3.1.1 Soil microorganisms and earthworms.

Soil organisms like bacteria, fungi and soil fauna are responsible for nutrient transformation, carbon cycling and degradation of organic pollutants. Depending on the amount and type of PPP, soil organisms and their activities can be positively or negatively affected. In this study, we will measure carbon and nitrogen mineralization, the activities of soil enzymes involved in C-, N- and P-cycling, quantify functional genes involved in PPP degradation, and sequencing soil microbiome (which will give information about the response of bacteria, fungi and archaea to PPP at high taxonomical resolution). PPP are also known to adversely affect and contribute to earthworm populations decrease [13]; the severity and subsistence of PPP effects depends strongly on the type of PPP and earthworm species considered [14]. Given earthworms ecosystem engineers role, in this study we will collect earthworms to assess diversity, PPP content, and gut microbiome.

3.1.2 Fish.

PPP may accumulate in aquatic organisms at concentrations much higher than the ones found in the water column [15]. In addition, several substances, including PPP, can sink into sediments and persist for long periods, which can constitute a secondary source of contaminants over time [16]. Hence, fish are considered an effective indicator to assess and monitor the PPP impacts over time and space in aquatic ecosystems [17, 18] as well as to infer the potential risks for human health due to consumption of contaminated fish. In this study, fishes of the most common species per CSS will be caught and used to assess PPP content and microbiome.

3.1.3 Benthic macro-invertebrates.

In this study, the macro-invertebrate communities are used as a proxy for aquatic biodiversity impairment. Benthic macro-invertebrates are added to this study to cover the link between exposure and ecological effects at the community level in the aquatic compartment, hence extending the primarily proposed monitoring of exposure, bioaccumulation and effects at the individual level of fish species. Since benthic macro-invertebrates are very sensitive to pollution and linked to calibrated metrics that can accurately reflect the ecological status of the waterbody, they are good indicators of disturbed aquatic ecosystems [19].

3.1.4 Bats.

PPP are known to affect bats; next to the potential direct skin contact in their roosts, bats in Europe are indirectly affected via insects, their main food source [20]. Better understanding the relations between the distribution of PPP in ecosystems, food availability and bats health is eminent for their protection. As all bat species are endangered species, sampling in CSS will only consist of the collection of faeces (used also to assess PPP content and microbiome).

3.1.5 Insects.

Non-target and beneficial insects are known to be negatively affected by PPP, with for example the effects of neonicotinoid insecticides receiving much attention in the last decade [21]. As insects are susceptible to PPP, insects are considered indicator species to quantify differences in the use of PPP between fields [22, 23]. In this study we will collect ground-dwelling and flying insects, in three 21-day cycles (for flying insects the cycles may be slightly longer depending on weather conditions) to assess biodiversity.

3.1.6 Cat.

In the study the cat is preferably a domestic ‘farm’ cat that lives outdoors and to some extent reflects the life of a (wild) predator species. It is supposed that the blood samples from the cat will reflect bio magnification of PPP from prey rodents and birds who in turn feed on left-overs in and around barns and on fields near the farm. Cat results will inform on exposures expected to occur in wild predator species higher up in the food chain.

3.1.7 Crops.

By focusing on most crop types in the EU, this study will contribute to PPP productivity and food safety discussions [12, 24]. Different parts of the CSS plants will be tested for PPP–these parts are crop type specific, being selected based on what is used or processed for the final product, or used as food or feed. PPP data will be complemented with crop yield, pest incidence, weed infestation, and plant diseases data.

3.3.1 Farmer families.

As mentioned above, two contrasting types of farm(er)s are studied: conventional and organic. The farmer is the central person involved in PPP use decisions; he/she may apply PPP and be therefore prone to occupational exposure. As farmers are often living on or near the farmland, the farmer and farmer family are a relevant target population [29] with a higher exposure likelihood than non-farmer families living in a rural environment [30]. The consumption of agricultural products by farmer families is also considered a potential source of PPP residues’ exposure. These products may be locally produced, imported from EU member states or to some extent also related to products imported from outside the EU.

3.3.2 Neighbour families.

Neighbours are those with a main residence adjacent to or at short distance from fields, with no professional involvement in farming. Members of these neighbour families may be exposed to PPP directly by spray drift when they are outside during spray applications. PPP may also disperse as vapours, aerosols or absorbed on the surface of airborne particulate matter that penetrates into the residence [31, 32]. Environmental exposures are then defined as ‘rural background’ and depend on the type of crop and type of farm systems in the region. Tracking back on footwear is another mechanism by which PPP residues may be translocated into the home environment [33, 34]. Again, consumption of agricultural products can be a potential source of PPP residues’ exposure, as some families may buy local products from neighbouring farms.

