*Article* **The E**ff**ect of Biochar Used as Soil Amendment on Morphological Diversity of Collembola**

**Iwona Gruss 1,\*, Jacek P. Twardowski 1, Agnieszka Latawiec 2,3, Jolanta Królczyk <sup>4</sup> and Agnieszka Medy ´nska-Juraszek <sup>5</sup>**


Received: 24 July 2019; Accepted: 15 September 2019; Published: 19 September 2019

**Abstract:** Biochar was reported to improve the chemical and physical properties of soil. The use of biochar as a soil amendment have been found to improve the soil structure, increase the porosity, decrease bulk density, as well increase aggregation and water retention. Knowing that springtails (Collembola) are closely related to soil properties, the effect of biochar on morphological diversity of these organisms was evaluated. The main concept was the classification of springtails to the life-form groups and estimation of QBS-c index (biological quality index based on Collembola species). We conducted the field experiment where biochar was used as soil amendment in oilseed rape and maize crops. Wood-chip biochar from low-temperature (300 ◦C) flash pyrolysis was free from PAH (polycyclic aromatic hydrocarbon) and other toxic components. Results showed that all springtail life-form groups (epedaphic, hemiedaphic, and euedaphic) were positively affected after biochar application. The QBS-c index, which relates to springtails' adaptation to living in the soil, was higher in treatments where biochar was applied. We can recommend the use of Collembola's morphological diversity as a good tool for the bioindication of soil health.

**Keywords:** biochar; biological soil quality; Collembola life-form groups; QBS-c index

### **1. Introduction**

One of the major threats to global agriculture is soil degradation, including decreased fertility and increased erosion [1]. The common problem is acidification and soil organic matter depletion, which decreases soil aggregate stability [2]. Therefore, the development of methods is needed to sustain soil resources by different remediation strategies. The application of organic materials like manure, compost, and biomass waste seems very promising, but a lot of attention has been paid to stable forms of organic carbon like biochars [3,4]. The main feature of biochar is the porous carbonaceous structure, which can contain amounts of extractable humic-like and fluvic-like substances [5]. Biochar was reported to improve the chemical and physical properties of soil [6]. The use of biochar as a soil amendment has been found to improve the soil structure, increase the porosity, decrease bulk density, as well increase aggregation and water retention [7–9]. On the other hand, the main concerns with respect to biochar use as a soil amendment is its potential contamination with heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs) [10].

Springtails (Hexapoda: Collembola) are a key group of soil arthropods with densities often reaching thousands of individuals per square meter [11,12]. They contribute mainly in substrate decomposition and nutrient cycling [12,13]. Moreover, these organisms are sensitive to environmental changes in soil and are therefore often used as indicators of soil quality [14]. For bioindication, Collembola species diversity is used [15,16]. The disadvantage of this method is the difficulty in the determination to the species level. An alternative could be the QBS-c index (biological quality index based on Collembola), which responds to the morphological diversity of springtails [17]. Using this index, each individual is evaluated in terms of different morphological traits, e.g., for antennal length, size of furca, presence of ocelli pigmentation, and the presence of hairs and/or scales along the body. The principle of this index is that the presence of individuals with better adaptation to live in soil (with reduced appendages or less pigmented) indicates better soil quality [17,18]. Also, on the basis of morphological traits, springtails can be divided into three main life-forms [19]. First, epedaphic Collembola are adapted to live on the soil surface. The major features of this group are a pigmented body, well developed eyes and appendages, as well a fast dispersal ability. In contrast to them, soil dwelling species (euedaphic), with a relatively small, less pigmented body and reduced eyes. Their dispersal ability is limited. Species showing adaptations between epedaphic and euedaphic species are classified as belonging to the hemiedaphic group [20]. The vertical stratification of springtails reflects their function in the ecosystem. For instance, only epedaphic springtails contribute in the early stages of organic matter decomposition [19]. Ponge et al. [21] suggested that Collembola living in the soil characterized by limited active dispersal, may suffer more from land use intensification, than species living on the soil surface. In contrast, Ellers et al. [22] showed stronger effects of intensive land use on epedaphic than on euedaphic Collembola. The majority of studies on biochar effect on springtails were conducted in laboratory conditions on one model species [23–25]. Considering the impact of biochar under field conditions some experiments have been made also on nematodes [26] and earthworms [27].

The potential of biochar for pH and nutrient availability changes or improvement of some physical properties like porosity, water retention, or temperature and impact on soil microbial life, are well documented [28–30]. Therefore, springtails can be affected directly by the changes in soil chemical properties [31,32] or indirectly from biochar-induced changes in microorganisms' biomass [33]. It has been reported that many Collembola species feed on bacteria or fungi [34,35]. Biochar particles might be considered analogous to soil aggregates in that their large internal surface areas and pores could be important for biological processes [36].

Within the presented study we aimed to estimate the effect of biochar on the morphological diversity of Collembola species and evaluate its potential for field application.

