Zn is abundantly present in the pig's body, with quantities in the order of 1.5 to 2.5 g for an animal of 100 kg live weight (LW). It is found mainly in the muscle tissue of the pig (about 60%) and in the skeleton (about 30%), with the bones constituting the main site of body reserve, i.e. storage and mobilisation of Zn when intakes vary. The highest concentrations of Zn are found in the bristles (200 mg.kg^-1 DM) and the liver (150 mg.kg^-1 DM). As in most mammals, the plasma Zn content is about 1 mg.L^-1 in pigs, corresponding to 0.1% of total body Zn (Swinkels et al., 1994).

The body content of Cu in pigs is lower, in the order of 200 mg for an animal of 100 kg body weight. Most of the Cu is found in the skeleton (40-46%), muscles (23-26%) and liver (8-10%) (Cromwell, 1997). The liver is the main site of Cu accumulation when requirements are exceeded. Liver Cu content measured after slaughter is used as an indicator of the Cu status of the animals and an indicator of feeding practices. Hodges and Fraser (1983) observed a large variability of Cu content in liver samples from pigs of different origin and age. In 9% of the cases, they observed a content lower than 12 mg.kg^-1 DM, indicating a risk of deficiency, but for the majority of animals the content exceeds 20 mg.kg^-1 DM, indicating supra-nutritional Cu intakes. Liver Cu content depends not only on the dietary concentration but also on the bioavailability of the intake (Romeo et al., 2018).

Zn is fundamental to the proper structural and functional integrity of almost 200 transcription factors. In addition, most metabolic pathways are dependent on one or more proteins that function through the presence of Zn, which is a cofactor in over 300 metalloenzymes (Suttle, 2010). In particular, it is a component of DNA and RNA synthetases and transferases, and many digestive enzymes (National Research Council, 2012).

Cu is an essential element involved in many biological functions, such as cellular respiration, protection against oxidative stress and iron (Fe) transport (Suttle, 2010), as it is present in a large number of enzymes, co-factors and proteins (Espinosa and Stein, 2021).

Zn and Cu play a role in protection against oxidative stress. This is due to their role as cofactors of proteins and enzymes that protect against oxidative stress (superoxide dimutase, ceruloplasmin ferroxidase) (Suttle, 2010). Zn deficiency increases the susceptibility of endothelial cells to this stress, as Zn is the activator of metallothioneins (see Box 1), which are involved in the detoxification of cells and have a high affinity for Zn and Cu. Moreover, metallothioneins also have an antioxidant character (Suttle, 2010).

At "pharmacological" intake levels, Zn and Cu can also act as "growth promoters" in pigs. This effect may be related to an improvement in feed digestibility or animal appetite (Suttle, 2010), a reduction in digestive disorders, in particular diarrhoea (Bikker et al., 2016), and also to their antibiotic activity and their effect in modulating the microbiota and improving the integrity of the intestinal barrier (Villagómez-Estrada et al., 2020a)

Two main approaches are used to assess mineral requirement, one empirical and one factorial (Schlegel, 2010), the former being the most common for trace metals (TMEs). This consists of experimentally testing the effects of a gradual increase in the amounts of Zn and Cu in the feed in order to evaluate the response of several zootechnical or physiological parameters. Statistical treatment of the results by a linear plateau model is then generally used to describe the response and define the breakpoint requirement (Kirchgessner, 1993).

A specific and representative response of one of the animal's functions for Zn or Cu must be observed in order to determine a criterion as characteristic of the requirement (Schlegel, 2010). For Zn, the most representative criterion generally used is its concentration in bone or plasma; for Cu, this criterion is its concentration in the liver (Jongbloed, 2010).

This is illustrated in figure 1 for Zn in post-weaning piglets, based on different literature references. Depending on the level of intake, we can define i) a deficiency zone (in red) in which the intake is not sufficient to maintain an adequate plasma level; ii) a homeostasis zone (in green) in which the plasma level remains constant thanks to the implementation of different homeostasis mechanisms (storage, excretion...) and iii) a zone of excess (in blue) in which the plasma concentration remains constant.) and iii) a zone of excess (blue) in which the plasma concentration increases with food intake, the homeostasis mechanisms being probably saturated. The breakpoint between the red and green zones defines the nutritional requirement, whereas the breakpoint between the green and blue zones indicates the appearance of a risk of excess associated with a drop in intake.