3.3.3 Consumers.

Consumers are citizens living at greater distances from the fields than neighbours, in surrounding villages or towns, and have no professional involvement in farming either. Consumers are considered the reference for exposed persons in terms of environmental exposure. The average consumer is expected to receive most of the PPP exposure through the diet. The mixture of PPP residues they are exposed to is dependent on the composition of their ‘market basket’ that may consist of local products but most likely also products bought from a grocery or supermarket, which may include EU and non-EU imported products. Consumers’ attribution to conventional and organic classes is performed based on the degree of consumption preferences for conventional or organic food.

4.1.1 Soil.

Soil acts not only as a sink of PPP residues, including persistent residues, but also as a source of PPP residues, when conditions change and via wind and water erosion [35]. 83% of 317 EU agricultural soil samples tested by Silva and co-workers [36] presented PPP residues, 58% presented mixtures.

4.1.2 Water.

PPP reach water bodies mainly by surface runoff, leaching and spray drift. Once in water bodies, PPP may impair (drinking) water quality and aquatic life survival and health. For instance, since several of the currently approved PPP active substances have endocrine disruption properties, the reproduction of fish and other aquatic animals is likely affected when exposed to these substances. Furthermore, PPP residues can be transferred from lower to higher trophic levels, with residues being biomagnified through the trophic chain [37].

4.1.3 Sediment.

PPP often end up in watercourses, since they are usually intermediate or final recipients of organic contamination [38]. Among the processes inherent in this system, storage mechanisms play a major role in the dissipation of PPP residues, particularly in bottom sediments. PPP sorbed onto suspended particles in the water column are modulated by different parameters such as the texture of the sediment particles and their physico-chemical parameters such as SOM [39].

4.1.4 Gas phase and airborne particulate matter.

During application, part of the applied PPP is dispersed into the atmosphere by volatilization or spray drift [40]. The fate of PPP is influenced by their partitioning between the gas phase and the particulate phase while the rate and the extent of the emission after application depends on the physical and chemical properties of the pesticide; the application parameters; the meteorological conditions during and after application; and the characteristics of the target [41]. In addition, fine airborne particulate matter derived by wind erosion from topsoil (usually referred to as ‘dust’) emitted by wind erosion or by tillage activities may act as a transporter for PPP, dispersing it over considerable distance. Once settled, these particulates may act as secondary sources of PPP.

4.3.1 Urine.

Most PPP active ingredients are readily bio-transformed and appear in urine as conjugated or free metabolites. In addition to these residues, urine will be analysed for a limited number of biochemical effect biomarkers. In this study we will ask the participants to collect a morning void. For density adjustment urinary creatinine will be determined.

4.3.2 Faeces.

Faeces collection is a non-invasive method for sampling the microbiome of the gastrointestinal tract. The gut microbiome plays an important role for host health, and can modify environmental chemicals in the intestinal tract, thereby influencing their biological activity, absorbance and distribution in the body. The composition of the microbiome can also be modified by exposure to environmental chemicals, which can have potentially adverse consequences for host health. In addition, PPP and their metabolites, either unabsorbed or excreted in the bile, can be eliminated via faeces. Part of the faeces samples will be analysed for the presence of PPP and their metabolites, and part is collected in tubes containing storage buffer that stabilizes the genetic material of the microbes in the sample, allowing extraction and sequencing of microbial DNA at a later stage.

4.3.3 Blood.

The levels of PPP in blood are a primary study outcome and will serve as input to physiologically-based pharmacokinetic (PBPK) modelling of target dose for organs and organ systems where adverse effects may occur. Further, blood is the sample medium of preference for many additional biochemical effect biomarkers (see section 6.3). Collection of a blood sample is done only once and conducted by venepuncture by trained and authorized healthcare professionals according to national and international standards [42]. We will use a vacutainer system with three types of tubes (for plasma separation, serum separation and whole blood) to support the analyses of interest. The blood will be fractionated on the same day, and the resulting material will be frozen until analysis.

4.3.4 Wristbands.

The wristband is a low-tech solution for personal passive sampling of chemicals stemming environmental exposure [43]. The silicon material has the capability to absorb pesticides from the environment reflecting direct contact with environmental chemicals, either solid, liquid or gas phase and has been used before to study exposures to pesticides [44]. The wristbands will be worn by farmers, neighbours and consumers continuously for seven days prior to the collection of urine and blood.

4.3.5 Nasal swab.

Nasal swabs will be used to sample the microbiome of the upper respiratory tract. Like in the gastrointestinal tract, the nasal microbes are important for human health. Microbial DNA will be extracted from the swabs, allowing the identification of potential shifts in composition and genomic potential of the respiratory tract microbiota.