It was hypothesized that:


#### **2. Materials and Methods**

#### *2.1. Experimental Design*

The field experiment was set up in mid-April 2014 in the south of Poland (50.5740 N, 17.8908 E) and continued until October 2016. The soil type was poor (sandy and weakly acidic) agricultural soil [37]. The climate of the area is moderately warm with an average annual temperature of 8.4 ◦C and an average annual rainfall of 611 mm. The biochar effect on soil dwelling springtails was explored in two crops (oilseed rape and maize) compared to control (two crops with no biochar application). Within each treatment (plot), three replicates (subplots) were established. The area of each subplot

was 3 × 3 m. The research area was previously (before 2014) used as conventionally agricultural field. The forecrop was maize. Biochar was applied up to a depth of 30 cm at a rate of 50 t/ha and was mixed by ploughing. No chemical protection was applied before or during experiment period. Weeds were removed manually upon occurrence. Weeds were harvested manually a few times during the vegetation season. The maize variety in two years of the study was P8745 (FAO 250, Pioneer Company) and oilseed rape variety Monolit. The only fertilizer used in oilseed rape was ammonium sulfate 34% in a dose of 300 kg/ha, and in maize ammonium phosphate (Polydap) in a dose of 25 kg/ha. The same amount of fertilizers was applied in biochar and control treatments.

#### *2.2. Biochar Characteristic and Soil Properties*

The biochar used in the experiment was industrial produced by Fluid S.A. Company (Poland). It was produced in the low-temperature flash pyrolysis (300 ◦C) of pine and spruce wood chips. Its heating value was 25 MJ/kg. During the experiment selected properties of biochar (pH, organic carbon content, cation exchange capacity, heavy metal content and total PAH's) were analyzed according to International Biochar Initiative (IBI) Standard Product Definition and Product Testing Guidelines [38]. The particle size fraction of biochar applied on the field was more than 2 mm (sieve method).

The tested biochar was alkaline (pH 8.2) and had 52.3% of carbon (Table 1). The surface area of the tested biochar was low (only 16.5 m2/g) and cation exchange capacity was also lower—39.5 cmol/kg, compared with biochars produced at higher temperatures and from other feedstock, like wheat straw, giant miscanthus, rice husk, or sewage sludge [39,40]. It was free from polycyclic aromatic hydrocarbon and the concentration of all tested toxic compounds was very low or even under the level of detection, passing fixed recommendations for acceptable levels [38].


**Table 1.** The chemical characteristic of biochar properties used in the experiment (sourced from Gruss et al. [41]).

For physicochemical analysis, soil samples were collected twice a year from topsoil, before each crop in rotation in five replicates from each plot. The pH, total organic carbon, CEC, exchangeable acidity, and water properties were measured. Soil was classified as Cambisol [38], with a typical sandy loam texture with the addition of medium fine gravel. Application of biochar significantly increased CEC values in both trials, due to the increase of exchangeable Ca2+, Mg2+, and H+ + Al3+ (exchangeable acidity) in the soil sorption complex and total organic carbon in biochar trials (Table 2).


**Table 2.** Soil properties after biochar application in oilseed rape and maize (sourced from Gruss et al. [41]).

\* The values in bold font differ significantly between treatments.

#### *2.3. Collembola Studies*

Soil samples for Collembola analysis were taken three times during each of the vegetation season (from May to July) in 2015 and 2016. The growth stages according to the BBCH (growth stages of plants) scale [42] in the sampling dates were: maize: 10–15, 32–37, and 61–67; oilseed rape: 60–69, 72–79, and 83–89. On each date, 12 samples were taken from each subplot (36 samples from one plot), and transported to the laboratory. The samples were taken with the use of a soil sampler (diameter 5 cm and depth 10 cm). The volume of one sample was 196 cm2. Collembola were extracted over 24 h from the soil samples with the use of Tullgren funnels modified by Murphy [43]. After the extraction the springtails were kept in 75% ethyl alcohol.

Springtails from each sample were counted and identified to the species or genus. Each individual was placed on permanent microscope slide and determined to the species level with the use of following keys [44–46]. Springtails were classified to three life-form groups (euedaphic, hemiedaphic and epigeic) according to Karaban [20]. Epedaphic forms have strong pigmentation, fully developed furca and other appendages, and pigmented eyes (8 + 8). Hemiedaphic have reduced body pigmentation, eye numbers, and a reduced furca. Euedaphic forms are characterized by an unpigmented body (or eyes' pigmentation) with eyes and furca not developed. The QBS-c (biological quality index based on Collembola species) is calculated as the sum of EMI values in each sample (Table 3). The springtails species were evaluated for seven morphometric traits according to the scale. The results were the sums of scores (EMI) obtained for each trait. Species which are well adopted to live in soil obtain more EMI scores in comparison to those with adaptation to live on the soil layer [17].



\* Size: >3 mm = 0; 2–3 mm = 2; <2 mm = 4.

Pigmentation: Fully pigmented=0; only strips on the body = 1; r = Reduced to appendages = 3; none = 6.

Structures on cuticle: Well developed chaeta or scales, present trichobothria = 0; relatively low number of structures on cuticle=1; Reduced number of chaetae, presence of PSO (pseudocelli) on cuticle = 3; Low number of chaeteae, other structures present only in selected parts of the body = 6.

Number of ocelli in the eye spot: 8 + 8 = 0; 6 + 6 = 2; form 5 + 5 to 1 + 1 = 3; absence of ocelli = 6. Antennae: antennae longer than the head = 0; antennae more or less the same length as the

head = 2; antennae shorter than the head = 3; antennae much shorter than the head = 6. Legs: Well developed = 0, Medium developed = 2; Short = 3; Reduced or with reduced claw and mucro =6.

Furcula: Well developed = 0; Medium developed = 2; Short with reduced number of chaetae = 3; the absence of mucro and modification of manubrium = 5; Furcula reduced in residual form = 6.
