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Urease activity in cultural layers at archaeological sites
Accepted Manuscript Urease activity in cultural layers at archaeological sites Elena V. Chernysheva, Dmitry S. Korobov, Tatiana E. Khomutova, Alexander V. Borisov PII:
S0305-4403(15)00039-4
DOI:
10.1016/j.jas.2015.01.022
Reference:
YJASC 4331
To appear in:
Journal of Archaeological Science
Received Date: 4 April 2014 Revised Date:
28 January 2015
Accepted Date: 30 January 2015
Please cite this article as: Chernysheva, E.V., Korobov, D.S., Khomutova, T.E., Borisov, A.V., Urease activity in cultural layers at archaeological sites, Journal of Archaeological Science (2015), doi: 10.1016/ j.jas.2015.01.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Urease activity in cultural layers at archaeological sites
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Elena V. Chernysheva a, ∗, Dmitry S. Korobov b, Tatiana E. Khomutova a, Alexander V.
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Borisov a
4 5 a
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Institute of Physicochemical and Biological Problems in Soil Science Russian Academy of b
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Institute of Archeology Russian Academy of Sciences, Dm. Ulyanov st., 19, 117036, Moscow, Russia
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Abstract
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Sciences, Institutskaya st.,2, 142290, Pushchino, Moscow region, Russia
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Urease activity in soils and cultural layers at medieval settlements located within the
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Kislovodsk basin (Northern Caucasus, Russia) was studied to reveal the sites of cattle keeping
15
and the areas of ancient manured lands. Input of various organic materials increases a microbial
16
biomass and enzymatic activity in soils. In particular, urease activity increases in soils with long-
17
term amendment of manure, compost, and other organic residues due to the improvement of soil
18
fertility and income of ureolytic bacteria together with organic fertilizers. Soil urease activity
19
was estimated in two different zones within Alanic settlement (AD 200–400). In cultural layers
20
of the zone with ruined walls remains urease activity was almost twice higher than in those of the
21
second zone without walls remains. The results demonstrated that buildings of the settlements
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were used as cattle pens. In the vicinity of other Alanic settlements (AD 500–800), urease
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activity decreased with distance from settlements. Comparison of urease activity, pottery
24
scattering, and soil phosphorus content made it possible to mark the boundaries of ancient
25
manured lands. The parameter of soil urease activity may be useful in revealing the infrastructure
26
of settlements, sites of cattle keeping, and areas of ancient arable lands.
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Keywords: anthropogenic soils, manuring, urease activity, Northern Caucasus, Middle
Ages
Introduction Human
33 34
activity
essentially
influences
many
soil
properties.
Most
significant
transformations occur under residential land use and agricultural management of the territory. ∗
Corresponding author. Institute of Physicochemical and Biological Problems in Soil Science Russian Academy of Sciences, Institutskaya st., 2, 142290, Pushchino, Moscow region, Russia. +7 916 6685465 E-mail address: [email protected]
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Human activity is accompanied by the input of specific organic materials into the soil. These
2
organic materials are subsequently lost due to their complete mineralization by soil
3
microorganisms. It is worth noting that despite the organic substances had been mineralized, the
4
information about their income is stored in the changes of soil microbial communities and
5
enzymatic activity; and these changes can be preserved up to now. High input of various organic
6
materials usually stimulates microbial activity, which leads to improvement of soil properties
7
and increase in microbial biomass and enzymatic activity (Klose, Tabatabai, 2000; Dodor,
8
Tabatabai, 2003; Marschner et al., 2003). A reliable increase in activity of urease, phosphatase,
9
β-glucosidase, and other enzymes has been shown following the amendment of arable soils with
10
manure and composts (Dick, 1992; Bandick, Dick, 1999; Antonius, 2003; Bol et al., 2003;
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Bohme et al., 2005). Increase in urease activity may occur due to the income of ureolytic
12
microorganisms together with manure (Gianfreda, Ruggiero, 2006).
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Changes in enzymatic pool in soil are preserved for an indefinitely long period of time due
14
to association of enzymes with clay minerals and soil organic matter (Burns et al., 2013).
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Enzymes bound to soil colloids are more sustainable to denaturation and proteolysis and may be
16
active even under conditions that limit microbial activity, and are not regulated by repressive
17
growth factors (Nannipieri et al., 2002). Urease and phosphatase activity was registered in
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several 9,000 year-old permafrost soils, phosphatase activity was observed in all layers of 13,000
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year-old lacustrine deposits (Skijins, 1976). Phosphatase activity was also observed in soils
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buried beneath kurgans in steppe zone over 4,500 years ago (Khomutova et al., 2012).
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Elevated levels of enzymes involved in the decomposition of certain organic materials can
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testify previous input of those materials into the soil. This approach seems very promising for
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studying cultural layers and ancient fields. To date only some initial steps in applying soil
24
microbiological assay to the study of soils and cultural layers in archaeological sites have been
25
taken. There are some studies on fungal microbiota of ancient cultural layers in medieval
26
settlements (Ivanova et al., 2006; Marfenina et al., 2001; Marfenina et al., 2008a; Marfenina et
27
al., 2008b). The microfungal communities in the cultural layers of medieval settlements were
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shown to differ in their composition and structure from undisturbed soils.
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The influence of ancient agriculture on soil microbiological community is much less
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studied. We can cite only a few publications in this field. Investigation of the microbiological
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properties of soils cultivated in cloisters in the 16th and 17th centuries were carried out, and
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increased biological activity in the buried arable layer, as well as changes in the taxonomic
33
structure of bacterial communities under soil cultivation were noted (Lysak et al., 2004;
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Novikov, Stepanov, 2000). It was demonstrated that even 400 years after organic fertilization,
35
increased amidase activity was registered in abandoned agricultural soil (Frankenberger, Dick,
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1983). In regard to studies of more ancient agriculture impact on soil, the only work we found
2
(Dick et al., 1994) showed that the soil in an ancient agricultural terrace (about 1500 years old)
3
had retained a high level of phosphatase and amidase activity, which exceeded the respective
4
levels in presently cultivated and uncultivated soils. It was recently shown that urease activity could be used to reveal the infrastructure of
6
ancient settlements (Borisov et al., 2013). This approach was based on the hypothesis that urea is
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one of the key organic substances entering soils of archaeological sites in large amounts. This
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concerns soils within settlements especially those associated with animals keeping, inner
9
habitation areas, and arable plots amended by organic fertilizers and plant residues.
