Biomonitoring of Persistent Pollutants: Autochthonous Material and Target Species

1. Biomonitoring of persistent pollutants by using autochthonous material • the target species; • the sampling; • sample processing and analysis; • difference among species; • how to compare different data sets: the naturality scale proposed by Nimis & Bargagli (1999).

2. LICHENS Foliose lichens are preferred, because relatively large quantities of material are necessary for the analytical work. Fruticose lichens are unfortunately very rare in urbanized areas. Epiphytic (= growing on plants, usually trees) lichens are preferred to epilithic (= growing on rocks) ones, because soil contamination is generally lower.

3. The selected species are rather resistant to airborne pollutants, and have a broad distributional range on the territory. This makes comparisons among different survey areas possible. Flavoparmelia caperata (L.) Hale Parmelia sulcata Taylor Punctelia subrudecta s. lat. Xanthoria parietina (L.) Th. Fr.

4. Sampling sites The stations must be homogeneous, selected far from roads with heavy traffic (or all of them should be near roads with heavy traffic...), in areas covered by meadows. Lichen sampling is carried out on trunks of deciduous trees, with inclination <10o, without decorticated areas, or evident injuries. The most common tree species is selected.

5. Sampling: from 1 to 2 m above ground, using a ceramic knife („Made in Japan“!), and putting the material in envelopes made with Whatman paper, and – when dry - in a numbered petri dish (preservation?). If there are more than three thalli, sampling must be random. 1 m 2 m

6. Sampling chard • photographs of the trees; • a short description of the area; • list of species sampled and list of accompanying taxa; • exposition on the truncks; • height above the ground; • lichen health indications (N: necrosis; D mechanical damage; S: bleaching; 0=absent; 1=low; 2=med-ium; 3=strong); • map (1:5000) of the area; • UTM coordinates; • Critical remarks.

8. WHEN - Notduringrains or at the end of a prolongeddrought season. Samplingshould be carried out in the shortest time, e.g. in one-twodays, withoutrainevents in between. Itispreferablethat a single operator carries out the sampling.

9. Sample preparation

10. Come preparare i campioniprima dell’analisi? Una preparazione accurata di campioni di Xanthoria parietina abbatte le concentrazioni di elementi ampiamente diffusi nel suolo quali Al e Fe, mentre per gli altri elementi la variabilità nell’intercettazione delle particelle di suolo non consente di fare previsioni affidabili. Loppi, Paoli, Frati, Baragatti (2004)

11. Zonation of trace metal accumulation in three species of epiphytic lichens belonging to the genus Parmelia Bargagli, Iosco, D’Amato (1987) Al, Cr, Cu, Fe, Mn, Pb, Se, Zn Confronto tra ambienti Confronto tra specie: • Flavoparmelia caperata • Parmelia saxatilis • Parmelia sulcata Confronto tra parte interna ed esterna del tallo Lavaggio dei campioni

12. Mean metal content in the peripheral (Pe) and central (C) parts of five thalli of Flavoparmelia caperata. I II III IV ppm (g g-1) 40 Pe Pi M C 35 30 25 20 15 10 5 0 Al Ba Cr Cu Fe Mn Ni Pb

13. Mean metal content in the external (Pe) and central (C) parts of five thalli of Parmelia caperata. I II III IV 35 Pe Pi M C 30 25 20 15 10 5 0 Mg Zn

14. The central part of foliose lichens (C), being older, has higher metal contents than the peripheral lobes (Pe). 3 3 2 2 1 Cr ppm 1 C 0 M 2 5.2 Pi Parte del tallo 5.6 Distanza dalla strada 7.3 Pe 16.2 (m)

15. Flavoparmelia caperata (L.) Hale

16. Foliose lichens Under the stereomicroscope... Approximately 2-4 hours are needed to clean a sample of 200 mg (!).

17. The fragments obtained are carefully homogenized in a agata mortar or are finely chopped using a ceramic knife. Great care must beusedtoavoidcontamination. A goodruleistodresslatticeglovesrinsed in distilledwater, anddried out.

18. The pulverized samples can be preserved for a long time in small envelepes of Whatman paper closed in petri dishes or in teflon tubes until use. Before analysis, they are dried out at low temperatures (40°C, although a thermal treatment at 120o does not reduce the Hg content!). A small aliquot is used to determine the water content of the material, which generally is below 5-6%.

19. Mineralization of samples The chemical analyis of the samples is wrongly considered by biologists the less important source of noise in studies of this type. On the contrary, this phase is very tricky, and several improvements have been suggested by analysts in the new protocol. Basically, the question concerns the mineralization of samples.

20. c. 200 mg of each sample are mineralized in a teflon tube at 150 bar, 120 oC for 8 hours with the addition of a mineralizing solution. In each series of tubes (generally 9 or 12, forming the “BOMB”), one is the blank: only the reagents are added, to check the quality of the reagents, and the “memory effect” of teflon. A second blank can contain a certifiedreference material, possibly of the same nature of the material under study. the “BOMB” After digestion, the material is left to cool, and bidistilled water added up to 10 ml. The solution is ready for the analysis.

21. The reference materials are of different provenence: they are provided by international agencies, are expensive, and are the result of a joint effort of several high-standard laboratories that analysed the same materials with different techniques and compared their analytical results. The reference materials are important in order to reduce the «matrix disturbance noise». For this reason it is important to select reference materials that correspond to the typology of material under analysis. The most popular among ecotoxicologists working with plants are «citrus leaves», «tomatoes leaves», «red cedar leaves». For cryptogams, reference materials are available both for lichens (CRM 482, Pseudevernia furfuracea), and mosses (two materials, not marketed).

