# Positive or neutral effects of biochar-compost mixtures on earthworm communities in a temperate cropping system

Nicolas Honvault, David Houben, Manhattan Lebrun, Charlotte Vedere, Cécile Nobile, Julien Guidet, Léa Kervroëdan, Marie-Liesse Aubertin, Cornelia Rumpel, Michel-Pierre Faucon, et al.

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## To cite this version:

Nicolas Honvault, David Houben, Manhattan Lebrun, Charlotte Vedere, Cécile Nobile, et al. Positive or neutral effects of biochar-compost mixtures on earthworm communities in a temperate cropping system. Applied Soil Ecology, 2023, 182, pp.104684. 10.1016/j.apsoil.2022.104684. hal-03810055

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1 **Positive or neutral effects of biochar-compost

2 mixtures on earthworm communities in a temperate

3 cropping system

4 Nicolas Honvault<sup>a,*</sup>, David Houben<sup>a</sup>, Manhattan Lebrun<sup>b</sup>,

5 Charlotte Vedere<sup>b</sup>, Cécile Nobile<sup>ad</sup>, Julien Guidet<sup>a</sup>, Léa

6 Kervroëdan<sup>a</sup>, Marie-Liesse Aubertin<sup>b,c</sup>, Cornelia Rumpel<sup>c</sup>,

7 Michel-Pierre Faucon<sup>a</sup>, Anne-Maïmiti Dulaurent<sup>a</sup>

8 <sup>a</sup> UniLaSalle, AGHYLE UP 2018.C101, 60000 Beauvais,

9 France

10 <sup>b</sup> National Institute for Agricultural Research, Ecosys

11 Soil INRAE-AgroParisTech, 78820 Thiverval-Grignon,

12 France

13 <sup>c</sup> Institute of Ecology and Environmental Sciences, UMR

14 7618, CNRS-UPMC-UPEC13 INRA-IRD, Sorbonne

15 University, 75005 Paris, France

16 <sup>d</sup> CIRAD, UPR Recyclage et risque, F-97743 Saint-

17 Denis, La Réunion, France

18 * Correspondence: nicolas.honvault@umontpellier.fr

19 **Abstract**

20 Biochar is increasingly considered an efficient

21 amendment to improve soil fertility and store carbon in

22 soils for climate change mitigation. However, few field

23 studies have evaluated biochar impacts on soil fauna and

24 especially earthworms in temperate areas despite their

25 importance for soil structure and laboratory-based
evidence of potential adverse effects. We investigated the effects of three biochars (from rapeseed, miscanthus and compost reject) in mixture with a green waste compost on the abundance and composition of earthworm communities during a three-year field experiment in northern France. Compost alone or biochar-compost mixtures were applied at 11.7 t dry matter ha⁻¹ and compared to equivalent mineral fertilization. Biochar-compost mixtures did not affect total earthworm density and biomass, while contrasting effects concerning specific ecological groups were observed. Compost addition increased juvenile endogeic earthworm density roughly one month after amendment while no adverse short-term effects of biochar-compost mixtures were observed. Two years after amendment application, anecic earthworm density was higher for the mixture with compost and biochar from compost rejects, which also resulted in the highest soil organic carbon concentrations. We conclude that biochar-compost mixtures at low but more economically viable biochar application rates could be used as an increasingly understood beneficial soil amendment under temperate climate conditions without adverse effects on earthworm communities.

Keywords
50 Earthworms, Biochar, Compost, Anecic, Endogeic,
51 Agroecology

52 **1. Introduction**

53 Biochar, the solid carbon-rich pyrolysis residue, can have
54 many beneficial effects on soil functioning, fertility and
55 carbon sequestration (Dai et al., 2020; Ding et al. 2016;
56 Majumder et al. 2019; Jenkins et al. 2017; Joseph et al.,
57 2021), contributing toward planning for future large scale
58 deployment (Joseph et al., 2021). One way to implement
59 biochar application at scale may be its addition to soil
60 organic amendments such as compost. In particular,
61 biochar may be produced at large scale composting
62 facilities from non-compostable material. However, the
63 effects of such materials on soil fauna and associated
64 ecosystem services have received little attention, with
65 most studies focusing on microbial communities
66 (Güereña et al., 2013; Lehmann et al., 2011).
67 Earthworms are of great importance for soil functioning
68 (Lavelle et al., 1997). They are often presented as "soil
69 engineers" owing to their effects on soil structure (Lavelle,
70 1988) and nutrient availability (Blouin et al., 2013) and
71 more generally biogeochemical cycling (Angst et al.,
72 2019). Despite recent efforts to decipher biochar effects
73 on earthworm communities (Ameloot et al., 2013;
74 Brtnicky et al., 2021; Domene, 2016), the impacts of
75 organic amendments including biochar and biochar co-
76 application with organic or mineral fertilizer on earthworm
77 communities remain poorly predictable (Whalen et al.,
78 2021). Further comprehensive research is thus required
79 before such strategies can be implemented at scale
80 (Malev et al., 2016; Oleszczuk et al., 2013; Sohi et al.,
81 2009).