Table

The main symptoms of Zn deficiency are initially loss of appetite, the onset of diarrhoea and growth retardation. Anorexia is one of the first signs observed and leads to reduced femur size and strength in deficient piglets (Suttle, 2010). Emergent diarrhoea is due to reduced enterocyte turnover (Revy, 2003). In addition, a decrease in plasma or serum Zn level is observed as well as alkaline phosphatase and albumin (Suttle, 2010; National Research Council, 2012). However, animals respond to Zn deficiency by regulating the various intestinal Zn transporters, but this is not sufficient to cover the premature decline in plasma and serum Zn concentration in deficient piglets (Suttle, 2010). The most prominent symptom of Zn deficiency and the last sign to appear is a hyperkeratinisation of the skin called parakeratosis (Suttle, 2010; National Research Council, 2012).

Cu deficiency situations in pigs are rarely observed and can occur in weanlings fed about 100 mg.kg^-1 MS Fe, 130 mg.kg^-1 MS Zn and 2 mg.kg^-1 MS Cu (Jondreville et al., 2002). Apparent signs of Cu deficiency are anaemia, limb bowing, the occurrence of spontaneous fractures and heart and vascular problems and depigmentation (National Research Council, 2012). Recent results (Dalto et al., 2021), however, indicate a risk of Cu deficiency developing in the case of Zn intake at pharmacological doses (2500-3000 mg.kg^-1 DM), confirming the reduction in plasma Cu observed by Hill et al. (2001) in this situation (see section 1.3.d).

Zn is not very toxic for most mammals. However, an excess of Zn (see Figure 1) in the food leads to a decrease in its absorption, an increase in its storage in the bones and in the enterocytes after binding to metallothioneins and therefore an increase in tissue turnover and endogenous Zn secretions. At pharmacological doses (see Box 1), excess Zn intake can be accompanied by a reduction in feed intake and therefore in growth rate (Hahn and Baker, 1983). This situation is accompanied by an alteration of Cu and Fe metabolism, with risks of deficiencies if the excess situation is prolonged (Dalto et al., 2021). Moreover, the susceptibility of the animal to excess Zn also depends on the Ca, Fe, Cu and cadmium content of the feed (Revy, 2003).

Cases of Cu poisoning are rare in pigs (Jondreville et al., 2002). However, a long-term distribution of more than 250 mg.kg^-1 DM of Cu can lead to a toxic effect and the signs observed are a reduction in haemoglobin levels and jaundice (National Research Council, 2012). This can lead to a reduction in Fe storage in the liver and therefore anaemia, due to a negative reaction of Cu on Fe, which has limited absorption (Jondreville et al., 2002). The tolerance of pigs to high dietary doses of Cu depends on the supply of Zn in the feed and the interactions of Zn and Cu with metallothioneins (Jondreville et al., 2002).

Finally, the capacity of a feed to cover the Zn and Cu requirements of pigs and the potential toxic effect of these elements depend on their concentration but also on their bioavailability (Hahn and Baker, 1983; Suttle, 2010); the absorption rate and the corresponding efficiency modify the total TME requirements of the animals (Männer, 2008).

The European regulations concerning the maximum permitted levels of Zn and Cu in pig feed have changed since 2003 (Figure 2).

Table

The basic ration of the pigs is mainly composed of cereals (mainly maize and wheat, but also barley, triticale, etc.), oil cakes (mainly soya, but also rapeseed and sunflower), oilseeds (peas, beans, etc.), cereal co-products (bran, middlings, draff, etc.), and vitaminised mineral supplements (feed additives).

Cereals and protein crops contain on average 20-30 mg.kg^-1 DM of Zn, with cereal co-products having a higher Zn content (between 70 and 90 mg.kg^-1 DM, Figure 3). Oilseeds and oilcakes contain between 30 and 90 mg.kg^-1 DM of Zn (figure 3).