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The hydrolysis of urea occurs under the urease enzyme produced by a number of soil
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microorganisms and, initially, by ureolytic bacteria (Paulson, Kurtz, 1970). We suppose that
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long-term habitation and animal keeping in ancient and medieval settlements resulted to increase
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in soil urease activity. Beyond settlement areas, a high level of urease activity may be associated
14
with manuring of ancient arable lands. Reconstruction of such agricultural techniques is the
15
subject of many studies. Attempts to reveal the manuring of ancient arable plots have been
16
undertaken using various methods such as estimating the levels of total phosphorus, δ13C, trace
17
elements and free soil lipids, and analysis of soil micromorphology (e.g. Bull et al., 1999a; Bull
18
et al., 1999b; Davidson, Carter, 1998; Davidson, 2002; Entwistle et al., 1998; Hjustrom B.,
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Isaksson S., 2009; Leonardi, 1999; Simpson, 1997; Simpson et al., 1999; Wilson et al., 2008).
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However, the level of urease activity as an indicator of manuring was not applied.
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Study area and site description
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manuring.
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In this study we used urease activity in soils as indicator of cattle keeping and ancient
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The study area is located within the Kislovodsk basin in the central part of the northern
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slope of the Great Caucasus Mountain Ridge (Fig.1). The territory of the basin is bordered by the
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Borgustansky Ridge (height up to 2000 m) to the north and the Dzhinalsky Ridge (1541 m) to
29
the east. It is composed of chalky limestones of the Late Cretaceous period. To the south and
30
south-east the territory of the basin is bordered by the cuestas of the Skalisty (Rocky) Ridge – the
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Kabardinsky Ridge (1526 m) and the Bermamyt Plateau – composed of calcareous sandstones of
32
the Early Cretaceous (Milanovsky, 1968).
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The climate of the region is temperate continental. The annual level of precipitation is about
34
600 mm and the average annual temperature is +8°C; the sum temperature above 10°C is 2400–
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2600° (Agroclimatic ..., 1971).
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Archaeological observations in the Kislovodsk basin have a long history, briefly described
2
by Korobov and Reinhold (Reinhold, Korobov, 2007). Our investigations of urease activity were
3
part of a research project on the terraced agriculture that was widespread in the region during
4
prehistoric and early medieval times (Korobov, 2012; Korobov, Borisov, 2013). For this
5
purpose, several settlements of the Alanic culture dating back to AD 200–400 and 500–800 were
6
chosen as key sites.
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Alanic settlement Podkumskoe-2
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The settlement of Early Alanic culture (AD 200–400) is located to the north of Kislovodsk
11
town at the foot of the Borgustansky Ridge (43º 57´ 06´´ N, 42º 36´ 22´´ E) on the first terrace of
12
the left bank of the Podkumok River, and occupies a cape area of general size 140 × 165 m. The
13
settlement area is uneven and covered by ruins, which can be traced on the surface by a number
14
of dishes and separate grassy hills from the ruined walls remains of buildings (Fig. 2A; Fig. 4).
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About one third of the settlement to the south-west has no visible surface traces of constructions.
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The soil cover around the settlement is composed by thin and medium deep-leached and
17
typical soddy calcareous soils (Rendzina) developed from the eluvium of limestone. At the
18
settlement the cultural layer is up to 90 cm in depth and is loamy, structureless, and ash grey,
19
enriched by pottery and bones (Fig. 2B).
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At the settlement Podkumskoe-2, soil properties were investigated in sections B-338 (first
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zone with ruined walls remains) and B-341 (second zone without ruined walls remains). In
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addition, a series of soil sections in the inner parts of buildings were examined.
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Background soil samples were taken in the area bounded by deep gully with steep slopes that excludes anthropogenic impact (Fig. 2C).
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Alanic settlements Podkumskoe-3 and Podkumskoe-7
Alanic settlement Podkumskoe-3 is located at the north-eastern part of the cape (43º 54´ 50´´
29
N, 42º 23´ 46´´ E) (Fig. 3A). To the south-west of the settlement there is a large area potentially
30
suitable for agriculture with a slope of about 3–5º. About 1000 m from the settlement the slope
31
increases to 10º and peaks at 2000–2500 m on the watershed. At the southern cape the fields of
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another Alanic settlement Podkumskoe-7 (43º 54´ 20´´ N, 42º 23´ 16´´ E) were located. Both
33
settlements occupy two rocky sites on the left bank of the Podkumok River. On the upper site of
34
settlements the ruins of stone towers, several houses, and walls are preserved.
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To study the changes of soil properties with distance from the sites Podkumskoe-3 and
2
Podkumskoe-7 several soil sections were analyzed. In the soil cover mountain chernozems
3
prevail. These are dark-coloured soils under meadow vegetation, very fertile and rich in soil
4
organic matter. In all sections considerable amounts of Alanic pottery were found (Fig. 3B-C). At the sites sampled the slope does not exceed 5º, and thick vegetation cover prevents
6
erosion of the soils. A reliable marker for the absence of clear erosion processes in this case is
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the pattern of distribution of pottery fragments within the soil profile. When the main mass of
8
fragments is located in the upper 0–15 (20) cm (sections B-345, B-344, B-353, B-354, B-355),
9
we believe that the erosion/accumulation processes does not occur. When the pottery is
10
concentrated in the 20–40 cm layer, ancient arable soils are covered by slope deposits (sections
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B-346, B-350, B-357, B-356, B-358).
