in the experimental treatments at the different experimental time points. The expected values of the treatments at the same observation time are connected with lines as a visual aid for identifying interactions between the observation time and the treatments. Bars indicate 95% CI. Different small letters indicate significant (p < 0.05) differences between treatments within the same sampling, while different capital letters indicate significant differences between 28 and 56 days. Abbreviations: C0: Control without slurry, UL: Untreated slurry, low rate, UH: Untreated slurry, high rate, AL: Acidified slurry, low rate, AH: Acidified slurry, high rate.
overall objective was to link the impact of acidified slurry to soil health, using a mesocosmos set-up and Collembola as bioindicators. The limitations of this approach when studying the effect of complex substrates such as slurry is recognized, and one alternative approach is to extract Collembola from field plots subject to different management [e.g. 14, 15]. However, seasonal and annual fluctuations in collembolan numbers and species severely limit the suitability of Collembola as indicator of soil health when based on field samplings [38–40].
their population abundances during the experiment, regardless of the treatment. Acidification of slurry is an important step towards higher N use efficiency and thereby improved sustainability in agricultural crop production. In this study, the low rate of slurry simulates soil conditions following surface application while the high rate was taken to simulate soil conditions in the injection slit. We conclude that the impact on Collembola of low rates of untreated and acidified slurry applied to agricultural soils is marginal while higher rates of slurry may subsequently form a favourable habitat for euedaphic species and preserve their decomposition potential.
5. Conclusions Using a mesocosmos set-up with a constructed population of six species of Collembola incubated for 4 and 8 weeks, we found that application of slurry at a rate corresponding to 30 Mg ha−1 had little impact on the collembolan community while a slurry rate corresponding to 90 Mg ha−1 showed a positive effect. The collembolan species adopted in this study were generally unaffected by slurry acidification. However, two epedaphic species strongly decreased in
Financial support This study was financially supported through a PhD grant from the Graduate School of Science and Technology (GSST) at Aarhus University.
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Table 4 15 N Atom % Excess (15N-APE) of Collembola exposed to untreated or acidified cattle slurry applied at low or high rate. Means ± SEM are given with number of samples in brackets. The slope coefficient Est. is the effect of a unit increase slurry concentration (in the range from 3 to 9 g per 100 g). 95% confidences intervals (CI) are reported in brackets and different letters indicate significant (p < 0.05) differences between harvest time and treatment combinations. “na” indicates “not available”. Time
Treatment
Low rate
High rate
Est. (95% CI)
Untreated Acidified Untreated Acidified
S. curviseta 0.034 ± 0.012 (5) 0.042 ± 0.004 (4) 0.038 ± 0.007 (5) 0.061 ± 0.01 (4)
0.108 0.116 0.095 0.124
0.01 0.01 0.01 0.01
Treatment
Low rate
High rate
Est. (95% CI)
Untreated Acidified Untreated Acidified
P. minuta 0.558 ± 0.106 (2) 0.600 (1) 0.629 (1) 0.648 (1)
1.039 1.007 0.832 0.969
0.12 0.12 0.10 0.12
Treatment
Low rate
High rate
Epedaphic species 28 days 56 days Time
Hemiedaphic species 28 days 56 days Time
Euedaphic species 28 days 56 days
Untreated Acidified Untreated Acidified
P. fimata 0.169 ± 0.221 ± 0.399 ± 0.336 ±
0.035 0.011 0.034 0.035
(2) (3) (5) (4)
0.435 0.445 0.542 0.656
(1) ± 0.012 (2) ± 0.016 (5) ± 0.02 (6)
± 0.059 (2) ± 0.086 (2) ± 0.019 (2) (1)
± ± ± ±
(0.01–0.02) (0.01–0.02) (0.01–0.01) (0.01–0.02)
(0.09–0.16) (0.08–0.15) (0.07–0.13) (0.07–0.17)
a a a a
(2) (2) (5) (2)
0.03 0.03 0.04 0.05
(0.01–0.04) (0.01–0.05) (0.03–0.06) (0.04–0.07)
Acknowledgement [13]
We gratefully acknowledge the technical assistan[1–3]ce of Mette Nielsen.
[14]
Appendix A. Supplementary data [15]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ejsobi.2019.103117.
[16]
References
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8
High rate
Est. (95% CI)
H. nitidus 0.077 ± 0.021 (3) 0.03 ± 0.028 (2) 0.112 (1) 0.120 (1)
0.235 0.278 0.199 0.212
0.03 0.03 0.02 0.03
Low rate
High rate
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F. fimetaria 0.266 (1) 0.340 (1) na 0.396 (1)
0.626 0.622 0.730 0.933
0.07 0.07 0.08 0.11
(1) ± 0.102 (2) (1) (1)
(0.01–0.04) (0.02–0.04) (0.01–0.04) (0.01–0.04)
a a a a
a a a a
Est. (95% CI)
0.011 0.003 0.034 0.139
Low rate
b b ab a
(1) (1) (1) ± 0.044 (2)
(0.05–0.09) (0.06–0.09) (0.06–0.10) (0.09–0.12)
a a a a
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