Our maps for agriculture and pasture subscenarios (Figure 1) reveal a heterogeneous “patchwork” reflecting the diverse influences on agricultural and livestock activities in Spain, which include bioclimatic, socioeconomic and policy factors [46, 76, 77].
Geographical areas under high manure load in our study correspond to high productive regions of arable land, pasture and meadows . In the agriculture scenario, areas under high manure load were located predominantly in the Iberian central high plateau and the Ebro River Basin, which feature mainly arable lands with herbaceous crops, especially cereals (Figures 1a, 1c and 1e). We also observed areas under high manure load in southern Spain and along the Mediterranean coast that were associated with rearing of chickens (Figure 1e). The wider distribution of chickens than of cattle or pigs may reflect the stronger economic constraint on chicken farms to be closer to markets, rather than the influence of climatic or other environmental factors .
In the cattle pasture scenario, geographical areas under high manure load were present in the humid northern provinces, especially along the north and central mountain ranges, which are characterised by rich pastures and meadows. Such areas lay also in the west and southwest of the Peninsula, where pasture lands known as “dehesas” are abundant . Dehesas, an agroforestry system that harmoniously combines pastures and trees, are one of the most ecologically valuable and economically important rural landscapes on the Iberian Peninsula . The distribution of dehesas coincides exactly with that of pig-rearing pastures in our study (Figure 1b), even though cattle also graze on dehesas .
The agriculture scenario showed extensive surface devoted to arable land, mainly for herbaceous crops such as barley and wheat, and to a lesser extent for woody crops, mainly olive, vineyards and fruit trees. In contrast, more than two thirds of the land surface in the pasture scenario was woody vegetation (forest and shrublands) and natural grass. Natural grasslands, which include meadows, rough grassland and rough grazing rangeland, are dominated by herbaceous species and are the main food source for extensively managed livestock in Spain . Shrublands and open forests also play an essential role in livestock farming when green grass is in short supply .
Plant toxicity assays are designed to be rapid, simple and reproducible, which helps explain why the OECD 208 method includes only herbaceous, not woody, species as sentinels for assessing risk of environmental effects due to antibiotics given to livestock. Indeed, herbaceous crop plants are the focus of efforts to fertilise arable lands with manure. In England and Wales, manure is applied to arable crops of cereals (wheat, barley and maize), sugar beet and potatoes, as well as to land for grazing and silage [59, 60]. In France, 36.5% of total manure used for fertiliser is spread onto grassland, 39.6% onto land for maize, 12.9% onto land for cereals and 7.9% onto land for oilseed or protein crops .
We found that in Spain, the Monocotyledonae group and concretely the Poaceae family dominated herbaceous crop species in the agriculture scenario (Table 4). This reflects that cereals cover more land in Spain than any other crop, occupying 71% of the herbaceous crop area. This broad distribution reflects, in part, the plants’ adaptability to different soils and climatic conditions. At the national level, cereals account for 7.1% of agrarian production and 12.3% of vegetable production . We found that barley, wheat and corn occupied the top positions in the agriculture scenario (Table 4), consistent with their status as the main cereals produced in Spain . The situation is likely to be similar at the level of the entire EU, which is the largest wheat and barley producer worldwide and which accounts for 6% of global corn production . Wheat, barley and corn are included in Annex 2 of the OECD method.
The cereals oat, triticale, rye and rice are also cultivated in Spain and all were identified in the agriculture scenario, but each of them accounted for less than 5% of the herbaceous crop area. A similar pattern occurs at the EU level, where rye accounts for 2.0% and rice for 0.4% of the total arable land . All these cereal species except triticale are listed in Annex 2 of the OECD 208 method. Triticale is cultivated for winter cereal-legume forage intercrops, but legumes are necessarily more abundant in such intercrops in order to maintain feeding values .
The Liliaceae family was found to occupy 0.5% of the herbaceous crop area in the agriculture scenario, with the most abundant crops being onion (0.25%) and garlic (0.21%). Onion is the only species of this family listed in Annex 2 of the OECD 208 method, and it is the second most cultivated fresh vegetable in the EU. However, animal manure is not suitable for fertilising onion or other horticultural crops because bacteria, parasites and viruses in manure can contaminate the food supply  and lead to bioaccumulation of contaminants such as veterinary antibiotics in stems and roots [85, 86]. Nevertheless, manure can be composted for a long period and the resulting fertiliser can be considered safe for use on vegetable crops .