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It is worth noting that all settlements studied had the one period of occupation, dating back
13
to AD 200–400 for the Podkumskoe-2 and AD 500–800 for the Podkumskoe-3 and
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Podkumskoe-7. Neither before nor after those periods was there any anthropogenic activity that
15
influenced soil properties, which facilitated the exceptional preservation of archaeological sites
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and therefore all anthropogenic transformations of soil properties are connected to the influence
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of the activities of medieval population.
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Methods
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To evaluate the effect of ancient anthropogenic impact on urease activity, samples were
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collected from A and C soil horizons (sections B-338, B-342, B-344, B-345, B-354, B-355, B-
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357) or from each 10 cm layers (sections B-341, B-346, B-352, B-353, B-356, B-358 and small
24
sections b-2, b-7, b-9, b-10) in representative square sections (1 m × 1 m). The depth of the
25
sections varied from 15 to 80 cm. Samples were taken in sterile plastic bags to exclude microbial
26
contamination. Sample preparation included air-drying, removal of large roots, and sieving (< 3
27
mm). Soils and cultural layers were sampled in mid-autumn 2012.
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Estimation of urease activity was carried out by the colorimetric method (Kandeler, Gerber,
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1988). Briefly the procedure was as follows: 1 g of soil was placed into a 50 ml flask and wetted
30
with 1.5 ml of aqueous urea solution (0.08M) and 5 ml borate buffer (0.05M Na2B4O7 x 10 H2O
31
and 0.2 M NaOH, pH 10). The flasks were sealed, placed at 37°C for 2 h, then 15 ml of 2M
32
NaCl were added. The resulting suspensions were filtered and the filtrates were analyzed for
33
ammonia content. For this, 1 ml of the filtrate was diluted to 10 ml with distilled water, then 5 ml
34
of sodium salicylate and 2 ml of 0.1% sodium dichlorisocyanurate were added. The sodium
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salicylate solution was prepared by mixing 100 ml of 0.12% aqueous sodium nitroprusside, 100
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ml of 17% aqueous sodium salicylate, and 100 ml of distilled water. After 30 min incubation at
2
room temperature the optical density was read at 690 nm. All values of urease activity are
3
reported on a dry weight basis. A total of 72 samples were analyzed.
4
The
bulk
content
of
phosphates
was
determined
with
an
X-ray fluorescent
spectrophotometer. This method is widely used in archaeology as an indicator of ancient
6
manuring (Nielsen, Kristiansen, 2014). The analyses were performed for Podkumskoe-3
7
(sections B-344, B-346, and B-352) and Podkumskoe-7 (sections B-356 and B-357).
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Results and discussion
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Alanic settlement Podkumskoe-2
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The results of estimation of urease activity at Podkumskoe-2 are shown in Fig. 4. In general,
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urease activity in cultural layer of the settlement was higher than in background soddy
15
calcareous soil in the area adjacent to the settlement. In the upper horizon of the latter it varied
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from 119 to 389 µg ammonium (NH4+) g–1 soil h–1. In the cultural layer of the first zone with
17
ruined walls remains (section B-338), in 0–10 cm layer urease activity did not exceed 305 µg
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NH4+ g–1 soil h–1; however, in deeper layers the level of urease activity was 466 µg NH4+ g–1 soil
19
h–1. Similar pattern of urease activity was observed in the cultural layer in small section b-10. In
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the upper cultural layer of the second zone without ruined walls remains (section B-341), higher
21
values of urease activity were observed, but further down the soil profile they significantly
22
decreased from 207 to 37 µg NH4+ g–1 soil h–1. Maximal values of urease activity in the upper
23
layer were found in small sections 9 and 7, where they reached 389 and 377 µg NH4+ g–1 soil h–1,
24
respectively. It is notable that in all soil sections at the settlement, urease activity in layers 10–20
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(20–40) cm exceeded that of the upper horizon of the background soil.
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The high level of urease activity in soils of the first zone (with ruined walls remains) most
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likely reflects the use of buildings for cattle keeping. The second zone (without wall remains) is
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probably habitable area that is supported by thick cultural layer, abundance of pottery and bones,
29
and presence of deep household pits.
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Alanic settlement Podkumskoe-3
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To study the changes in urease activity in soils outside archaeological sites, a set of soil
34
sections located at varying distances from Podkumskoe-3 (AD 500–800) was analysed. The
35
following soil sections were investigated: B-345 was located at 60 m, B-344 at 120 m, B-346 at
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250 m, B-350 at 600 m and B-353 at 1200 m from the break-ups of external fortification wall; B-
2
352 was at the watershed plot about 2 km from the site. At sections B-345 and B-350 sizeable
3
amounts of pottery fragments from Alanic time were found. In our study we deal with so called
4
“manure” pottery. This is small fragments up to 3–4 cm, because large fragments are cracked
5
during the plowing and management of soil. The analysis of pottery scattering is often applied in
6
archaeology to reveal the boundaries of ancient agricultural plots (Wilkinson, 1982). The
7
fragments of pottery had entered the soil with manure and wastes that allowed us to consider this
8
territory as fertilized arable lands. An adjacent area to the south was probably also under
9
ploughing, but without manuring, because here only a few pottery fragments were found (section
10
B-353). The plot most distant from the settlement (2000 m) was most probably not subjected to
11
anthropogenic impact.
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The results of estimation of urease activity are shown in Fig. 5 and Fig. 6. Urease activity in
13
soils decreased with distance from the settlement. The highest urease activity was observed in
14
the most vicinal soil at 60 m from the settlement: in the upper horizon it was 403 µg NH4+ g–1
15
soil h–1 and decreased almost twice in the lower horizon. With distance from settlement a sharp
16
decrease in soil urease activity in the upper horizon was observed: down to 143 µg NH4+ g–1 soil
17
h–1 at 120 m, and to 67 µg NH4+ g–1 soil h–1 at 250 m. In the zone of irregular manuring during
18
Alanic time (section B-353), urease activity down the soil profile decreased from 43 to 5 µg
19
NH4+ g–1 soil h–1. Minimal activity was determined in the soil at the top of the watershed at 2000
20
m distance from settlement; in the upper horizon it did not exceed 22 µg NH4+ g–1 soil h–1.