82 While biochar effects on earthworm communities were
83 found to be very inconsistent (Lehmann et al., 2011;
84 Weyers and Spokas, 2011), many studies reported a
85 negative short-term (<6 months) effect of biochar on
86 earthworm density and activity (Li et al., 2011; Weyers
87 and Spokas, 2011). Moreover, the presence of biochar
88 reduced microbial activity during organic matter
89 decomposition in the presence of earthworms (Barthod et
90 al., 2016). This deleterious effect can be attributed to the
91 presence of contaminants in biochar, such as polycyclic
92 aromatic hydrocarbons (PAH) (Malev et al. 2016; Ren et
93 al. 2018). PAH can be issued from direct carbonization
94 and aromatization at temperature below 500 °C or free
95 radical pathways at temperature above 500 °C, with
96 widely differing concentrations between biochar (Wang et
97 al. 2017). Decreased soil matric potential following
98 biochar addition (Tammeorg et al., 2014), or biochar
99 liming effects (Domene, 2016) can also contribute to
100 deleterious effects. Other authors have suggested that
101 physical protection of the microbial communities
102 inhabiting biochar particles would result in shifts in
103 microbial grazers including earthworms (Briones et al.,
104 2020). Studies on longer term effects (periods superior to
105 six months to one year) are scarce and inconsistent as
106 negative (Briones et al., 2020), neutral (Whalen et al.,
107 2021) or positive (Husk and Major, 2010) effects have
108 been reported. Earthworm diverse behaviours per
109 ecological groups, such as deep burrowing and mixing of
110 mineral and organic soil layers for endogeic earthworms,
111 limited shallow soil mixing for epigeic earthworms or
112 vertical burrowing and litter burying for anecic
113 earthworms (Bouché 1977; Bohlen et al. 2004) may in
114 part explain these inconsistencies. Nonetheless,
115 detrimental effects of biochar on earthworms have been
116 predominantly reported after biochar application at high
117 rates, ranging from 25 to 68 t ha⁻¹ (Briones et al., 2020;
118 Liesch et al., 2010; Malev et al., 2016), relative to the
119 recommended rates ranging from 5 to 20 t ha⁻¹ (Joseph et
120 al., 2021). In addition, most studies have focused on
121 tropical climates while temperate areas have been much
122 less investigated (Tammeorg et al., 2014), and the wide
123 heterogeneity of biochar properties (Dai et al., 2020;
124 Xiang et al., 2017) was often omitted by approaches
which used only one biochar type. Given the considerable effects of organic matter on earthworm communities (Leroy et al., 2008, 2007; Sizmur et al., 2017) and especially the strong response of endogeic earthworms to soil available C (Tiunov and Scheu, 2004), biochar co-application with organic fertilization such as compost could importantly impact its effects (Tammeorg et al., 2014). Mixture with compost may also enhance other benefits of biochar (Jeffery et al., 2017; Ye et al., 2020). The large majority of lab-based evidence of biochar impact on earthworm communities underlines the need for more field-based experiments to confirm tendencies observed ex-situ and their evolution over longer time periods (Briones et al., 2020; Whalen et al., 2021).

In order to investigate the effects of different types of biochar co-applied with organic fertilization on earthworm communities we performed a field experiment with three contrasting biochar-compost mixtures as well as compost alone and a mineral control in northern France. Biochar-compost mixtures were applied at a rate of 11.7 t ha⁻¹ with a 30:70 biochar compost ratio in order to investigate realistic and economically viable practices. We aimed to evaluate if contrasting biochar properties alter the response of earthworm communities to biochar-compost mixture addition. To this end, we monitored earthworm
density and biomass during three years. We examined
how earthworm diverse behaviour and especially feeding
behaviour influenced their response to amendment
addition by identification of earthworm species and their
classification into ecological group (i.e. anecic, endogeic
and epigeic earthworms) (Bouché, 1977). We
hypothesised contrasting effects of the different biochar-
compost mixtures on earthworm communities because of
their diverse properties induced by the different biochar
types.