Cereals, their co-products, oilseeds and oilcakes contain between 5 and 15 mg of Cu per kg DM (Figure 3).

Table

Two experimental approaches exist to measure the bioavailability of a mineral. The first is to measure the response of different physiological criteria to increasing intakes of the mineral, while remaining below the requirement (red area in Figure 1). The second is to measure this response with intakes well above requirement and measure their accumulation in the blood or in different tissues such as bone or liver (Spears and Hansen, 2008). In both cases, these responses are assessed relative to a reference source (CuSO₄ and ZnSO₄ ) whose bioavailability is set at 100%. Bioavailability is then calculated by comparing the response slopes of the different sources tested. Generally speaking, the bioavailability of minerals is influenced by the type of mineral supplementation provided, by factors such as the physiological status of the animal or its mineral status, and also by interaction effects with other agonistic or antagonistic elements of the ration (O′Dell, 1989; Männer, 2008). Different types of possible interactions are the formation of non-absorbable complexes in the gut, competition between cations for the transport of non-specific divalent cations or even the induction of proteins by non-specific metals (Suttle, 2010). The difference in bioavailability of Zn and Cu between sources could be largely explained by these phenomena that affect absorption, as well as their different physicochemical properties that affect their solubility.

Phytates form non-absorbable complexes with Zn, which reduces the bioavailability of Zn (Suttle, 2010). The hydrolysis of phytates allows the release of Zn, but monogastric animals do not naturally produce phytase, or in very limited quantities. The addition of microbial phytase to the ration can therefore improve Zn availability. Revy et al (2004) showed that 700 units of phytase can replace 32-43 mg of Zn in the form of sulphates. Similarly, Bikker et al. (2012) estimated that 500 phytase units can replace 27 mg of Zn as sulphate. According to the meta-analysis conducted by Schlegel and Jondreville (2011), only the Zn naturally present in the feed is influenced by phytates and phytase, and not the Zn added to the ration in the form of mineral or organic supplements. This antagonistic effect of phytates is increased after adding Ca to the ration (Suttle, 2010). A phytate-Ca-Zn complex is formed and precipitates Zn in the digestive tract (Revy, 2003). The antagonistic effect of phytates on Zn is all the more important in young animals, which are fed diets richer in Ca than their needs (Suttle, 2010).

Cu is less affected by phytates and Ca in the ration, due to its higher affinity for free amino acids, with which it forms chelates, allowing Cu to retain its solubility (Jondreville et al., 2002). However, soluble sources of Cu can interact in the digestive tract with phytates and form zinc-calcium-copper-phytate or copper-calcium-phytate complexes (Oberleas, 1973), which are resistant to phytase hydrolytic activity.

Zn has an antagonistic effect on Cu, i.e. it inhibits its absorption. This effect is due to the inducing effect of Zn on the synthesis of metallothioneins, which have a strong affinity with Cu, preventing its transfer to the intestinal epithelium (Revy, 2003).

Numerous studies have determined the body retention of Zn and Cu by pigs, which makes it possible to calculate the quantities excreted and their content in the effluent by difference with the ingestion. The retention is relatively low and is estimated in the literature for piglets and growing pigs at about 22 mg.kg^-1 of weight gain for Zn (Dourmad et al., 2002) and less than 2 mg.kg^-1 of weight gain for Cu (Jondreville et al., 2002). In case of intake close to the requirement, about 80 to 90% of the Zn and Cu ingested by pigs are found in pig faeces of which only 1 to 2% are excreted in urine (Dourmad et al., 2002).

The composition of the slurry will mainly depend on the composition of the feed given to the pigs. As the digestibility of DM and Organic Matter (OM) of the feed is around 70-80%, and therefore higher than that of Zn and Cu (around 1-2%), these TMEs are much more concentrated in the manure than in the feed. Manure can thus contain up to 2,000 mg of Zn per kg DM and 1,000 mg of Cu per kg DM, considering all types and stages of animals and feed (Dourmad et al., 2002; Jondreville et al., 2002; Marcato, 2007).