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The soil phosphorus content also decreased along the transect, from 0.51–0.42% at 60 m from the settlement to 0.18–0.11% in the background soil on the watershed (Fig. 7). We carried out the correlation analysis of pottery scattering and urease activity (Fig. 6),
24
proposing that the pottery fragments had been introduced into the soil together with manure. To
25
confirm this, the total number of pottery fragments per square metre was calculated and
26
compared to the mean values of urease activity in the soil profile. A strong positive correlation (r
27
= 0.90, P = 0.05) was revealed. Comparison of the data on urease activity, pottery fragment
28
content, and, partly, phosphorus content allowed us to make reliable conclusions on the proposed
29
boundaries of agricultural lands during Alanic time. The boundary of manuring zone was located
30
at 500 m from the settlement, and the boundary of zone with irregular manuring at 1000 m. The
31
plot most probably untouched by anthropogenic activity was located at about 2000 m.
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Alanic settlement Podkumskoe-7
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At Podkumskoe-7 a set of soil sections located at varying distances from the site was
2
studied: B-357 at 60 m, B-354 at 150 m, B-355 at 180 m, B-356 at 500 m, and B-358 at 700 m.
3
Similar to Podkumskoe-3, the urease activity decreased with distance from settlement (Fig. 5;
4
Fig. 6). Highest level of urease activity was observed in the nearest to the site soils: 94 and 119
5
µg NH4+ g–1 soil h–1 at sections B-357 and B-354, respectively. Along the transect, a sharp
6
decrease in urease activity down to 27 µg NH4+ g–1 soil h–1 was observed in the layer 2–15 cm.
7
In the soil located at 500 m from the settlement, urease activity increased again to 349 µg NH4+
8
g–1 soil h–1 and in subsurface layers this parameter decreased from 91 to 21 µg NH4+ g–1 soil h–1.
9
Elevated values of urease activity at this area evidently are connected with erosion processes. In
10
particular, at section B-356 pottery scattering indicates accumulation of erosion material. In soil
11
at 700 m from the settlement, minimal urease activity was found: in the upper layer it did not
12
exceed 45 µg NH4+ g–1 soil h–1. Besides, in the profile of this soil we observed an increase of
13
activity at 50–60 cm depth.
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The content of phosphates decreased from 0.50 to 0.43% in the soil nearest to the settlement, and to 0.33–0.21% in the most distant one (Fig. 7).
In soils adjacent to Podkumskoe-7 we recorded lot of pottery fragments, and observed the
17
strong correlation (r = 0.92, P = 0.05) between urease activity and numbers of fragments (Fig. 6).
18
At Podkumskoe-7 the boundary of the manured zone was located at 500 m from the
19
settlement similar to that at Podkumskoe-3. However, marking of the boundaries of arable lands
20
here is more complicated due to erosion process.
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Conclusions
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Anthropogenic impact on the soils of archaeological sites associated with human habitation,
25
cattle keeping, and agricultural activity resulted in changes soil urease activity that have been
26
preserved to present day.
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The level of urease activity is considered as a novel feature of cultural layers and soils of
28
ancient manured lands and may be applied for their characterization. We believe that increased
29
values of urease activity in certain parts of a settlement may indicate the location of cattle pens.
30
High urease activity in soils within large areas around ancient settlements is associated with
31
additional income of urea via manure fertilization of arable plots. This is confirmed by the close
32
relationship between the level of urease activity and the number of pottery fragments in soils.
33
To date, archaeologists had no readily available and simple way to reveal the locations of
34
cattle keeping at archaeological sites. Such locations were conditionally determined by specific
35
form and size of buildings as well as peculiarities of artifacts distribution in soils. In other case
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fixation of cattle pens was impossible. In our work we offer simple and effective method for
2
such fixation. It may be also valuable for revealing the infrastructure of settlements and
3
determination of residential and cattle keeping zones. The application of the method is promising
4
for indication ancient manured lands around settlements.
5 6
Acknowledgments
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The work was supported by Russian Foundation for Basic Research (projects No 12-06-
9
00272; 14-06-00200) and the Program for Fundamental Research of the Presidium of Russian
10
Academy of Sciences.
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Eurasian Soil Sci. 39 (1), 53–62.
Kandeler E, Gerber H., 1988. Short-term assay of urease activity using colorimetric determination of ammonium. Biol. Fertil. Soils 6 (1), 68–72.
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Khomutova T.E., Demkina T.S., Kashirskaya N.N., Demkin V.A., 2012. Phosphatase activity in
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the surface and buried chestnut soils of the Volga-Don interfluve. Eurasian Soil Sci. 45 (4),
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423–429.
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Klose S., Tabatabai M.A., 2000. Urease activity of microbial biomass in soils as affected by cropping systems. Biol. Fertil. Soils 31, 191–199.
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in Cultivated Soils of Monasteries in the Taiga Zone. Eurasian Soil Sci. 37 (8), 853–863.
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Marfenina O.E., Gorbatovskaya E.V., Gorlenko M.V., 2001 Mycological Characterization of the
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Occupation Deposits in Excavated Medieval Russian Settlements. Microbiology 70 (6),
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738–743.
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Marfenina O.E., Ivanova A.E., Kislova E.E., Sacharov D.S., 2008. The mycological properties of
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medieval culture layers as a form of soil ‘biological memory’ about urbanization. J. Soils
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Sediments 8, 340–348.
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Marfenina O.E., Ivanova A.E., Kislova E.E., Zazovskaya E.P., Chernov I.Y., 2008. Fungal
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Communities in the Soils of Early Medieval Settlements in the Taiga Zone. Eurasian Soil
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Sci. 41 (7), 749–759.