## 2. Material and methods

### 2.1 Organic amendments

The compost used in the experiment was a green waste
compost issued mostly from grass, poplar and conifers
branches provided by Fertivert (Seine-Maritime, France).
Three compost-biochar mixtures were examined,
containing biochar produced with diverse temperature
conditions and from different feedstocks. Three
feedstocks for biochar production were examined
miscanthus straw, rapeseed straw and green waste
compost reject, issued from reject material
undecomposed after composting. Biochars were
produced using an industrial pyrolysis reactor (Biogreen®
Technology, ETIA, Oise, France) with pyrolysis
temperatures of 550 °C for miscanthus biochar, 650 °C
for rapeseed straws biochar and 450 °C for green wastes
compost rejects biochar and a 10 minutes pyrolysis time
for all biochars. Biochars were selected based on their
contrasting properties and effects on plant growth (higher
biomass after miscanthus biochar addition than rapeseed
straws biochar and intermediate for green wastes
compost rejects biochar) during a preliminary experiment
(Honvault et al., 2022). Compost-biochar mixtures and
compost characteristics are presented in Table 1 and
biochar properties are presented in Table 2

2.2 Field experiment

The field experiment was carried out in Beauvais
(UniLaSalle farm), northern France (49°25'49"N,
2°04'51"E). The soil was classified as an Haplic Luvisol
(IUSS Working Group WRB, 2015), with 21 % clay, 69%
silt and 10% sand (Table 3). Soil properties were
determined following protocols published by the French
Association for Normalization (AFNOR, 1996), such as
dry combustion on decarbonated soil for organic carbon
(NF ISO 1423) (Table 3). The climate was oceanic with
minimum and maximum temperatures averaging at 6.5
°C and 14.9 °C and the average annual precipitation was
655 mm year⁻¹. The experiment started in spring 2019
and lasted three cropping cycles until autumn 2021. The
crop rotation consisted of maize in 2019, wheat in 2020
and barley in 2021. The experimental design consisted in
five treatments applied in four replicates for a total of 20
randomly distributed plots of 32 m² (3.2 m x 10 m).
Treatments consisted of a green waste compost either
alone ("Compost") or mixed with biochar issued from rape
straws ("Compost/BiocharRap"), miscanthus straw
("Compost/BiocharMis") or green waste compost rejects
biomass ("Compost/BiocharRej"). Compost alone was
applied at a rate of 19.6 t fresh matter ha⁻¹ corresponding
to approximately 7.8 t dry matter, while compost-biochar
mixtures were applied at a rate of 24.5 t fresh matter ha⁻¹
consisting of 7.8 t dry matter ha⁻¹ compost and 3.9 t dry
matter ha⁻¹ biochar. A mineral control ("Control") adding
the same quantity of phosphorus and potassium as in the
Compost treatment as triple superphosphate and KCl
respectively was also part of the experiment. Nitrogen
was not included as standard recommended nitrogen
fertilization was applied (in several doses for a total of 60
kg ha⁻¹ in 2019, 164 kg ha⁻¹ in 2020 and 180 kg ha⁻¹ in
2021) to ensure optimum nitrogen nutrition. No additional
mineral fertilisation or organic fertilisation was applied
during the whole duration of the experiment. The
amendments were applied manually to the soil once in
2019 before mechanical incorporation in the first 5 cm using rotary harrow. Control plots were prepared as the other plots, i.e. by mechanically mixing inorganic fertilizer within the first 5 cm. The tillage was then very reduced and superficial throughout the experiment.

## 2.3 Earthworm sampling

Earthworms were sampled twice every year in spring and autumn, except in autumn 2019 and in spring 2020 (Table 4). Earthworms were extracted with a formaldehyde solution (10 mL 37 % in 10 L water) on three 0.5 m² (0.7*0.7m) areas per plot (ISO 2018). Each 0.5 m² area was watered 3 times, each time followed by a 10 minute-collection period. Harvested areas were identified with a plastic picket and progressively shifted within the plots to avoid previously harvested areas. Due to the long-term character of the still ongoing experiment, earthworms were collected on the soil surface without soil excavation. Earthworms were stored in 70% ethanol until determination shortly after collection. Total earthworm biomass was measured for spring samplings, as well as earthworm biomass per ecological group in 2021. They were classified by species and ecological group (i.e. epigeic, endogeic, anecic) (Bouché, 1977).

## 2.4 Statistical analysis
All tests were performed with R v 3.6.0 and the package Rcmdr 2.5-3 with a significance level of 0.05 (R Core Team, 2013). The effects of year, season and treatments were first examined via generalised linear models (GLM) (quasi-poisson distribution, log link) with specified interactions between year, season and treatment to understand the overall effects of biochar-compost mixtures. GLM with quasi-poisson distribution were selected due to the skewed and slightly over dispersed density (count) data overall. The proportion of juvenile to adult earthworms was calculated and compared for each ecological group as well as the proportion of each ecological group to the total density. In order to examine plausible effects on earthworm diversity the Shannon diversity index was calculated as presented below (Shannon, 1948).

$$H = - \sum [(pi) \times \ln(pi)]$$

With pi proportion of each individual species in the community.

