Work package number


Lead Beneficiary


Work package title

Assessment of the impact of MNP and combined effects with other stressors on soil biodiversity

Participant number








Short name of participant








Person/months per participant








Start month


 End month


Objectives of the work package:

The main objective of WP4, fully aligned with SO4 and SO5, will be to assess the effects of MNP (alone and in combination with other soil stressors) on the function and diversity of the soil biota. Specific experiments will be performed (T4.1) so that analysis can be run at different scales and supported by WP5 and WP6 (SO2).

WP4 specific objectives are:

(a) to determine the effects of MNP of different, size chemistry and origin on the function (T4.2) and diversity (T4.3) of the soil microbial community, including endophytes and rhizosphere microorganisms

(b) to determine the effects of MNP of different chemistry, size and origin on the diversity and functions of soil micro- and macrofauna (T4.4)

(c) to define effects on food web interactions and soil network configuration with links to function (T4.5) and

(d) to evaluate the interactive effects of MP linked with their additives and other pollutants, often associated with MNP in agricultural soils (i.e. pesticides, veterinary drugs, additives), on the soil microbiota and soil fauna (all tasks). 

Results will be integrated, with data from WP5 and WP6, into an overall sustainability analysis (WP7) and stored as described in WP2.

Task 4.1 Implementation of multi-scale experimental plan  

Task leader: UTH| Participant(s): WU, FIBL-CH, UCSC, FUB, INRAE

To determine the effects of MNP on soil biota a set of experiments will be performed at three scales. Soil biota measurements, especially at field level (and for specific pot experiments), will be supported by soil physicochemical and plant measurements (WP5) and MNP levels determination (WP6):

1) Soil lab experiments (no plants). 3-4 soils (ca. 100 g from soils sampled at CSS in WP3 or characterized control soils in WP5) with variable physicochemical characteristics will be used. For each soil, three different MNP types selected based on WP3 data (to cover >90% of the type of plastics identified in WP3 inventory and CSS), applied at rates (plus a no plastics control) either alone or in combination with three pesticides/veterinary drugs will be tested. FUB, UTH, INRAE, and WU will undertake these experiments;

2) Pot experiments (co-analysed WP4, 5 and 6). 3-4 soils (as in lab experiments) with plants (common with WP5) will be tested. 3 different MNP types (as in lab experiments) applied at 3 rates (plus a no plastics control), either individually or in combination with three pesticide/veterinary drugs will be tested. These experiments will be performed by FUB, UCSC and UTH questioning effects on soil microbiota and by WU, USCS addressing effects on soil fauna.

3) Field experiments (co-analysed WP4, 5 and 6). 3 experimental platforms will be involved: Wageningen (NL, Atlantic region, managed by WU), Dijon (FR, Continental region, managed by INRAE) and Crete (EL, Mediterranean region, managed by CHQ). Effects of 2 MNP types will be tested at two rates (plus an untreated control) in combination or not with one pesticides/veterinary drug in a completely randomized plot design with 3 reps per treatment (summing to 24 + 3 untreated plots). Each field experimental site will be supported by at least two groups for the measurements of soil fauna (WU, UCSC) and soil microbiota (UTH, UCSC - CHQ site / FUB, UTH - WU site / INRAE, FUB - INRAE site).

Task 4.2 Assessment of the impact of MNP and other stressors on functional attributes of soil microorganisms

Task leader: FUB | Participant(s): INRAE,UTH,UCSC,FiBL-CH| 

We will assess effects on key functional microbial attributes including:
(a) N-cycling microbial groups: like (i) all AOM, including ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and complete ammonia-oxidation bacteria (comammox), via q-PCR (amoA genes), measurement of NH4, NO3 and potential nitrification (UTH); (ii) denitrifying microorganisms via q-PCR of microbial genes involved in the four steps of the denitrification process (i.e. nirS/nirK, nosZ, narG) (INRAE);

(b) AMF, via measurement of root colonization, P plant uptake (FUB, UTH);

(c) Carbon compounds decomposers via q-PCR of pcaH and catA genes (INRAE);

(d) Plant growth promoting microbes (PGPM) via q-PCR of microbial genes involved in N fixation (nifH), auxin biosynthesis (ipdC, ppdC), P solubilization (gcd, gad, phnX) (UCSC);

(e) microbial enzymatic activities involved in C-, N-, and P- cycling by fluorescence plate assays (UCSC);

(f) metabolic fingerprinting of microbial communities via MicroResp® (FiBL-CH).;