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Marschner P., Kandeler E., Marschner B., 2003. Structure and function of the soil microbial community in a long-term fertilizer experiment. Soil Biol. Biochem. 35, 453–461.
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Milanovsky E.E. 1968. Newest tectonics of the Caucasus. Nedra Publishing House, Moscow.
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Nannipieri P., Kandeler E., Ruggiero P. 2002. Enzyme activities and microbiological and
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biochemical processes in soil. In: Burns R.G., Dick R.P. (Ed.). Enzymes in the environment.
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Activity, ecology and applications. New York, pp. 1–33.
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Nielsen N.H., Kristiansen S.M., 2014. Identifying ancient manuring: traditional phosphate vs. multi-element analysis of archaeological soil. J. Archaeol. Sci. 42, 390–398.
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Paulson K.N., Kurtz L.T., 1970. Michaelis constant of soil urease. Soil Sci. Soc. Am. J. 34 (1), 70–72.
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Reinhold S., Korobov D., 2007. The Kislovodsk basin in the North Caucasian piedmonts –
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archaeology and GIS studies in a mountain cultural landscape. Preistoria Alpina 42, 183–
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Simpson I.A., 1997. Relict Properties of Anthropogenic Deep Top Soils as Indicators of Infield Management in Marwick, West Mainland, Orkney. J. Archaeol. Sci. 24, 365–380.
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Simpson I.A., van Bergen P.F., Perret V., Elhmmali M.M., Roberts D.J., Evershed R.P., 1999
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Lipid biomarkers of manuring practice in relict anthropogenic soils. The Holocene 9 (2),
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223–229.
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Skujins J.J., 1976. Extracellular enzymes in soil. Crit. Rev. Microbiol. 4, 383–421.
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Wilkinson T.J., 1982. The definition of ancient manured zones by means of extensive sherd-
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sampling techniques. J. Field Archaeol. 9, 323–333. Wilson C.A., Davidson D.A., Cresser M.S., 2008. Multi-element soil analysis: an assessment of
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its potential as an aid to archaeological interpretation. J. Archaeol. Sci. 35 (2), 412–424.
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Fig. 1. Study area location.
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Fig. 2. Alanic settlement Podkumskoe-2 (A); cultural layer – section B-341 (В), background
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walls).
Fig. 5. Urease activity at the sites Podkumskoe-3 and Podkumskoe-7 (A – settlement, B – section).
Fig. 6. The amounts of pottery fragments and the urease activity in soils along the transect at the sites Podkumskoe-3 (A) and Podkumskoe-7 (B).
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Fig. 4. Urease activity at the site Podkumskoe-2 (A – section, B – small section, C – ruined
Fig. 7. Phosphorus content at the sites Podkumskoe-3 (A) and Podkumskoe-7 (B).
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Podkumskoe-3 - B-346 (B) and B-344 (C).
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Fig. 3. Alanic settlements Podkumskoe-3 and Podkumskoe-7 (A); soil sections at the site
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soil – section B-342 (C).
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Anthropogenic impact on soil results in an increase in soils urease activity. Elevated level of urease activity have been preserved to present day. High level of urease activity was observed in soils of ancient cattle pens. Urease activity may be used as diagnostic feature of ancient manured lands.
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S0305-4403(15)00039-4
DOI:
10.1016/j.jas.2015.01.022
Reference:
YJASC 4331
To appear in:
Journal of Archaeological Science
Received Date: 4 April 2014 Revised Date:
28 January 2015
Accepted Date: 30 January 2015
Please cite this article as: Chernysheva, E.V., Korobov, D.S., Khomutova, T.E., Borisov, A.V., Urease activity in cultural layers at archaeological sites, Journal of Archaeological Science (2015), doi: 10.1016/ j.jas.2015.01.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Urease activity in cultural layers at archaeological sites
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Elena V. Chernysheva a, ∗, Dmitry S. Korobov b, Tatiana E. Khomutova a, Alexander V.
3
Borisov a
4 5 a
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Institute of Physicochemical and Biological Problems in Soil Science Russian Academy of b
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Institute of Archeology Russian Academy of Sciences, Dm. Ulyanov st., 19, 117036, Moscow, Russia
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Abstract
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Sciences, Institutskaya st.,2, 142290, Pushchino, Moscow region, Russia
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Urease activity in soils and cultural layers at medieval settlements located within the
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Kislovodsk basin (Northern Caucasus, Russia) was studied to reveal the sites of cattle keeping
15
and the areas of ancient manured lands. Input of various organic materials increases a microbial
16
biomass and enzymatic activity in soils. In particular, urease activity increases in soils with long-
17
term amendment of manure, compost, and other organic residues due to the improvement of soil
18
fertility and income of ureolytic bacteria together with organic fertilizers. Soil urease activity
19
was estimated in two different zones within Alanic settlement (AD 200–400). In cultural layers
20
of the zone with ruined walls remains urease activity was almost twice higher than in those of the
21
second zone without walls remains. The results demonstrated that buildings of the settlements
22
were used as cattle pens. In the vicinity of other Alanic settlements (AD 500–800), urease
23
activity decreased with distance from settlements. Comparison of urease activity, pottery
24
scattering, and soil phosphorus content made it possible to mark the boundaries of ancient
25
manured lands. The parameter of soil urease activity may be useful in revealing the infrastructure
26
of settlements, sites of cattle keeping, and areas of ancient arable lands.
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Keywords: anthropogenic soils, manuring, urease activity, Northern Caucasus, Middle
Ages
Introduction Human
33 34
activity
essentially
influences
many
soil
properties.
