Work package number

6

Lead Beneficiary

UCSC

Work package title

Degradability, mobility and fate of MNP and other stressors in the soil environment

Participant number

1

2

3

4

5

6

7

Short name of participant

WU

UBERN

FiBL-CH

UTH

UL

UCSC

FiBL-AT

Person/months per participant

15

19

0

11

0

34

0

Participant number

8

9

10

11

12

13

14

Short name of participant

UoG

FUB

INRAE

POLITO

CHQ

DTU

AGES

Person/months per participant

0

4

10

12

15

1

0

Participant number

15

16

17

18

19

20

Short name of participant

NVM

WR

EQY

CAMPO

SPOT

EMU

Person/months per participant

2

8

0

0

0

0

Start month

6

 End month

60

Objectives of the work package:

The main aim of WP6 will be to assess the environmental fate of MNP, bioplastics and other stressors in terrestrial environments (SO5), with a focus on degradation mechanisms, microbial processes, environmental transport and effects on non-terrestrial targets (SO6). Research activities will be conducted to: (a) assess at fine scale the degradation level of MNP of different chemistry and origins (synthetic and biological) using both newly synthesized ¹³C labelled plastics and aged plastics from CSS (T6.1); (b) characterize the microbiological composition of the plastisphere, focusing on the isolation and characterization of plastic-degrading microorganisms, the evolution of novel catabolic traits for pollutants sorbed or interacting with the plastics (i.e., pesticides and antibiotics), and the presence and dispersal of human and plant pathogens and/or antibiotic resistance genes (T6.2); (c) assess the soil and aerial transport of MNP, for the latter focusing on pollinating insects as non-terrestrial targets of ecotoxicological relevance (T6.3).

Task 6.1 Assessment of the degradation and disintegration of MNP and other stressors in soils

Task leader: UBERN-GIUB | Participant(s): POLITO, WR, NVM, UBERN-CDEUB | Timing: M9-50

We aim to assess the degradation and disaggregation of MNP in soils based on WP3 findings in the CSS. We will select the most common polymer types found at the CSS (biodegradable materials according to the main standard and non-biodegradable materials) and investigate their decomposition in microcosms, pot and field experiments as described in T4.1. In addition, we will undertake dedicated lab experiments to assess 1) plastic disaggregation, 2) MNP degradation and 3) the state of plastic degradation in CSS as follows:

(1) To evaluate plastics disaggregation soil lab experiments (as described in T4.1) will be employed where soils will be fortified with known size classes of MeP and MP for different time periods; microplastics will be extracted from soils and their size compositions before and after the experiment will be compared.

(2) To assess MNP degradation processes a general characterization in terms of molecular, thermal and chemical properties will be carried out to define the nature of plastic material and the proofs of degradation in lab experiments. A dedicated microcosm experiment, based on ¹³C-labelled polymers produced in house, will be implemented, to monitor plastic degradation and disaggregation. Partners responsible: GIUB (lab experiment, isotope analysis, NanoSIMS)

(3) The extent of plastics degradation in CSS will be assessed by retrieving plastic samples from CSS and conduct analysis of plastic molecular structure, crystallinity and morphology and size. Partners responsible: UBERN-GIUBN (MP extraction from soil, µFTIR, SEM), POLITO (FTIR, SEM), WR (GPC, DSC, FTIR, SEM).

Task 6.2 Microbial interactions in the plastisphere

Task leader: UCSC | Participant(s): UTH, INRAE, FUB, CHQ | Timing: M6-60

Working with non-labelled aged and spiked plastics, UCSC in collaboration with UTH, INRAE and FUB will determine the composition and diversity of prokaryotes and fungi on the plastisphere using amplicon sequencing analysis. We will focus on microorganisms known to act as plant or human pathogens, with MP facilitating their transportation in soil. Based on the outcome of the microbiome measurements, UCSC and UTH will employ shotgun metagenomics to determine the metabolic and genetic potential of the plastisphere microbiota. In addition, the role of plasmidome in the evolution of novel catabolic traits against organic pollutants or resistance to antibiotics will be determined via plasmidome-targeted metagenomic analysis (UTH). This will be carried out in targeted small-scale soil lab microcosms inoculated with aged MP samples (that have an established community, composition determined at T0). These MP will be placed in soil repeatedly treated with the same pesticides/antibiotics as in the original soil from where they originate. At regular intervals MP will be extracted from soil, and their metagenome and plasmidomes will be determined enabling the identification of metagenomic assembled genomes (MAGs) with interesting traits like plant/human pathogenicity, antibiotic resistance and associated transposable elements etc. Parallel culturomic approaches will lead to the isolation of microorganisms with plastic-degrading abilities following two-step enrichment protocols using ¹³C labeled polymers (UCSC): INRAE, UCSC and UTH will isolate by density-gradient centrifugation the ¹³C-DNA of the plastisphere, and analyse it by amplicon sequencing to define the diversity of the most relevant plastic degraders in the plastisphere. Bacterial (UCSC and CHQ) and fungal (FUB) strains will be retrieved and screened; those showing high degradation abilities will be subjected to genome sequencing (UCSC, FUB), and analysed for genes coding for pesticide/antibiotic catabolic enzymes, by UTH and INRAE, who will perform tests for the degradation of these xenobiotics.