In 2016, the official references (CORPEN, 2016) setting the amounts of N, P, Zn and Cu excreted by pigs have been updated ( RMT Élevage et Environnement, 2015). They are reported in Table 1, incorporating values calculated taking into account the most recent EU regulations on maximum permitted levels in feed (Regulation (EU) 2016/1095, Regulation (EU) 2018/1039).

Table

titre du tableau
Physiological stages	Zn ingested	Zn excreted	Cu ingested	Cu excreted
Breeding sow, g/year	180 1441	173 1371	30,0 30,0 1	29,7 29,71
Post-weaning, g/pig	5,8 5,81	5,3 5,3 1	6,6 4,91	6,6 4,91
Post weaning with 3000 mg /kg feedDM, g/pig	19,2	18,7	6,6	6,6
Feeding, g/pig	36 28,81	34,1 26,91	6,0 6,01	5,9 5,91

¹According to the new regulations (EU) 2016/1095, (EU) 2018/1039.

The levels of Zn and Cu excreted by pigs, calculated taking into account the new regulations, are lower than the current CORPEN references, which were defined taking into account the old regulations (Regulation (EC) No 1334/2003). This is due to the reduction of the maximum permitted levels in feed following the new regulations of 2016 and 2018. An update of these references could take into account these new values.

Although the most common way of managing manure is storage followed by spreading (Nicholson and Chambers, 2008), almost 12% of French pig farms were already using an alternative or complementary manure treatment technique to 'storage - spreading' in 2008 (Loyon, 2017). The treatments applied can be grouped into different categories: mechanical (phase separation), biological (aerobic treatment or anaerobic digestion, composting), chemical and thermal. These different technologies can be combined in different ways and are part of the overall manure management system (Figure 6 and Table 2).

Table

Table 3 summarises the main effects observed on Zn and Cu during the treatments.

Table

titre du tableau
Management/Processing	Treatment effects on Zn and Cu	Parameter to be taken into account	References
Storage	- Increase in the influence of the distribution of Zn and Cu between the different classes of particles : the binding of metals to the particles	Type of storageDuration Temperature	Popovic and Jensen (2012)
Phase separation	- Concentration of Zn and Cu in the solid phase as a function of separation efficiency	Scraping methodin the building	Loussouarn et al (2014) Likiliki et al (2020)
		Type of separator	Møller et al (2007) Nicholson and Chambers (2008) Pantelopoulos and Aronsson (2020)
Aerobic treatment	- Increased concentration of Zn and Cu, relative to OM and DM- Redistribution of Zn and Cu between solid (screenings) and liquid phases (supernatant and sludge)	Type of separators	Levasseur (2003)Béline et al (2004)
Methanisation / Anaerobic digestion	- Increase in Zn and Cu concentration, relative to OM and DM- Increase in the share of Zn and Cuin solid particles- Increased concentration of Zn and Cu of Zn and Cu in the poorly bioavailable fractionavailable fraction(unrecognized symbol) Decrease in the phytoavailability of these elements	Temperature pHRetention timehydraulicOxidation-reductionreduction	Marcato (2007)Marcato et al (2009)Amaral et al (2014)Matheri et al (2016)Legros et al (2017)Yang et al (2020)
Thermal treatment (incineration)	- Cu accumulation in bottom ash- Concentration of Zn and Cu in particles > 30 µm		Møller et al (2007)Kuligowski et al (2008)
Composting	- Increased concentration ofof Zn and Cu, relative to OM and DM- Increase of soluble part at the beginning of the process and then - Decrease of Cu in this form at the end of the composting process- Decrease in bioavailability factorof Zn and Cu	Moisture pH Nitrogen contentDissolved organic carbon organic carbon content Humic mattercontent	Hsu and Lo (2001)He et al (2009)Santos et al (2018)Li et al (2019)

- Phase separation in buildings

Phase separation directly in the building, e.g. by the V-scraping process, is fairly recent and the characterisation of the products obtained is still poorly documented. Zn and Cu follow mostly the solid fraction (between 90 and 95%) and this fraction is mainly treated by composting or anaerobic digestion. (Loussouarn et al., 2014; Likiliki et al., 2020).