Dicotyledonae herbaceous crop species covered approximately 25% of the agriculture scenario (Table A4). The families Fabaceae and Asteraceae clearly predominated (19%), occupying the top four positions by land cover: an Asteraceae species (sunflower), followed by three Fabaceae species of alfalfa, vetch, and dried pie (Table 4). All these species except alfalfa are included in Annex 2 of the OECD 208 method.
Sunflower is an oilseed species, so it is used for human and animal consumption, fuel and industrial purposes, giving it high economic value . Oilseed species come from different Dicotyledonae families: rape, which accounts for 59% of total oilseed production in the EU, comes from the Brasicaceae family; sunflower, from Asteraceae; and soybean, from Fabaceae (EC, 2021). Of these oilseed species, only sunflower and rape have a notable presence in Spain ; rape occupied 0.92% of the agriculture scenario, placing it at 15th position in terms of land cover.
Alfalfa and vetch are fodder crops, although vetch is also used for human consumption. Fodder crops comprise legumes, cereals, sown meadows and other crops (Eurostat, 2020b). Fodder crops are important for extensively managed livestock in Spain . Among the four most abundant fodder species in our analysis, alfalfa is by far the most important in Spain (Table 4) and the EU . Vetch species are also frequently used in combination with a cereal such as oat or barley or with a pasture grass such as Italian ryegrass [51, 83].
Dried pie is a protein-rich crop, and such crops are a major source of amino acids for human and animal nutrition. Dried pie ranked eighth for land cover in the agriculture scenario (Table 7), and it is the most widespread protein-rich crop in Spain . At the EU level, pea, bean and lupine are major cultivated protein crops .
Among the other dicot families and crop species listed in Annex 2 of the OECD 208 method, only the Chenopodiaceae family and sugar beet species have some importance in Spanish agriculture, accounting for 0.5% of herbaceous crops (20th position). The EU is the world’s leading producer of sugar beet, accounting for around 50% of global production . Most sugar beet in the EU is grown in northern Europe, where the climate is more suitable for this crop .
The European Medicines Agency recommends that assessments of risk from veterinary antibiotics include plant species that are grown on agricultural land that are fertilised with manure . It would also be advisable to take into account the intrinsic susceptibility of plant species to antibiotics , but such data are nearly absent from the literature . Thus, given the currently available data, the best species for risk assessments may be those that are abundant on agricultural land and that receive manure fertiliser.
Given that Poaceae is the crop family with the largest area in the agriculture scenario, it would be advisable to consider at least two monocot species from among barley, wheat or corn in the risk assessment of antibiotics used to treat livestock species. Such assessment should also consider the dicot species of sunflower (Asteraceae) as soilseed, dried pie (Fabaceae) as a protein-rich crop, as well as alfalfa and vetch (Fabaceae) as fodder crops. It may also be helpful to consider the oilseed rape (Brassicaceae) and sugar beet (Chenopodiaceae), since both are widely cultivated in the EU.
The Mediterranean Basin is one of the 25 biodiversity hotspots around the globe where many endemic species are losing their habitat . The only hotspots of importance in Europe are the Mediterranean Basin and the Caucasus, both of which are extremely vulnerable to climate change . As a representative region of the Basin, Spain has among the greatest diversity of ecosystems, habitats and natural species in the EU, housing more than half of the Union’s species of vertebrates and vascular plants as well as 65% of its priority habitats .
Most pasture areas serve for grazing by livestock and/or wild ungulates, but they also provide other valuable regulation and cultural ecosystem services . Spanish pasture lands contain an abundance of Poaceae species as well as numerous other families catalogued in the Habitats of Community Interest under the EU Habitats Directive (92/43/EEC) (San Miguel et al., 2016). Our results highlight Lysimachia arvensis (L.) U. Manns & Anderb. (Primulaceae) and Galium aparine (Rubiaceae) as the most frequent species (Table 5). Lysimachia arvensis has been reported to cause cattle death in Uruguay and potentially also sheep death in Australia . In contrast, cleaver (Galium aparine) may support plant biodiversity . The following species appear in Annex 3 of the OECD method and have been described as most abundant in Spanish pasture lands , which may make them useful for assessing risk from veterinary antibiotics: Agrostis, Bromus and Festuca within the Poaceae family, as well as Trifolium and Lotus within the Fabaceae family. However, in the pasture scenario, species from this genera accounted for only 2-5% of all wild species, while Festuca species accounted for <1%. These species may therefore not be sufficiently representative. In other words, Annex 3 does not include most of the dominant plant species on Mediterranean pasture lands.