23. For preparation of the multielement lichen reference material, 25 kg of the epiphytic lichen Pseudevernia furfuracea (L.) Zopf was collected in 20 randomly chosen remote areas of Switzerland. • The material was ground to a mesh size ≤125 μm. The material homogeneity was evaluated by determining Na, K, Mn, Br, As, La and Sm on two samples of 100 mg taken at each of 20 locations of the bulk material. • The material is homogeneous for fractions ≥100 mg, as shown by the evaluation of the heterogeneity of the sub-sampling operation in the 20 locations and by the results of a one-way analysis of variance. • The composition varies neither with the region where it grows nor with the date of collection.

26. Risultati di analisi di materiale certificato in fase di calibrazione strumentale (A) e durante analisi routinarie (B) in confronto ai valori certificati del materiale CRM 482 (C), e rispettivi valori percentuali di recupero (D, per A; E, per B). *: elemento certificato; +: elemento dichiarato. Tretiach et al. (2014) Biomonitoraggio di elementi in traccia mediante licheni nel territorio di Monfalcone. http://www.arpa.fvg.it/cms/tema/aria/stato/biomonitoraggio/licheni/Studio-licheni-centrale-a2a.html

27. Until now, most authors used as mineralization agent(s) a mixture of HNO3 , H2O2and/or other acids different from HF (obtaining the so-called „partial digestion“). HNO3 HNO3-H2O2 HNO3-HCl HNO3-H2O2-HCl HNO3-HClO4-HCl HNO3-HClO4 HNO3-HClO4-H2SO4

28. This procedure was criticized because the recovery (quantity of the element solubilized with respect to the total present in the material) is rather variable, depending on: • the element concerned; • the minerals involved (e.g. soil versus anthropogenic particulate); • the conditions of mineralization.

30. Some anthropogenic particulates (es. combustion dusts) are not mineralized in a satisfactory way by „partial digestion“, and the same is true for many soil particles, particularly those rich in silicate (SiO2) minerals. Furthermore, the „partial digestion“ suffers for small, apparently negligible changes in the process, and therefore it negatively influences both  repeatability (= I obtain the same result if I repeat the work that I did, using the same instruments);  reproducibility (=other people obtain the same result if they repeat the work according to my protocol, using their own instruments) (Bettinelli et al., 2002).

31. HNO3 HNO3 + H2O2 Al 68% [1] 7411% [2] 54% [3] Cr 75% [1] 53% [3] Ti* 7515% [2] 48% [3] Recoveryby„partial digestion“ is not problematicforhighly soluble metals (Cd, Cu, As, Mn, Pb, Se, andZn), but itiscriticalforsomeelementsusedtocalculatethe„enrichmentfactor“, veryusefultoestimatesoilcontamination (seeinfra). Per cent recovery of selected metals from references materials. [1] SRM 1672 Citrus leaves (Bargagli, 1998) [2] CRM 482 Pseudevernia furfuracea (Minganti et al., 2003) [3] CRM 482 Pseudevernia furfuracea (Bettinelli et al., 2002) * CRM 482 Pseudevernia furfuracea, problematic

32. The alternative solution is to use a mixture which contains the strongest inorganic acid available, HF: HNO3-HF HNO3-HCl-HF HNO3 + H2O2 + HF

33. It is potentially dangerous to work with HF. You need many precautions against fortuitos accidents: not many labs are properly equipped. Finally, if HFl will be adopted, comparison with old data will be very difficult, if not impossible.

34. Element (method of analysis) HNO3 + H2O2 HNO3+ H2O2+HF Al(ICP-OES) 4532  505 7378 682 Cr(ICP-OES) 6,35  1,20 9,15  0,82 Ti(ICP-OES) 68,7  13,7 412 19 V(ICP-OES) 10,4  0,7 14,1 0,5 +163% +144% +600% +136% Mean (± S.D.) values of metal content in thalli of Pseudevernia furfuracea collecting in a mining area of Southern Sardinia (Bettinelli et al. 2002).

35. The Enrichment Factors (EF) with respect to crustal composition or to the composition of surface soils of the study area have often been used to estimate anthropogenic contributions to the elemental composition of lichens and mosses. The general formula is: [x]/[reference element] in lichen/moss EF= [x]/[reference element] in soil Where X is the element under investigation, and the reference element is selected among those that characterize soils, typically aluminium or titanium.

36. ([x]/[reference element] in biological sample) E.F. = ([x]/[reference element] in soil) E.F.  1 arricchimento di origine non terrigena E.F. ˂ 1 arricchimento di origine terrigena.

37. 1 2 3 4 5 6 7 8

39. Chromium Valori medi del cromo standardizzati rispetto al massimo relativo riscontrato nell'area di studio nei licheni Flavoparmeliacaperatae Xanthoriaparietina(Figura a) e gli stessi corretti in base al fattore di arricchimento per l'Alluminio (Figura b) per le tre principali tipologie di suolo (vedi slide precedente).

40. Mercury Valori medi del mercurio standardizzati rispetto al massimo relativo riscontrato nell'area di studio nei licheni Flavoparmeliacaperatae Xanthoriaparietina(Figura a) e gli stessi corretti in base al fattore di arricchimento per l'Alluminio (Figura b) per le tre principali tipologie di suolo (vedi slide precedente).