- Duration and type of storage

Few studies exist on the effect of storage on Zn and Cu. According to Popovic and Jensen (2012), storage has no significant effect on Zn and Cu concentrations but increases the binding of Zn and Cu to less soluble solid particles.

- Mechanical phase separation

As only about 10% of Zn and Cu are soluble, these elements are mainly found in the solid fraction after separation from the slurry (Nicholson and Chambers, 2008). This redistribution will however depend on the type of separator or combination of separation processes used (Møller et al., 2007; Pantelopoulos and Aronsson, 2020). The separation of Zn and Cu to the solid phase is highest for chemical treatment with filtration (99.5% Zn and 94.9% Cu in the solid phase) followed by centrifugation (38.1% Zn and 31.1% Cu) and then screw compacting (7.1% Zn and 6.6% Cu) (Møller et al., 2007; Popovic et al., 2012; Pantelopoulos and Aronsson, 2020). Several studies evaluate the effect of combining different types of separation with an aerobic biological treatment plant (Levasseur, 2003; Béline et al., 2004). The separation results in lower concentrations in the liquid phase, thus reducing the annual soil load of Zn and Cu after the liquid fraction is used as fertiliser by spreading, compared to spreading raw manure (Béline et al., 2004; Møller et al., 2007; Popovic et al., 2012). The solid fraction can then be exported and used as organic fertiliser, usually after further treatment (drying or composting).

- Methanisation

As several nutrients such as N, P, K, Zn, Cu are retained during anaerobic digestion, the resulting digestate can be used as fertiliser (Marcato, 2007; Marcato et al., 2009; Amaral et al., 2014). However, the transformation of part of the OM into biogas leads to an increase in Zn and Cu contents in relation to the DM and an effect on their speciation (see Box 1) which is important to take into account. Furthermore, Zn and Cu can also stimulate or inhibit the bacteria responsible for anaerobic digestion (Matheri et al., 2016), thus modifying biogas production.

- Drying by incineration

After incineration of the solid fraction of slurry or digestate from anaerobic digestion, two types of ash are obtained, a bottom ash (which tends to be richer and to accumulate Cu) and a so-called "fly ash" (Møller et al., 2007).

- Composting

Composting leads to the production of humic acid, the main constituent of humus, and Cu has a better affinity for this humic acid than Zn, which influences their distribution (He et al., 2009) in the final product of composting: compost. Hsu and Lo (2001) observed an increase in the soluble Cu content during the first 18 days of the composting process (up to 16% of Cu in this form) and then a decrease in this content to 3% of Cu in soluble form in the final compost. A lesser effect was observed on the soluble part of Zn, which reached a maximum of 2%. Composting therefore seems to have a significant effect on the risk of Cu leaching, but less on that of Zn. Hsu and Lo (2001) observed that most of the Zn and Cu were in the low bioavailable fraction of the compost. The addition of material, such as straw or other organic substrates, or mineral additives, reduces the availability of heavy metals during composting and facilitates their transfer to more stable forms. This is related to the formation of compounds with affinity to the humic matter of the compost (Santos et al., 2018; Li et al., 2019). Furthermore, the addition of OM is also accompanied by a reduction in Zn and Cu content through dilution.

One of the purposes of these different treatments is the production of organic fertilisers or soil improvers (see Box 1) that are exported outside the livestock farms to crop systems where OM is lacking, such as market gardening. This reduces the risk of accumulation of elements such as Zn and Cu in pig farm soils by optimising their use. However, this requires that these products be standardised and take into account the impact of these treatments on the speciation of the TMEs and therefore their bioavailability to plants.

The Zn and Cu present in soils can come from natural or exogenous sources (e.g. fertiliser inputs). Indeed, metals are found naturally in soils after alteration of the parent rock (Legros, 2008; Kumar et al., 2021). The composition of Zn and Cu and their behaviour depend on the type of soil and the parent rock on which it develops (Legros, 2008). Exogenous inputs are also sources of soil enrichment in Zn and Cu: phosphate fertilisers, inputs of manure and slurry, spreading of sewage sludge, 'urban' composts and phytosanitary treatments (Baize, 1997). It is therefore important to control Zn and Cu inputs, which can become toxic for plants or soil micro-organisms.