Task 6.3 MNP and other stressors transportation and their effects on non-terrestrial biota

Task leader: WU | Participant(s): WU, UBERN-GIUB, FUB, UCSC, DTU UBERN-CDEUB | Timing:M6-M60

In this task we will measure biotic and abiotic MNP transportation in soil at lab and field scale (subtask 6.3.1), and we will evaluate aerial transportation of MNP through bees and the ecotoxicological effects of plastics on these non-terrestrial bioindicators (subtask 6.3.2.). In this frame targeted mesocosm and field experiments will be undertaken as described below.

Subtask 6.3.1. Biotic and Abiotic transportation of MNP and other stressors in soil

Mesocosm experiments: Vertical and horizontal, biotic and abiotic MNP transportation will be determined in mesocosms by WU. Regarding biotic transportation, we will assess the movement of different MNP types, with and without organic stressors, by endogeic and anecic earthworms, and ants in 3 different soil types (texture-based). In collaboration with T6.2 MNP degradation will be assessed inside earthworms’ guts and burrows. MNP transportation by ants will be determined in 300x405x300 mm double-connected formicarium for observing how worker ants move MNP from one site to another; response variables will be distance of MNP transportation and amount of MNP transported. Regarding vertical abiotic transportation, we will determine the leaching of at least 3 types of MNP, with and without organic stressors, through earthworms biostructures in 60x12cm metal columns in controlled environmental conditions in 3 soil types (same as for biotic transportation), simulating rainfall at different intensities. Response variable will be the leaching of MNP (particles per ml). Data will be used by T5.2 for modelling purposes. WU, POLITO and GIUB will support WPs 4 and 5 in the analysis of MNP in dedicated pot experiments described in T4.1

Field experiments: we will measure MNP transportation in tillage and no tillage CSS with 3 different kind of plastics and different soil types, with and without organic stressors (pesticides, antibiotics). Eroded soil sediments will be collected by WU with a portable wind tunnel. The depth of MP transport in the CSS will be analyzed by UBERN-GIUB complementary to the lab experiments in this task and T5.2. The same sites used for the lab experiments in NL and CH, as well as selected CSS (WP3, selection depending on plastic use and soil properties) will be analyzed for general transport and along preferential flow path. The results will be combined with the results from T5.2 about preferential flow and outcomes of lab experiments to assess possible biotic and abiotic depth transport. Data from farmer’s questionnaires (WP3) will be evaluated for the time of plastic use at the individual sites and compare it to the depth of plastic transport to assess temporal dynamics of the transport. At sites with shallow ground water table, groundwater will be sampled and analysed for MP concentrations. The data from soil depth transport and groundwater will be combined to assess a possible threat of MP leaching from agricultural soil to groundwater. In addition, WU, POLITO and UBERN-GIUB will support WPs 4 and 5 in the analysis of MNP in the dedicated field studies described in T4.1.

Subtask 6.3.2. Aerial transport and effects of MNP and other stressors on non-terrestrial biota

Mesocosm/Microcosm experiments: The potential for aerial transportation of MNP through pollinators and the effects of MNP on honeybees (Apis mellifera) will be determined by UCSC. The most common polymer types found at the CSS will be tested at 3 levels with and without organic stressors. Brood frames from experimental apiary at UCSC will be selected and placed in incubators. Newly eclosed adults will be randomized into experimental and control groups. Bees will be exposed via controlled oral administration of MNP. Acute and chronic oral exposure of MNP at sublethal levels will be tested. MNP ingestion and NP translocation in tissues and organs, i.e. gut, haemolymph, and in faeces, will be analysed. Effects on the ultrastructure of the gut epithelium of honeybees will be also investigated. Effects on the bee gut microbiome following MNP ingestion will be analysed at UCSC by amplicon sequencing targeting bacteria, archaea and fungi (T6.2). Moreover, since locomotion is essential for flight and foraging ability, and its impairment may affect pollination, behavioural testing on bees treated with MNP will be performed.

Field experiments: Bees exposed to environmental MNP levels will be used as bio-monitors (Bee-O-monitors) by UCSC. MNP aerial transport will be assessed on the bee body (i.e. on the fore wings) and through translocation in tissues and organs. Chemicals contained in MNP will be characterized for their general toxicity on terrestrial, aquatic and pollinating insect species in support of classification and prioritization of MNP types with respect to their ecotoxicological relevance

Deliverable 6.1: Degradability report of MNP in soils (M48, UBERN-GIUB, R, PU)

Deliverable 6.2: List of microorganisms composing the plastisphere microbiome per soil and MP type including culture collection and genomes of selected isolates [M52, UCSC, FUB, OTHER, PU]

Deliverable 6.3: Assembled metagenome and plasmidome of the plastisphere [M52, UTH, OTHER, PU]

Deliverable 6.4: Biotic and abiotic MNP transportation characterization and monitoring of sublethal effects on bees - Report [M56, WU, R, PU]