As data per year and season was normally distributed, differences among earthworm density, biomass, species richness and diversity were examined with one-way ANOVAs followed if necessary by Tukey post hoc tests.
270 **3. Results and discussion**

271 **3.1 Earthworm total density and biomass**

272 Earthworm total density at the beginning of the
273 experiment was very low between 7 and 14 individual m⁻²
274 and more than doubled two years later with densities
275 between 24 and 44 individuals m⁻² (Fig. 1 c and f). This
276 was also reflected in earthworm biomass which increased
277 from 5.7 to 11.9 g m⁻² on average in spring 2019 to 16.0
278 to 35.2 g m⁻² on average in spring 2021 (Supplementary
279 material S1). During the three cropping cycles, the
280 different organic amendments did not show any effect on
281 total earthworm abundance, richness or diversity (Table
282 5). Strong seasonal effects were underlined with
283 earthworm density, species richness and diversity being
284 higher in spring than autumn, likely due to dry summers
285 months reducing earthworm densities (Holmstrup, 2001;
286 Whalen, 2004). Strong temporal effects were also
287 underlined with earthworm density, species richness and
288 diversity increasing with time since mechanical
289 incorporation of the amendments (Table 5). Despite
290 significant interactions between treatment, year and
291 season (Table 5) no significant differences were
292 observed in earthworm species richness and diversity
293 (Supplementary material S2). Increasing earthworm
294 density in the first years after the establishment of the
295 field experiment is consistent with results of Denier et al.
296 (2022), who observed a similar two fold increase in
297 earthworm density after three years without tillage in a
298 field experiment adjacent to ours. This increase may thus
299 be related to initial mechanical disturbances and reduced
300 mechanical disturbances of soil during the experiment,
301 which is known to strongly affect earthworm communities
302 in agricultural fields (Karaca, 2011). Our results showed
303 that mechanical disturbances were the predominant
304 factor driving earthworm density over time irrespective of
305 the treatment. We thus overall highlight neutral effects of
306 biochar-compost mixtures on earthworm total densities
307 compared to equivalent fertilisation, in agreement with
308 recent observations (Whalen et al., 2021) but in contrast
309 with numerous studies showing adverse short term (<6
310 months) effects of biochar application on earthworm
311 density and activity (Li et al., 2011; Weyers and Spokas,
312 2011). We indeed observed no short-term negative
313 effects of biochar roughly one month after addition (Fig. 1
314 a, b and c) and up till three years after application.

315 3.2 Earthworm community composition

316 The earthworm communities from this cropped soil were
317 composed of two to five species and dominated by two
318 species, *Lumbricus terrestris* and *Allolobophora chlorotica* (see Supplementary material S3 for a list of
319
species observed). Despite overall neutral effects of organic amendments on total earthworm density, significant differences between treatments were noted per ecological group within the community, i.e. for anecic earthworm density. Significant interactions between treatment, year and season were also observed for endogeic earthworm density (Table 6). Further investigations highlighted significant differences in juvenile endogeic earthworm density for organic amendments compared to the mineral control and anecic earthworm density between organic amendments in spring 2019 and 2021 respectively, while no significant differences were observed in autumn, likely due to lower density (Supplementary material S4). Significantly higher juvenile endogeic earthworm density was observed for the compost treatment compared to the mineral control in spring 2019 (Fig. 1 b), despite very low densities. All organic amendments in turn tended to have higher, albeit not significantly, juvenile endogeic earthworm density than the mineral control in spring 2019 (Fig. 1 b). In spring 2021, anecic earthworm density was higher for the Compost/BiocharRej treatment (23 ± 3 individuals m⁻²) compared to the Compost treatment (12 ± 2 individuals m⁻²) and to the other organic treatments (Fig. 1 d) but not to the mineral control. Anecic earthworm biomass was
also significantly higher for the Compost/BiocharRej with 345 32 g m⁻² as compared to 14 g m⁻² for the 346 Compost/BiocharRap treatment. 347