The functional endpoints to be measured at each experimental scale as defined in T4.1 are:

1) Soil lab experiments: AOM and denitrifiers abundance, NH4, NO3, potential nitrification, C compound-decomposers abundance, activity of soil microbial enzymes and MicroResp®;

2) Pot experiments: In addition to the measurements described in the lab experiments, we will measure AMF P uptake and colonization, PGPM abundance and plant growth;

3) Field experiments: AMF P plant uptake and colonization level; AOM, C compound-decomposer and denitrifiers

Task 4.3 Assessment of the impact of MNP and other stressors on the diversity of soil microorganisms

Task leader: INRAE | Participant(s): FUB, UTH, UCSC, WU|

The methodology to determine the quantity and the quality (e.g. polyethylene, polypropylene, polystyrene, polyurethanes, synthetic oxo- and photo-degradable plastics, and biodegradable plastics) of plastic residues in soil will be standardized and included in the monitoring plan (T2.4).

We will determine effects on soil microbial diversity via amplicon sequencing targeting:

(a) prokaryotic (bacterial and archaeal), fungal and protists diversity (INRAE, FUB, UTH, UCSC, WU)

(b) AMF intraradical diversity (FUB, UTH)

(c) AOM diversity (i.e. AOA, AOB, commamox bacteria) (UTH).

Data will be analysed by FUB, UTH, INRAE, UCSC and WU with appropriate bioinformatic tools using relevant databases (Silva, UNITE, Abell et al., Alves et al.) and biometric tools (R software). Responsive groups of microorganisms and their relevance for the soil ecosystem homeostasis will be identified. Functional endpoints to be
measured are:

1) Soil lab experiments: prokaryotic and fungal diversity

2) and 3) Pot and field experiments: prokaryotic, protist, fungal and AOM soil diversity, and AMF intraradical diversity.

Task 4.4 Assessment of the impact of MNP and other stressors on the soil fauna

Task leader: UCSC | Participant(s): WU,UCSC|

We will determine in pot and field experiments the MNP effects on micro-, meso and macro-fauna targeting:
(a) soil ecosystem engineers (earthworms): Effects on the mortality and reproduction rates of endogeic and anecic earthworms. Ingestion rate, cast production, avoiding behaviour, number and size of burrows, and transport of MNP into deeper soil layers through earthworm burrows (WU);

(b) litter fragmenters (earthworms): Effects on biomass, reproduction, density, and mortality of epigeic earthworms (WU);

(c) bioindicators of pollution: Mortality acute tests of coleopteran (larvae) (WU);

(d) Interphase soil-litter organisms: Effects on the community of micro-arthropods and edaphic, hemiedaphic and epiedaphic collembola (mesofauna) (UCSC);

(e) nematodes: effects on nematodes diversity determined by amplicon sequencing (WU).

Task 4.5: Effects of MNP and other stressors on soil food web interactions and network configuration

Task leader: WU | Participant(s): WU|

In this task, biodiversity data obtained in all tasks in WP4 will be merged to test whether MNP:

(a) have increasing effect sizes on larger soil animals than on smaller microorganisms. We will comparatively assess differences in community metrics such as diversity and community composition between different groups of organisms. We will use different statistical approaches, to investigate potential differences of treatments on soil biodiversity groups;

(b) change the configuration of soil biodiversity networks. We will combine all data obtained to construct co-occurrence networks across trophic levels and calculate different metrics on the network scale;

(c) affect interactions between different (trophic) groups of soil organisms.

We will use structural equation modelling to examine the magnitude of causal effects of MNP on trophic food-web interactions. Targeted multi-trophic in vitro tests (WU), including bacteria, fungi, protists, nematodes and larger soil animals in the presence of MNP will validate data observed in (a) and (b) and identify routes
of MNP from one trophic level to the other.

Deliverable 4.1.: Protocol for multi-scale experiments [M11, UTH, R, PU]

Deliverable 4.2: Guidelines on the assessment of the toxicity of MNP and other stressors on soil microbial functioning [M46, FUB, R, PU]

Deliverable 4.3: Guidelines on the assessment of the toxicity of MNP and other stressors on soil microbial diversity [M49, INRAE, R, PU]

Deliverable 4.4: List of soil biota indicators affected by MNP and other stressors [M52, WU, R, PU]

Deliverable 4.5: Guidelines on the assessment of the toxicity of MNP and other stressors on soil fauna [M56, UCSC, R, PU]

Deliverable 4.6: Guidelines for assessing effects of MNP and other stressors on the entire soil food web [M60, WU, R, PU]