Sampling

1. Background
1.1. Objectives

The objectives pursued with the development of this research methodology are to:

Know the metal content in native and cultivated plants: risk assessment

Have and accurate risk estimation assessment based on single chemical extraction:
bioavailability

Know the origin of metals: isotopic studies

Select plants for phytostabilisation that are well adapted to the harsh conditions of
the area

1.2. Sampling area

The study area is Rambla del Avenque. Within the area there are zones that have been not mined and
a number of tailings ponds with different conditions. In addition, this area is going to be extensively
studied along other GTs which is an advantage in terms of supporting information and exchanging
data.

As per Figure 1, we defined two areas, the one to the right defined as not mined and the one to the
left that has been mined and has naturally growing plants.

Figure 1. Proposed study areas in Rambla del Avenque for sampling native plants

2. Sampling description

Not mined area

 Start, for example, with the not mined. Divide the space available visually in 5 areas. For each
area we will select 5 subareas where the plant species highlighted in green in the next Table
will be sampled: Arisarum simorrhinnum (candil), Asparagus horridus (esparragera), Lavanda
multifolia (lavanda), Olea europea sylvestris (acebuche), Pistacia lentiscus (lentisco), Rhamnus
Lycioides (Espino negro). In the selection of these subareas we will consider the existence of
bare areas for the non-rhizosphere soils. If any of the plants selected is not present in the
selected subareas we will sample as close as possible. As a rule of thumb, the whole plant will
be taken and will be divided in the lab but it is important to have clear which are the most
important parts for each plant (see Table 1)

Arisarum simorrhinnum (candil) Asparagus horridus (esparragera)

Lavanda multifolia (lavanda) Olea europea sylvestris (acebuche)

 Pistacia lentiscus (lentisco) Rhamnus Lycioides (Espino negro)

Table 1. List of plant species available in the Not mined area, palatability and fructification dates

Soil sampling: rhizosphere and non-rhizosphere soil will be taken. Rhizosphere soil will be
taken for each plant. Within each area, 5 subsamples of non-rhizosphere soil will be enough
(ideally, one per sample point)
o Rhizosphere soil: soil adhered to the root
o Non-rhizosphere soil: 0-30 cm, which comprises A and C horizons down to the parent
material. From the bare area at a given distance from the plant
Other samples/information
o Plant richness and cover at each sampling point
o Possibility of taking animal faeces
Mined area

Divide in 5 areas
Within each area take 3 replicates (because of the lower expected variability) of the following
species:

Phagnalon saxatile (manzanilla yesquera) Helichrysum decumbens (siempreviva)

Piptatherum miliaceum (triguera) Atriplex halimus (salao)

 Dittrichia viscosa (olivarda)

Proceed in the same way to that described for the not mined area

3. Sample manipulation (lab)

3.1.1. Plants

Sample preparation is crucial because it conditions further analyses. It is also important in terms of
coordination with other GTs since there will be subsamples that will be sent to other laboratories. For
plants, the following steps will be followed:

Separation of plants in the different parts

Thoroughly mix
Keep a subsample fresh
Dry the rest and grind
o Send an aliquot (>600 nanograms) for isotopic analysis to Jrme/Eva (CNRS
Toulouse)
3.1.2. Soils
For soil samples, an aliquot will be kept fresh
The remaining to be dried at 40C, sieved and ground
o Send an aliquot (>600 nanograms) to CNRS Toulouse for isotopic analysis. In GT4 and
link with GT2
o Send an aliquot (> 10 g) to Marilyne and Emmanuel (University of Limoges): IR and
XRF, but also gastric. Link with GT 2
o Keep an aliquot (amount not specified) to Montse/Irene (they decided not to analyse
but just in case): inhalator bioavailability (risk due to wind dispersion). Link with GT 2
4. Material to prepare
Mochila
 Bolsas: que no falten.
o Zona no minera: plantas (5 reas * 5 rplicas * 6 especies = 150), suelos (150
rizosfricos + 25 no rizosfricos)
o Zona minera: plantas (5 reas * 3 rplicas * 5 especies = 75), suelos (75 rizosfricos +
15 no rizosfricos)
Gomas
Azada, Pala y tijeras de podar
Bolgrafos, permanentes
Cuaderno de campo
Gua de plantas: aunque llevamos la gua de plantas andante, Ral
Cinta mtrica y barra graduada
Equipacin protectora: guantes, mascarilla, gorra, crema solar
Cmara de fotos: est cargada?
GPS: creo que estara bien marcar los puntos
Comida, agua