Increased endogeic earthworm in spring 2019 may be 348 explained by boosting effects of organic carbon additions 349 on earthworm communities (Leroy et al., 2008, 2007), 350 which are expected to particularly affect endogeic 351 earthworms. Indeed endogeic earthworms feed on more 352 humidified substrates and are mostly limited by available 353 C in soil (Tiunov and Scheu, 2004), which can be directly 354 enriched by the carbon contained in organic fertilizers 355 (Briones et al., 2020). Moreover, endogeic earthworm 356 communities have been observed to decline less or even 357 increase after tillage (Chan, 2001; Ernst and Emmerling, 358 2009). Since the soil was slightly tillied to allow 359 amendment incorporation roughly one month before the 360 first sampling in spring 2019, the weaker sensitivity to 361 tillage of endogeic earthworms might explain why only 362 endogeic earthworms responded quickly positively to 363 compost addition. By contrast, tillage may have overrode 364 the very short-term amendment effect on the other 365 ecological groups which are more sensitive to mechanical 366 disturbances (i.e.anecic earthworms) (Denier et al., 2022; 367 Simonsen et al., 2010). Anecic earthworm indeed 368 represented a lower proportion of the total community at 369
the beginning of the experiment (Fig. 1 a, c, d, f).

However, no significant differences in total earthworm
density and anecic earthworm density were observed
compared to the mineral control, highlighting low short-
term impacts of organic inputs in our system.

Intermediate juvenile endogeic earthworm densities were
observed in Spring 2019 for compost-biochar mixtures
compared to compost alone and to the mineral control
(Fig. 1 b). The lack of adverse short-term effects of
biochar may be related to the low biochar application rate
which was well below the threshold rates reported for
negative effects such as 16 t ha⁻¹ in Malev et al. (2016) or
68 t ha⁻¹ in Liesch et al. (2010). Moreover, simultaneous
co-application with organic matter appears to largely
compensate short-term negative effects of biochar
(Weyers and Spokas, 2011), suggesting that co-
application of compost could have contributed to alleviate
possible negative effects of biochar. Slightly lower
juvenile endogeic earthworm density for biochar-compost
mixtures compared to compost alone could then be
related to the higher nutritional value of compost alone
(Leroy et al., 2008). Earthworms have indeed been
shown to prefer more labile and less humidified sources
of carbon (Leroy et al., 2008) and biochar is considered
to be of low nutritional value for earthworms (Briones et
al., 2020). Lower average nutritional value for biochar-compost mixtures as well as protection of labile carbon sorbed on biochar particles may thus have contributed to slightly lower juvenile endogeic densities for biochar-compost mixtures compared to compost applied alone. Biochar liming effect and PAH content may also have contributed, although low biochar application rates would suggest minor effects.

Longer term effects of organic amendments contrasted with the earlier observations, showing significantly higher anecic earthworm density for the Compost/BiocharRej treatment compared to compost alone and no significant differences in endogeic earthworm density between organic amendments. Endogeic (and total) earthworm density did not significantly differ for the organic amendments compared to the mineral control, showing progressively decreasing effects of organic inputs. The approximately two fold increase in anaecic earthworm density for treatments amended with Compost/BiocharRej as compared to the compost treatment is consistent with observations by Husk and Major (2010) of an approximately two times increase in earthworm density one year and half after biochar addition as well as observations by Tammeorg et al. (2014) of an 1.8 times increase in earthworm density four months after biochar
addition (even though not significantly different for the later). Biochar application can lead to positive effects on earthworm density after several months (Husk and Major, 2010; Tammeorg et al., 2014). However, these effects are scarcely reported with neutral effects or negative effects being reported more frequently (Briones et al., 2020; Weyers and Spokas, 2011; Whalen et al., 2021). Increased anecic earthworm density could hardly be related to increased crop biomass productivity, soil fertility or soil pH which did not show significant differences among treatments (data not shown, in preparation). We, however, observed increased organic carbon concentration in soils amended with biochar-compost mixtures (Fig. 2). Specifically, organic carbon concentration was higher for all soils amended with organic amendments compared to the mineral control in 2019 (Fig. 2 a), for the Compost/Biochar treatments compared to the mineral control in 2020 (Fig. 2 b) and for the Compost/BiocharRej treatment compared to the mineral control in 2021 (Fig. 2 c). To our knowledge, this is the first observation of a plausible long-term (i.e. more than one year) change in earthworm communities due to biochar application in mixture with compost, simultaneous to an increase in soil organic carbon concentration. However, no significant correlation was observed
between soil organic carbon concentration and anecic earthworm density, and differences in anecic earthworm density were observed between organic treatments rather than with the mineral control. Increased anecic earthworms density after two years seems to contrast with the increased juvenile endogeic earthworm density observed roughly one month after amendment incorporation and with the higher preference of endogeic earthworms for humidified C possibly increased by organic amendments (Tiunov and Scheu, 2004). Recent evidence nonetheless points toward higher assimilation of biochar carbon by anecic earthworms compared to endogeic earthworm (Briones et al., 2020). However, *Lumbricus terrestris*, the dominant specie in our communities, appear to poorly assimilate carbon originating from biochar (Briones et al., 2020). Moreover, anecic earthworm density significantly differed between organic amendments but not compared to the mineral control despite increases in organic carbon concentration, prompting further investigations. We thus overall underline a mostly neutral long term (i.e. more than one year after addition) effect of biochar-compost mixtures on earthworm total density, coherent with the low biochar rate applied and fertile temperate soils (Weyers and Spokas, 2011), while also highlighting scarcely reported
470 changes in earthworm community composition that
471 require further investigations.