In view of the risks associated with the input of Zn and Cu through the application of organic fertilisers and soil improvers, maximum levels of these elements have been set for their marketing (Table 4). However, these standards do not apply to raw livestock manure spread as part of a land application plan.

Table

titre du tableau
Type of fertilizer	Zinc	Copper
Organic fertilizer
Ecolabel1	300	100
Afnor2	600	300
Methanation digestate 3	1 500	600

¹ Decision (EU) 2015/2099. ²AFNOR standard NF U44-051 (April 2006). ³Order of 13 June 2017 (NF U44-051 of April 2006)

Pig manure for fattening contains about 955 mg.kg^-1 DM of Zn and 300 mg.kg^-1 DM of Cu when this type of animal is fed a feed that complies with current European regulations (120 mg.kg^-1 Zn and 25 mg.kg^-1 Cu), these values being slightly higher if the entire post-weaning/fattening period is considered (figure 5). However, effluent treatment will generally concentrate these elements to a high degree and thus increase their content. Thus, given the current Zn and Cu levels in feed, these levels exceed Afnor standards and even more so the European Ecolabel for products resulting from certain treatments. A specific rule was recently established for methanisation digestates with higher thresholds set at 1,500 and 600 mg.kg^-1 of DM respectively for Zn and Cu (NF U44-051). However, even with these different regulations, the quantities of Zn and Cu applied remain higher than the needs of the crops.

Zn and Cu are essential minerals for good plant growth and are therefore taken up by plants. Plants contain between 10 and 100 mg.kg^-1 MB of Zn (Gräber et al., 2005). They require on average between 5 and 20 mg.kg^-1 MB of Cu (Legros, 2008; Kumar et al., 2021). Cu deficiency in plants leads to reduced plant growth, promotes leaf chlorosis and causes cytotoxicity (Kumar et al., 2021). In some regions there is a risk of Zn and Cu deficiency (GIS Sol, 2001), with significant variability depending on the presence of pig or poultry farms and vineyards. Amendments rich in Zn and Cu may therefore be of interest. Pig manure has a higher Zn content than Cu, which leads to a more rapid increase in Zn than Cu content in soils (De Conti et al., 2016). There is no risk of Cu contamination in the human food chain, as it is not very mobile in the plant and therefore rarely reaches the consumable parts of plants (Marcato, 2007).

Zn and Cu accumulated in the soil after spreading can subsequently be leached and transferred to the water system. This depends on the speciation of these elements which will be more or less soluble depending on the physico-chemical characteristics of the environment. These transfers to aquatic ecosystems are also favoured by erosion phenomena, with Zn and Cu being transferred with soil or OM particles. In this way, Zn and Cu can be taken up by marine organisms and contribute to the contamination of the food chain (Jondreville et al., 2002; Legros et al., 2013).

Many studies highlight the fact that Cu is a much less accessible and less mobile element than Zn (Gräber et al., 2005; Mallmann et al., 2014; Marszałek et al., 2019). Indeed, Cu has a higher affinity for OM (Kumar et al., 2021) and is therefore more sensitive to soil properties such as OM content or pH. It can therefore become more or less mobile when these characteristics vary, but will mainly accumulate in the soil. Zn is also a poorly mobile element in soils, but it is less sensitive to variations in soil characteristics (Marszałek et al., 2019). In the soil solution (water circulating in the open spaces of the soil), Zn will be mostly in the free form Zn²⁺ while Cu will be mostly in the form complexed with OM (De Conti et al., 2016; Kumar et al., 2021).