Most
significant
transformations occur under residential land use and agricultural management of the territory. ∗
Corresponding author. Institute of Physicochemical and Biological Problems in Soil Science Russian Academy of Sciences, Institutskaya st., 2, 142290, Pushchino, Moscow region, Russia. +7 916 6685465 E-mail address: [email protected]
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Human activity is accompanied by the input of specific organic materials into the soil. These
2
organic materials are subsequently lost due to their complete mineralization by soil
3
microorganisms. It is worth noting that despite the organic substances had been mineralized, the
4
information about their income is stored in the changes of soil microbial communities and
5
enzymatic activity; and these changes can be preserved up to now. High input of various organic
6
materials usually stimulates microbial activity, which leads to improvement of soil properties
7
and increase in microbial biomass and enzymatic activity (Klose, Tabatabai, 2000; Dodor,
8
Tabatabai, 2003; Marschner et al., 2003). A reliable increase in activity of urease, phosphatase,
9
β-glucosidase, and other enzymes has been shown following the amendment of arable soils with
10
manure and composts (Dick, 1992; Bandick, Dick, 1999; Antonius, 2003; Bol et al., 2003;
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Bohme et al., 2005). Increase in urease activity may occur due to the income of ureolytic
12
microorganisms together with manure (Gianfreda, Ruggiero, 2006).
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Changes in enzymatic pool in soil are preserved for an indefinitely long period of time due
14
to association of enzymes with clay minerals and soil organic matter (Burns et al., 2013).
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Enzymes bound to soil colloids are more sustainable to denaturation and proteolysis and may be
16
active even under conditions that limit microbial activity, and are not regulated by repressive
17
growth factors (Nannipieri et al., 2002). Urease and phosphatase activity was registered in
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several 9,000 year-old permafrost soils, phosphatase activity was observed in all layers of 13,000
19
year-old lacustrine deposits (Skijins, 1976). Phosphatase activity was also observed in soils
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buried beneath kurgans in steppe zone over 4,500 years ago (Khomutova et al., 2012).
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Elevated levels of enzymes involved in the decomposition of certain organic materials can
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testify previous input of those materials into the soil. This approach seems very promising for
23
studying cultural layers and ancient fields. To date only some initial steps in applying soil
24
microbiological assay to the study of soils and cultural layers in archaeological sites have been
25
taken. There are some studies on fungal microbiota of ancient cultural layers in medieval
26
settlements (Ivanova et al., 2006; Marfenina et al., 2001; Marfenina et al., 2008a; Marfenina et
27
al., 2008b). The microfungal communities in the cultural layers of medieval settlements were
28
shown to differ in their composition and structure from undisturbed soils.
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The influence of ancient agriculture on soil microbiological community is much less
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studied. We can cite only a few publications in this field. Investigation of the microbiological
31
properties of soils cultivated in cloisters in the 16th and 17th centuries were carried out, and
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increased biological activity in the buried arable layer, as well as changes in the taxonomic
33
structure of bacterial communities under soil cultivation were noted (Lysak et al., 2004;
34
Novikov, Stepanov, 2000). It was demonstrated that even 400 years after organic fertilization,
35
increased amidase activity was registered in abandoned agricultural soil (Frankenberger, Dick,
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1983). In regard to studies of more ancient agriculture impact on soil, the only work we found
2
(Dick et al., 1994) showed that the soil in an ancient agricultural terrace (about 1500 years old)
3
had retained a high level of phosphatase and amidase activity, which exceeded the respective
4
levels in presently cultivated and uncultivated soils. It was recently shown that urease activity could be used to reveal the infrastructure of
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ancient settlements (Borisov et al., 2013). This approach was based on the hypothesis that urea is
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one of the key organic substances entering soils of archaeological sites in large amounts. This
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concerns soils within settlements especially those associated with animals keeping, inner
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habitation areas, and arable plots amended by organic fertilizers and plant residues.
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The hydrolysis of urea occurs under the urease enzyme produced by a number of soil
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microorganisms and, initially, by ureolytic bacteria (Paulson, Kurtz, 1970). We suppose that
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long-term habitation and animal keeping in ancient and medieval settlements resulted to increase
13
in soil urease activity. Beyond settlement areas, a high level of urease activity may be associated
14
with manuring of ancient arable lands. Reconstruction of such agricultural techniques is the
15
subject of many studies. Attempts to reveal the manuring of ancient arable plots have been
16
undertaken using various methods such as estimating the levels of total phosphorus, δ13C, trace
17
elements and free soil lipids, and analysis of soil micromorphology (e.g. Bull et al., 1999a; Bull
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et al., 1999b; Davidson, Carter, 1998; Davidson, 2002; Entwistle et al., 1998; Hjustrom B.,
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Isaksson S., 2009; Leonardi, 1999; Simpson, 1997; Simpson et al., 1999; Wilson et al., 2008).
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However, the level of urease activity as an indicator of manuring was not applied.
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Study area and site description
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manuring.
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In this study we used urease activity in soils as indicator of cattle keeping and ancient
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The study area is located within the Kislovodsk basin in the central part of the northern
27
slope of the Great Caucasus Mountain Ridge (Fig.1). The territory of the basin is bordered by the
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Borgustansky Ridge (height up to 2000 m) to the north and the Dzhinalsky Ridge (1541 m) to
29
the east. It is composed of chalky limestones of the Late Cretaceous period. To the south and
30
south-east the territory of the basin is bordered by the cuestas of the Skalisty (Rocky) Ridge – the
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Kabardinsky Ridge (1526 m) and the Bermamyt Plateau – composed of calcareous sandstones of
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the Early Cretaceous (Milanovsky, 1968).
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The climate of the region is temperate continental. The annual level of precipitation is about
34
600 mm and the average annual temperature is +8°C; the sum temperature above 10°C is 2400–
35
2600° (Agroclimatic ..., 1971).
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Archaeological observations in the Kislovodsk basin have a long history, briefly described
2
by Korobov and Reinhold (Reinhold, Korobov, 2007). Our investigations of urease activity were
3
part of a research project on the terraced agriculture that was widespread in the region during
4
prehistoric and early medieval times (Korobov, 2012; Korobov, Borisov, 2013). For this
5
purpose, several settlements of the Alanic culture dating back to AD 200–400 and 500–800 were
6
chosen as key sites.