472 **4. Conclusion**

473 This study intended to gain insight into the sparsely
474 investigated effect of biochar-compost mixtures on
475 earthworm community in an agricultural field under
476 temperate climate. Our experiment indicated overall
477 neutral effect of biochar-compost mixtures. We conclude
478 that biochar addition to compost at low application rates
479 (4 t dry matter ha⁻¹) has no adverse effects on the
480 abundance of earthworm communities both on the short-
481 and the long-term. However, we also observed a group-
482 specific influence of the biochar type on the amendment
483 effect, as anecic earthworm density and biomass were
484 greater for compost containing compost rejects biochar
485 as compared to the other organic amendments. This
486 mixture also induced a simultaneous lasting increase in
487 organic carbon concentration, while our results don't
488 support a direct relationship, prompting further
489 investigations. To our knowledge, our results thus
490 constitute one of the very few evidences of a multiannual
491 impact of biochar compost mixtures on earthworm
492 communities in a temperate setting. We conclude that
493 biochar type could influence the effect of such mixtures.
494 However, as biochar interaction with soil environments
495 and its effect on soil biological response tend to be very

496 variable depending on biochar and soil properties, further

497 investigations are required in order to generalize our

498 observations.
499 Acknowledgements

500 The authors thank Bpifrance and the Région Hauts-de-
501 France for funding the project (FUI Biochar 2021). CR
502 acknowledges funding from ANR under the U2 worm
503 project (ANR-20-CE01-0015-01). All members of the FUI
504 biochar are acknowledged for their contribution to the
505 project. The authors also thank Vincent Hervé for his
506 technical assistance. We thank ETIA and Agriculteurs
507 Composteurs de France for providing compost and
508 biochars. We thank the “ASET 160-161-162” students for
509 technical assistance.

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<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 1</figcaption></figure>

791

792

793
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>Fig. 2</figcaption></figure>
796 **Fig. captions**

797 **Fig. 1:** Mean total earthworm density and density per ecological group (± SE) in spring 2019 and 2021.

799 Rap: mixture of green waste compost and biochar issued from rape biomass; Mis: mixture of green waste compost and biochar issued from miscanthus biomass; Rej: mixture of green waste compost and biochar issued from green wastes compost rejects biomass; Comp: green waste compost; Min: Mineral control. Different letters above the bars indicate statistically significant differences. a Anecic earthworm density in spring 2019, b Endogeic earthworm density in spring 2019, c Total earthworm density in spring 2019; d Anecic earthworm density in spring 2021, e Endogeic earthworm density in spring 2021, f Total earthworm density in spring 2021.

811 **Fig. 2:** Mean soil organic carbon concentration (± SE) in 2019, 2020 and 2021.

813 a: Organic carbon concentration in 2019; b: Organic carbon concentration in 2020; c: Organic carbon concentration in 2021. Different letters above the bars indicate statistically significant differences. Rap: mixture of green waste compost and biochar issued from rape biomass; Mis: mixture of green waste compost and biochar issued from miscanthus biomass; Rej: mixture of
820 green waste compost and biochar issued from green

821 wastes compost rejects biomass; Comp: green waste

822 compost; Min: Mineral control.
<table><caption>**Table 1**</caption><thead><tr><th>Amendment</th><th>Organic C<sup>†</sup><br>(g kg<sup>-1</sup>)</th><th>Total N<sup>‡</sup><br>(g kg<sup>-1</sup>)</th><th>C:N</th><th>P<sup>§</sup><br>(g kg<sup>-1</sup>)</th><th>K<sup>§</sup><br>(g kg<sup>-1</sup>)</th><th>Ca<sup>§</sup><br>(g kg<sup>-1</sup>)</th><th>Mg<sup>§</sup><br>(g kg<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Compost</td><td>292</td><td>27</td><td>10</td><td>4.2</td><td>17</td><td>29</td><td>2.8</td></tr><tr><td>Compost/BiocharMis</td><td>337</td><td>18</td><td>18</td><td>3.4</td><td>16</td><td>24</td><td>2.3</td></tr><tr><td>Compost/BiocharRej</td><td>344</td><td>22</td><td>15</td><td>5.3</td><td>19</td><td>31</td><td>2.9</td></tr><tr><td>Compost/BiocharRap</td><td>429</td><td>21</td><td>20</td><td>3.5</td><td>20</td><td>32</td><td>2.2</td></tr></tbody></table>