Zn and Cu from applied livestock manure accumulate overwhelmingly in the topsoil (0-20cm), i.e. the surface layer of soils (Jondreville et al., 2002; Revy, 2003; Marszałek et al., 2019). According to McGrath (1981), only 2-10% of the Cu brought in by land application is subsequently leached or taken up by plants. In soils that have received pig manure for many years, De Conti et al. (2016) observed an accumulation of carbon in the surface layer, having a high affinity with metals, in particular with Cu. This may explain the accumulation of these elements in this soil layer. Moreover, Zn and Cu are not very mobile and are therefore transferred in small quantities to the deeper soil layers. Zn and Cu can be transported by humic acid after formation of chelate complexes and these complexes are more stable when formed with Cu than with Zn (Gräber et al., 2005). This complexation of Zn and Cu in soils reduces the content of these elements in free form and thus their phytotoxicity (De Conti et al., 2016). The soil pH is also a factor that has a strong influence on the solubility of Zn and Cu. Its increase improves the adsorption of minerals on OM and thus decreases their mobility (Levasseur, 1998; De Conti et al., 2016). Acidic soils, as is often the case in Brittany, therefore have a higher solubility of Zn and Cu. Mallmann et al (2014) assessed the effect of tillage practices on the distribution of Zn and Cu following pig manure inputs over a long period. They showed that no-till practices amplify the accumulation of Zn and Cu at the soil surface and reduce their mobility to depths exceeding 20 cm.

Many studies have evaluated the effect of long-term application of pig manure on Zn and Cu accumulation (Coppenet et al., 1993; L'Herroux et al., 1997; McGrath et al., 2000; Gräber et al., 2005; De Conti et al., 2016; Formentini et al., 2016; Benedet et al., 2019; Benedet et al., 2020). According to Coppenet et al. (1993), between 1973 and 1988, i.e. after 15 years of regular application of pig manure, the soils of Finistère (France) were enriched in Zn by nearly 0.37 mg.kg^-1 .an^-1 and in Cu by nearly 0.22 mg.kg^-1 .an^-1 . They also showed that this increase is higher the more manure is applied. L'Herroux et al (1997) were interested in the speciation of Zn and Cu in the soils of Brittany (France), after 6 years of application of pig manure, and in their distribution in the different fractions determined according to their bioavailability: in the long term, Cu is mainly found in the poorly bioavailable fraction and with time the share of Zn increases in the slightly more bioavailable fractions These observations on the speciation of Zn and Cu or on their behaviour in soils are important to predict their effect on the environment (Formentini et al., 2016). Indeed, the toxic effect of Cu in soils is a long-term environmental problem (López Alonso et al., 2000). De Conti et al. (2016) evaluated this effect of Zn and Cu accumulation on soils and highlighted that the phytotoxic effect of Zn and Cu does not appear even after several years of pig manure application, because plants modify soil conditions so that the chemical species can complex with dissolved organic carbon. A certain stability of Zn and Cu is then observed. Plants generally tolerate Zn levels ranging from 10 to 100 mg.kg^-1 DM and Cu levels in soils ranging from 2 to 40 mg.kg^-1 DM (Gräber et al., 2005). According to Coppenet et al. (1993), some Zn-sensitive plants, such as maize, can suffer from deficiencies in this element when the soil content is below 1.5 mg.kg^-1 DM and a phytotoxic effect is observed when this content exceeds 120 mg.kg^-1 DM. In Breton soils, with a pH close to 6, this phytotoxic effect is observed when the accumulated content of Zn and Cu exceeds 120 mg.kg^-1 DM. Cu tends to accumulate in root tissues and can be transferred to the stem; Cu toxicity in plants thus affects root growth and plant morphology (Kumar et al., 2021). McGrath et al. (1995) reported that soil microbial activity is disrupted when soil Zn levels exceed 100 to 200 mg.kg^-1 DM. Zhang et al (2016) studied the effect of pig manure application on soil microbial community and put forward that Zn and Cu can improve soil fertility by allowing the development of soil biomass. According to them, Zn has the greatest influence on the soil microbiota.

The activity of earthworms in soils is reduced when the Cu concentration exceeds 50 mg.kg^-1 DM (Gräber et al., 2005). Toxicity of Zn and Cu can also affect animal species after spreading pig manure on pastures. López Alonso et al. (2000) showed that in regions with intensive pig farming, more than 20% of the livestock have liver Cu concentrations exceeding the potentially toxic concentration of 150 mg.kg^-1 . Sheep in particular are extremely sensitive to excess Cu and cases of poisoning of sheep grazing on grassland fertilised with Cu-rich pig manure have been reported (Poulsen, 1998).