Study sites and sampling
Riga, Latvia (Fig. 1) is located within the boreo-nemoral climate zone
(56.9489° N, 24.1064° E) and its green infrastructures amounts to 8% of
downtown percentage area (Nikodemus et al., 2003). During winter, most
precipitations (126 mm) fall in the form of snow and, given the 90 days
per year with freezing temperatures, mostly cheap NaCl salts are
extensively used for deicing the street pavement (Table 1). Soils in
Riga’s downtown have evolved from Baltic Ice Lake sandy deposits and
show a primarily sandy texture (>85% sand, 1-12% silt,
<5% clay). Those supporting street lines of trees are weakly
structured, compacted, highly heterogeneous with low to medium
proportions of anthropogenic artefacts and they show a low biological
activity with a maximum of 6.5% organic matter in the topsoil (Bouraoui
et al., 2019), overall typical for anthroposoils (IUSS Working Group
WRB, 2014). In addition, salt sludge splashing and salt-contaminated
snow heap melting at the foot of trees cause salt pollution more than 20
times higher than away from pavement, especially by the end of winter
(Cekstere et al., 2008, Cekstere & Osvalde, 2013). Given the high
heterogeneity of latter contamination in the soils of Riga’s downtown,
even within the same street section, peak Na/Cl concentrations in March
can thus range between 132-1568/26-745 mg kg-1,
decreasing then during the vegetation season and away from pavement. Air
pollution forms another environmental issue in Riga’s downtown, with
still increasing levels of
NO2/NO/O3/CO/PM10
(Anonymous, 2014).
For studying salt pollution and its effects in Riga’s street greenery,
seven street sites with salt contamination levels in foliage
representative of the full range of Na and Cl concentrations measured at
downtown roadside sites were selected (Cekstere & Osvalde, 2013). Two
sites showed slight (about 900 mg kg-1), two others
medium (about 4500 mg kg-1) and the last three severe
(about 9000 mg kg-1) salt contamination (Fig. 1).
Additionally, an eighth site in a National Botanical Garden (NBG) 20 km
southeast of Riga was added to the sampling list (uncontaminated site).
The selected street sites were planted with 5 to 20 Tilia x
vulgaris trees, 9.4 + 0.2 m high (range: 8.1 – 11.2 m) and
around 100-year-old (approximate age range: 90-110; NBG site: 18.7+ 0.4 m high trees, range: 18.0 – 19.1 m, older than 110 years).
At each site, three trees with similar crown condition were randomly
selected. By the end of vegetation season, soil and foliage material
were collected with a view to characterize the 1) nutrient and salt
contamination spectrum, 2) foliar allocation of salt contaminants and 3)
structural responses in foliage.
For the analysis of soil chemistry, three 0.3 L soil cores were
extracted on September 12 2014 from the topsoil (0-35cm) at 0.5-1.5 m
distance from the stem of each selected roadside and NBG tree and
thoroughly mixed prior to transfer to the laboratory. For the chemical
and structural assessments in foliage, one unshaded branch per street or
NBG tree, about 50 cm long, with around 60-70 leaves and located at 3-5
m high in the lower part of crown canopy, was pole-pruned on September
16, 2014. The average percentage area of necrosis per leaf was estimated
visually in the field and the scores attributed to one out of 6 leaf
injury classes (asymptomatic: 0%, starting injury: 1-5%, moderate
injury: 6-15%, intermediate injury: 16-30%, severe injury; 31-50%,
very severe injury: 51-100%). About 50 leaves were gathered for
chemical analysis whereas disks 10 mm in diameter were excised from
green tissues next to the leaf rim and apart main central vein in the
2nd or 3rd leaf from branch apex,
with a view to microscopy assessments and given the location of salt
injury (Fig. 2). These samples were immediately fixed using 2.5% EM- or
LM-grade glutaraldehyde, buffered at pH 7.0 with 0.067 M Soerensen
phosphate buffer. Back to the laboratory, they were fully evacuated
prior to storage in renewed fixing solution at 4 °C, waiting for further
processing.
With a view to the microlocalisation of salt contaminants within leaf
tissues and cells, the remaining branch material was stored in a cooler
and transferred on the same day to the Centre for Microscopy and Image
Analysis (ZMB) of the University of Zurich, Switzerland. On the
following two days, 2 mm disk samples excised at the aforementioned leaf
center and rim location (Fig. 2) were fixed by means of high-pressure
freezing (HPF) in liquid nitrogen (LN2) using a LEICA EM HPM 100 (Leica
Microsystems), after 10 min evacuation in 1-Hexadecene 92% and
insertion in 3 mm Al sample carrier (0.2 mm recess). The fixed leaf
samples were stored in LN2 until further processing.