**Table 1: Organic amendment properties**

†Dry combustion; ‡Dumas method; § Total, chemical digestion followed by ICP measurement (NF EN 13650, ICP MS NF EN ISO 17294) (EN 13650, 2002). NF and NF ISO determined following protocols published by the French Association for Normalization (AFNOR, 1996). Compost/BiocharMis: mixture of green waste compost and biochar issued from miscanthus biomass; Compost/BiocharRej: mixture of green waste compost and biochar issued from green wastes compost rejects biomass; Mixture Compost/BiocharRap: mixture of green waste compost and biochar from rape biomass; Compost: green waste compost.
<table><caption>Table 2</caption><thead><tr><th rowspan="2"></th><th rowspan="2">Unit (Dry Mass)</th><th colspan="3">Miscanthus Rapeseed Compost</th></tr><tr><th>BC</th><th>BC</th><th>BC</th></tr></thead><tbody><tr><td>Porosity<sup>†</sup></td><td>%</td><td>92.5</td><td>95.9</td><td>86.3</td></tr><tr><td>Density<sup>†</sup></td><td>g L<sup>-1</sup></td><td>112</td><td>78</td><td>291</td></tr><tr><td>Conductivity<sup>‡</sup></td><td>dS m<sup>-1</sup></td><td>1.30</td><td>1.22</td><td>3.54</td></tr><tr><td>CEC<sup>§</sup></td><td>cmol<sub>c</sub> kg<sup>-1</sup></td><td>31.0</td><td>2.9</td><td>8.5</td></tr><tr><td>pH<sup>#</sup></td><td></td><td>10.4</td><td>11.2</td><td>11.4</td></tr><tr><td>Ash 550°C<sup>††</sup></td><td>%</td><td>18.6</td><td>26.4</td><td>33.7</td></tr><tr><td>C<sup>††</sup></td><td>%</td><td>74.0</td><td>62.3</td><td>55.5</td></tr><tr><td>Total N<sup>§§</sup></td><td>%</td><td>0.53</td><td>0.81</td><td>0.88</td></tr><tr><td>C/N</td><td></td><td>141</td><td>77</td><td>63</td></tr><tr><td>P<sup>##</sup></td><td>g kg<sup>-1</sup></td><td>0.31</td><td>0.38</td><td>0.37</td></tr><tr><td>K<sup>##</sup></td><td>g kg<sup>-1</sup></td><td>2.52</td><td>1.82</td><td>1.78</td></tr><tr><td>Ca<sup>##</sup></td><td>g kg<sup>-1</sup></td><td>0.21</td><td>6.57</td><td>3.38</td></tr><tr><td>Mg<sup>##</sup></td><td>g kg<sup>-1</sup></td><td>0.06</td><td>0.41</td><td>0.50</td></tr></tbody></table>

**Table 2**: Chemical and physical properties of the biochars used.

<footnote>† (EN 13041, 2006) ; ‡ (EN 13038, 1999) ; § (NF X31-130, 1999) ; # (EN 13037, 1999) ; †† (ISO, 2016) ; f (EN 13039, 2011) ; §§ (EN 13654-1, 2002) ; ## total extracted with aqua regia, (EN 13650, 2002).</footnote>
<table><caption>Table 3</caption><thead><tr><th>Soil depth (cm)</th><th>Clay (%)</th><th>Silt (%)</th><th>Sand (%)</th><th>pH<sup>†</sup><br>H<sub>2</sub>O</th><th>Carbonates<sup>‡</sup><br>(%)</th><th>OM<sup>§</sup><br>(g kg<sup>-1</sup>)</th><th>CEC<sup>#</sup><br>(cmolc kg<sup>-1</sup>)</th><th>Total N<sup>††</sup><br>(g kg<sup>-1</sup>)</th><th>P<sup>‡‡</sup><br>(g kg<sup>-1</sup>)</th><th>K<sup>§§</sup><br>(g kg<sup>-1</sup>)</th><th>Mg<sup>§§</sup><br>(g kg<sup>-1</sup>)</th><th>Ca<sup>§§</sup><br>(g kg<sup>-1</sup>)</th></tr></thead><tbody><tr><td>0-15</td><td>21 ± 0.5</td><td>69 ± 0.8</td><td>10 ± 0.7</td><td>7.93 ± 0.03</td><td>0.8 ± 0.11</td><td>17.67 ± 0.2</td><td>109.17 ± 2.18</td><td>1.13 ± 0.01</td><td>0.07 ± 0</td><td>0.12 ± 0.01</td><td>0.07 ± 0</td><td>3.95 ± 0.36</td></tr><tr><td>15-30</td><td>21 ± 0.5</td><td>69 ± 0.8</td><td>10 ± 1.1</td><td>7.92 ± 0.03</td><td>0.83 ± 0.08</td><td>17.6 ± 0.26</td><td>109.83 ± 2.65</td><td>1.12 ± 0.01</td><td>0.07 ± 0</td><td>0.13 ± 0.01</td><td>0.07 ± 0</td><td>4.15 ± 0.38</td></tr></tbody></table>