Arable field margins provide a wide range of ecosystem services in rural landscapes and limit the negative effects of competitive and dominant species on crop productivity . Plant diversity in these habitats has declined in recent decades due to agricultural intensification [97, 99–102]. Thus, one of the main challenges of agriculture worldwide is to balance crop productivity and biodiversity maintenance.
In typical Mediterranean cereal cropping systems in the Guadalquivir river basin in southern Spain, Pallavicini et al. (2020) identified 306 weed species, of which only 10 are catalogued in the OECD Annex 3. Among these catalogued species, the four most abundant were also among the five most frequent in our agriculture scenario: Lysimachia arvensis, Galium aparine, Papaver rhoeas L. and Chenopodium album L. Similarly, in cereal fields of the Ebro basin in northeastern Spain, Cirujeda et al. (2011) identified 175 weed species. Only 6 of the most abundant species (occurring in >10% of the surveyed fields) are included in the OECD Annex 3, with Papaver rhoeas the most abundant. This similarity in most abundant species between our mapped scenarios and previous fieldwork suggests that our analysis based on the GBIF data is reliable.
In addition, our results indicate the relevance of Hypericum perforatum L., the fourth most frequent wild species in Spain among those in Annex 3 (Table 5). This species was not inventoried by Pallavicini et al. (2020) or Cirujeda et al (2011), which may indicate that this moisture-loving species does not prefer the hot, dry conditions in the Ebro and Guadalquivir river basins.
Cirujeda et al. (2011, 2019) identified several extremely abundant weed species in Spanish cereal fields not included in the OECD 208 method, including Avena sterilis, Convolvulus arvensis, Dactylis glomerata L., Lolium rigidum and Sonchus oleraceus L. The role of weeds in supporting biological diversity within crop fields has been studied by Marshall (2003) and Storkey (2006). From a total of 32 species identified in those studies as important for biodiversity, only five are included in Annex 3 of the OECD 208 method. All of them were present in our agriculture scenario: Chenopodium album (Chenopodiaceae), Galium aparine (Rubiaceae) and Papaver rhoeas (Papaveraceae) accounted for 5-8% of the total occurrence records of all targeted species in this scenario, while Persicaria maculosa and Fallopia convolvulus (Polygonaceae) accounted for 1.5-2.7% of the total occurrence records (Table 5).
Our analysis suggests that assessments of risk from veterinary antibiotics should take into account Papaver rhoeas, Galium aparine and Chenopodium album as sentinel wild species. These species are important for biodiversity and they are abundant among targeted wild species in our agriculture scenario.
Our analysis of the pasture scenario suggests that risk assessments should not take into account Lysimachia arvensis. Even though this species belongs to the most abundant family (Primulaceae), the plant has been linked to livestock death (Roche et al., 2012). Instead, our analysis suggests that risk assessments should include Bromus tectorum or Agrostis capillaris from the Poaceae family, as well as Trifolium pratense or Lotus corniculatus from the Fabaceae family. All these species are relatively abundant among targeted wild species and they are dominant in Mediterranean grasslands . The species Galium aparine (Rubiaceae) could also be considered, since it was the second most frequent species in our pasture scenario and it can support biodiversity.
While all the above mentioned species have been listed in Annex 3 of the OECD 208 method, there are other weed species at the margins of arable fields and on pasture lands that could be useful in assessments of risk from veterinary antibiotics but are not listed in Annex 3. These species may include those identified as the most frequent in surveys of field boundaries [99, 100, 102] and pasture lands (San Miguel et al., 2016) and that support biodiversity or food security. For example, Sonchus oleraceus is one of the most frequent weed species at field boundaries in Spain , and it is important for biodiversity . Some Poaceae species, such as Avena sterilis, Dactylis glomerata, Hordeun murinum and Lolium rigidum, are also frequent and are included on the Prioritised Spanish Checklist of Crop Wild Relatives , which compiles alternative crop species crucial to ensure food security.
The identification of sentinel species may be especially beneficial for biodiversity conservation within the Spanish Sites of Community Importance network and Biosphere Reserves, given that up to 75% of these protected areas may be vulnerable to phytotoxic effects from veterinary antibiotics, according to our pasture scenario. Future work to consider new “sentinel” species not currently in Annex 3 will also need to evaluate how feasible it is to develop standardised testing protocols akin to the OECD 208 method. This will depend on the commercial availability of seeds and whether they meet the conditions for laboratory assays (e.g. germination rate ≥ 70% and survival rate ≥ 90%).