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Alanic settlement Podkumskoe-2
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The settlement of Early Alanic culture (AD 200–400) is located to the north of Kislovodsk
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town at the foot of the Borgustansky Ridge (43º 57´ 06´´ N, 42º 36´ 22´´ E) on the first terrace of
12
the left bank of the Podkumok River, and occupies a cape area of general size 140 × 165 m. The
13
settlement area is uneven and covered by ruins, which can be traced on the surface by a number
14
of dishes and separate grassy hills from the ruined walls remains of buildings (Fig. 2A; Fig. 4).
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About one third of the settlement to the south-west has no visible surface traces of constructions.
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The soil cover around the settlement is composed by thin and medium deep-leached and
17
typical soddy calcareous soils (Rendzina) developed from the eluvium of limestone. At the
18
settlement the cultural layer is up to 90 cm in depth and is loamy, structureless, and ash grey,
19
enriched by pottery and bones (Fig. 2B).
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At the settlement Podkumskoe-2, soil properties were investigated in sections B-338 (first
21
zone with ruined walls remains) and B-341 (second zone without ruined walls remains). In
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addition, a series of soil sections in the inner parts of buildings were examined.
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Background soil samples were taken in the area bounded by deep gully with steep slopes that excludes anthropogenic impact (Fig. 2C).
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Alanic settlements Podkumskoe-3 and Podkumskoe-7
Alanic settlement Podkumskoe-3 is located at the north-eastern part of the cape (43º 54´ 50´´
29
N, 42º 23´ 46´´ E) (Fig. 3A). To the south-west of the settlement there is a large area potentially
30
suitable for agriculture with a slope of about 3–5º. About 1000 m from the settlement the slope
31
increases to 10º and peaks at 2000–2500 m on the watershed. At the southern cape the fields of
32
another Alanic settlement Podkumskoe-7 (43º 54´ 20´´ N, 42º 23´ 16´´ E) were located. Both
33
settlements occupy two rocky sites on the left bank of the Podkumok River. On the upper site of
34
settlements the ruins of stone towers, several houses, and walls are preserved.
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To study the changes of soil properties with distance from the sites Podkumskoe-3 and
2
Podkumskoe-7 several soil sections were analyzed. In the soil cover mountain chernozems
3
prevail. These are dark-coloured soils under meadow vegetation, very fertile and rich in soil
4
organic matter. In all sections considerable amounts of Alanic pottery were found (Fig. 3B-C). At the sites sampled the slope does not exceed 5º, and thick vegetation cover prevents
6
erosion of the soils. A reliable marker for the absence of clear erosion processes in this case is
7
the pattern of distribution of pottery fragments within the soil profile. When the main mass of
8
fragments is located in the upper 0–15 (20) cm (sections B-345, B-344, B-353, B-354, B-355),
9
we believe that the erosion/accumulation processes does not occur. When the pottery is
10
concentrated in the 20–40 cm layer, ancient arable soils are covered by slope deposits (sections
11
B-346, B-350, B-357, B-356, B-358).
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It is worth noting that all settlements studied had the one period of occupation, dating back
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to AD 200–400 for the Podkumskoe-2 and AD 500–800 for the Podkumskoe-3 and
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Podkumskoe-7. Neither before nor after those periods was there any anthropogenic activity that
15
influenced soil properties, which facilitated the exceptional preservation of archaeological sites
16
and therefore all anthropogenic transformations of soil properties are connected to the influence
17
of the activities of medieval population.
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Methods
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To evaluate the effect of ancient anthropogenic impact on urease activity, samples were
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collected from A and C soil horizons (sections B-338, B-342, B-344, B-345, B-354, B-355, B-
23
357) or from each 10 cm layers (sections B-341, B-346, B-352, B-353, B-356, B-358 and small
24
sections b-2, b-7, b-9, b-10) in representative square sections (1 m × 1 m). The depth of the
25
sections varied from 15 to 80 cm. Samples were taken in sterile plastic bags to exclude microbial
26
contamination. Sample preparation included air-drying, removal of large roots, and sieving (< 3
27
mm). Soils and cultural layers were sampled in mid-autumn 2012.
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Estimation of urease activity was carried out by the colorimetric method (Kandeler, Gerber,
29
1988). Briefly the procedure was as follows: 1 g of soil was placed into a 50 ml flask and wetted
30
with 1.5 ml of aqueous urea solution (0.08M) and 5 ml borate buffer (0.05M Na2B4O7 x 10 H2O
31
and 0.2 M NaOH, pH 10). The flasks were sealed, placed at 37°C for 2 h, then 15 ml of 2M
32
NaCl were added. The resulting suspensions were filtered and the filtrates were analyzed for
33
ammonia content. For this, 1 ml of the filtrate was diluted to 10 ml with distilled water, then 5 ml
34
of sodium salicylate and 2 ml of 0.1% sodium dichlorisocyanurate were added. The sodium
35
salicylate solution was prepared by mixing 100 ml of 0.12% aqueous sodium nitroprusside, 100
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ml of 17% aqueous sodium salicylate, and 100 ml of distilled water. After 30 min incubation at
2
room temperature the optical density was read at 690 nm. All values of urease activity are
3
reported on a dry weight basis. A total of 72 samples were analyzed.
4
The
bulk
content
of
phosphates
was
determined
with
an
X-ray fluorescent
spectrophotometer. This method is widely used in archaeology as an indicator of ancient
6
manuring (Nielsen, Kristiansen, 2014). The analyses were performed for Podkumskoe-3
7
(sections B-344, B-346, and B-352) and Podkumskoe-7 (sections B-356 and B-357).