**Table 3: Soil properties**

†1:5 soil: water ratio NF ISO 10 390; ‡ Total carbonates NF ISO 10693; § dry combustion on decarbonated soil NF ISO 14235; # CEC Metson NFX 31-130; †† Dumas method; ‡‡Available P, Joret Hebert NFX 31-161; §§Exchangeable cations NFX 31-108. NF and NF ISO determined following protocols published by the French Association for Normalization (AFNOR, 1996).
<table><caption>**Table 4**</caption><thead><tr><td></td><td>Date</td><td>Days since amendment</td></tr></thead><tbody><tr><td>Soil amendment</td><td>23/04/2019</td><td>0</td></tr><tr><td>Spring sampling 2019</td><td>20/05/2019</td><td>27</td></tr><tr><td>Autumn sampling 2019</td><td>19/09/2019</td><td>149</td></tr><tr><td>Autumn sampling 2020</td><td>22/09/2020</td><td>518</td></tr><tr><td>Spring sampling 2021</td><td>19/04/2021</td><td>727</td></tr><tr><td>Autumn sampling 2021</td><td>22/09/2021</td><td>883</td></tr></tbody></table>

**Table 4:** Earthworm sampling dates
<table><caption>**Table 5**</caption><thead><tr><th>Variable</th><th>Source of variability</th><th>p value</th></tr></thead><tbody><tr><td rowspan="5">Total</td><td>Year</td><td><strong>&lt;0.01</strong></td></tr><tr><td>Treatment</td><td>0.06</td></tr><tr><td>Season</td><td><strong>&lt;0.01</strong></td></tr><tr><td>Year*Treatment</td><td>0.09</td></tr><tr><td>Treatment*Season</td><td>0.58</td></tr><tr><td rowspan="5">Richness</td><td>Year</td><td><strong>&lt;0.01</strong></td></tr><tr><td>Treatment</td><td>0.57</td></tr><tr><td>Season</td><td><strong>&lt;0.01</strong></td></tr><tr><td>Year*Treatment</td><td><strong>&lt;0.05</strong></td></tr><tr><td>Treatment*Season</td><td>0.35</td></tr><tr><td rowspan="5">Shannon</td><td>Year</td><td><strong>&lt;0.01</strong></td></tr><tr><td>Treatment</td><td>0.99</td></tr><tr><td>Season</td><td><strong>&lt;0.01</strong></td></tr><tr><td>Year*Treatment</td><td><strong>&lt;0.01</strong></td></tr><tr><td>Treatment*Season</td><td><strong>&lt;0.01</strong></td></tr></tbody></table>

**Table 5:** Result of GLMs for the effect of year, season, treatment and their interaction on total earthworm density, species richness and diversity.

GLM quasipoisson with log link. Significant (α<5%) p value in bold. Total : Total earthworm density; Richness : Species richness; Shannon : Shannon diversity index.
<table><caption>**Table 6**</caption><thead><tr><th>Variable</th><th>Source of variability</th><th>p value</th></tr></thead><tbody><tr><td rowspan="6">Anecic</td><td>Year</td><td><strong>&lt;0.01</strong></td></tr><tr><td>Treatment</td><td><strong>0.01</strong></td></tr><tr><td>Season</td><td>0.40</td></tr><tr><td>Year*Treatment</td><td>0.77</td></tr><tr><td>Treatment*Season</td><td>0.73</td></tr><tr><td>Year</td><td><strong>&lt;0.01</strong></td></tr><tr><td rowspan="4">Endogeic</td><td>Treatment</td><td>0.24</td></tr><tr><td>Season</td><td><strong>&lt;0.01</strong></td></tr><tr><td>Year*Treatment</td><td><strong>0.03</strong></td></tr><tr><td>Treatment*Season</td><td>0.91</td></tr></tbody></table>

**Table 6:** Result of GLMs for the effect of year, season, treatment and their interaction on total earthworm density, species richness and diversity.

GLM quasi-poisson with log link. Significant (α<5%) p value in bold. Anecic: Anecic earthworm density; Endogeic: Endogeic earthworm density.