4.3.6 Food and beverages (meals).

One participant from each subgroup (farmers, non-farmers and consumers) will be asked to collect complete duplicate portions of food and beverages consumed during one day, and to collect all urine (a 24-h sample) on the same day. The duplicate food and beverage portions will be processed and analysed for pre-selected PPP residues and these will be compared with the ones recovered from urine. This information will be used to verify to what extent the diaries and questionnaire data provide an accurate reflection of the dietary intake of PPP.

4.4.1 Milk.

After uptake, PPP are metabolized, excreted via the urine or distributed to internal organs. PPPs are known residues in milk [45]. Usually, the presence of PPP in feed is the main source of their occurrence in the dairy products. Milk is an important source for human exposure since it is a valuable nutritional product in early life and also feedstock for a range of dairy products.

4.4.2 Feed.

Feed originates from several crops such as grass, maize, cereals, rape seed and soya beans. It was shown that fodder maize and sorghum retains residues even during harvesting although these were sprayed during early stages of growth. Nag and Raikwar [46] reported feed as an important source of pesticide residue intake by animals.

6.2.1 Human exposure modelling.

For human exposure modelling, the OBO exposure module will be used. This module consists of an a priori defined set of equations [29] and includes both human specific input and some more general and specific inputs from CSS. A brief summary of the model input is presented below:

• Human specific input: age, sex, bodyweight, body height, time spent indoors, farmer/neighbour/consumer role.
• General and CSS specific input: season of sampling, output from the environmental modelling to be used in the human exposure assessment, estimates of PPP concentration in different food items, daily lifestyle.

6.2.2 Ecosystems, plants and animal exposure modelling.

To estimate animal exposure, we will use the MERLIN-Expo tool [54], that involves an extensive list of inputs; some fixed, some following a probability density function [55], supplied by literature/existing databases and CSS data. For other non-target organisms we will use the MERLIN-Expo tool and the spatio-temporal exposure description by Roeben, Oberdoerster [56]. This approach covers environmental parameters, data on the behaviour of the species, information on toxicokinetics on organisms. There is still not yet a well-established method for the calculation of ecosystems exposure. The use of risk quotients appears a valid approach as it considers estimated environmental concentrations from fate modelling and (no-observed) effect concentrations of the most sensitive species in each ecosystem [57].

6.3.1 Exposure biomarkers.

As biomarkers of exposure, we will use the PPP components, i.e. active substances, degradation products and metabolites. If possible and if relevant we will also target known co-formulants. The a priori selection of these analytes is described in section 8. The results of these exposure biomarker analyses can be compared with the results of PPP residue analysis of environmental samples collected on an individual basis (e.g. diffusive sampling devices like wristbands) and samples collected in the micro-environment (e.g. indoor sample collection in homes of farmers, neighbours and consumers). This can lead to inferences regarding the potential source and route of exposures [30].

6.3.2 Biochemical effect biomarkers.

Biomarkers are applied in population-based studies to find support for the involvement of certain molecular or cellular mechanisms to explain adverse effects. However, none of these biomarkers can be used to reflect disease prevalence and incidence directly and, in this study, we will not be collecting data on the occurrence of disease. We include biomarkers of possible value to study associations between exposure and health risk in future large-scale epidemiological studies (S1 Table in S1 File). The evidence to support associations between exposure and predictors of effect will be explored by systematic searches of previous studies.

8.1.1 Study protocol for ethics approval.

A study protocol version was developed specifically for the ethics approval process. This protocol was dedicated to the recruitment of human participants and included details such as inclusion and exclusion criteria for eligibility, the contribution expected from study participants, and information about the potential benefits and burden for each individual participants of joining the study. Both males and females 18 years or older on the day of recruitment can enrol. We will strive for a gender balance within all populations. Individuals who are not able to speak and/or read the local/regional/national language will be excluded from participation. The protocol was submitted to the regional or national ethics committees linked to the CSS, and approved thereafter. Participant recruitment started shortly after the ethics approval.

8.1.2 Personally identifiable information.

All personal data will be pseudonymised before any treatment by replacing participant names with a code and protecting all electronic and paper records from unauthorised access (only designated researchers have access to these data). All results will be reported in a strictly non-identifiable format to protect the privacy of every subject. Personally identifiable information will be deleted after completion of the study. The biological specimen and data will be destroyed at the end of the project. The handling of personal data complies with the General Data Protection Regulation (GDPR) and the national legislation on data protection requirements in the country of the CSS.

8.1.3 Participant information and informed consent.

Every person invited to participate in the SPRINT study will be informed about the study, before they decide whether to participate or not. Every person who is invited to participate in the SPRINT study gives informed consent before enrolment in the study and can withdraw at any time. For all study outcomes the study leaders will adhere to the right to know and the right not to know as an overarching principle.