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Results and discussion
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Alanic settlement Podkumskoe-2
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The results of estimation of urease activity at Podkumskoe-2 are shown in Fig. 4. In general,
14
urease activity in cultural layer of the settlement was higher than in background soddy
15
calcareous soil in the area adjacent to the settlement. In the upper horizon of the latter it varied
16
from 119 to 389 µg ammonium (NH4+) g–1 soil h–1. In the cultural layer of the first zone with
17
ruined walls remains (section B-338), in 0–10 cm layer urease activity did not exceed 305 µg
18
NH4+ g–1 soil h–1; however, in deeper layers the level of urease activity was 466 µg NH4+ g–1 soil
19
h–1. Similar pattern of urease activity was observed in the cultural layer in small section b-10. In
20
the upper cultural layer of the second zone without ruined walls remains (section B-341), higher
21
values of urease activity were observed, but further down the soil profile they significantly
22
decreased from 207 to 37 µg NH4+ g–1 soil h–1. Maximal values of urease activity in the upper
23
layer were found in small sections 9 and 7, where they reached 389 and 377 µg NH4+ g–1 soil h–1,
24
respectively. It is notable that in all soil sections at the settlement, urease activity in layers 10–20
25
(20–40) cm exceeded that of the upper horizon of the background soil.
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The high level of urease activity in soils of the first zone (with ruined walls remains) most
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likely reflects the use of buildings for cattle keeping. The second zone (without wall remains) is
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probably habitable area that is supported by thick cultural layer, abundance of pottery and bones,
29
and presence of deep household pits.
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Alanic settlement Podkumskoe-3
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To study the changes in urease activity in soils outside archaeological sites, a set of soil
34
sections located at varying distances from Podkumskoe-3 (AD 500–800) was analysed. The
35
following soil sections were investigated: B-345 was located at 60 m, B-344 at 120 m, B-346 at
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250 m, B-350 at 600 m and B-353 at 1200 m from the break-ups of external fortification wall; B-
2
352 was at the watershed plot about 2 km from the site. At sections B-345 and B-350 sizeable
3
amounts of pottery fragments from Alanic time were found. In our study we deal with so called
4
“manure” pottery. This is small fragments up to 3–4 cm, because large fragments are cracked
5
during the plowing and management of soil. The analysis of pottery scattering is often applied in
6
archaeology to reveal the boundaries of ancient agricultural plots (Wilkinson, 1982). The
7
fragments of pottery had entered the soil with manure and wastes that allowed us to consider this
8
territory as fertilized arable lands. An adjacent area to the south was probably also under
9
ploughing, but without manuring, because here only a few pottery fragments were found (section
10
B-353). The plot most distant from the settlement (2000 m) was most probably not subjected to
11
anthropogenic impact.
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The results of estimation of urease activity are shown in Fig. 5 and Fig. 6. Urease activity in
13
soils decreased with distance from the settlement. The highest urease activity was observed in
14
the most vicinal soil at 60 m from the settlement: in the upper horizon it was 403 µg NH4+ g–1
15
soil h–1 and decreased almost twice in the lower horizon. With distance from settlement a sharp
16
decrease in soil urease activity in the upper horizon was observed: down to 143 µg NH4+ g–1 soil
17
h–1 at 120 m, and to 67 µg NH4+ g–1 soil h–1 at 250 m. In the zone of irregular manuring during
18
Alanic time (section B-353), urease activity down the soil profile decreased from 43 to 5 µg
19
NH4+ g–1 soil h–1. Minimal activity was determined in the soil at the top of the watershed at 2000
20
m distance from settlement; in the upper horizon it did not exceed 22 µg NH4+ g–1 soil h–1.
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The soil phosphorus content also decreased along the transect, from 0.51–0.42% at 60 m from the settlement to 0.18–0.11% in the background soil on the watershed (Fig. 7). We carried out the correlation analysis of pottery scattering and urease activity (Fig. 6),
24
proposing that the pottery fragments had been introduced into the soil together with manure. To
25
confirm this, the total number of pottery fragments per square metre was calculated and
26
compared to the mean values of urease activity in the soil profile. A strong positive correlation (r
27
= 0.90, P = 0.05) was revealed. Comparison of the data on urease activity, pottery fragment
28
content, and, partly, phosphorus content allowed us to make reliable conclusions on the proposed
29
boundaries of agricultural lands during Alanic time. The boundary of manuring zone was located
30
at 500 m from the settlement, and the boundary of zone with irregular manuring at 1000 m. The
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plot most probably untouched by anthropogenic activity was located at about 2000 m.
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Alanic settlement Podkumskoe-7
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At Podkumskoe-7 a set of soil sections located at varying distances from the site was
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studied: B-357 at 60 m, B-354 at 150 m, B-355 at 180 m, B-356 at 500 m, and B-358 at 700 m.
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Similar to Podkumskoe-3, the urease activity decreased with distance from settlement (Fig. 5;
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Fig. 6). Highest level of urease activity was observed in the nearest to the site soils: 94 and 119
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µg NH4+ g–1 soil h–1 at sections B-357 and B-354, respectively. Along the transect, a sharp
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decrease in urease activity down to 27 µg NH4+ g–1 soil h–1 was observed in the layer 2–15 cm.
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In the soil located at 500 m from the settlement, urease activity increased again to 349 µg NH4+
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g–1 soil h–1 and in subsurface layers this parameter decreased from 91 to 21 µg NH4+ g–1 soil h–1.
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Elevated values of urease activity at this area evidently are connected with erosion processes. In
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particular, at section B-356 pottery scattering indicates accumulation of erosion material. In soil
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at 700 m from the settlement, minimal urease activity was found: in the upper layer it did not
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exceed 45 µg NH4+ g–1 soil h–1. Besides, in the profile of this soil we observed an increase of
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activity at 50–60 cm depth.
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The content of phosphates decreased from 0.50 to 0.43% in the soil nearest to the settlement, and to 0.33–0.21% in the most distant one (Fig. 7).
In soils adjacent to Podkumskoe-7 we recorded lot of pottery fragments, and observed the
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strong correlation (r = 0.92, P = 0.05) between urease activity and numbers of fragments (Fig. 6).
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At Podkumskoe-7 the boundary of the manured zone was located at 500 m from the
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settlement similar to that at Podkumskoe-3. However, marking of the boundaries of arable lands
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here is more complicated due to erosion process.
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Conclusions
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Anthropogenic impact on the soils of archaeological sites associated with human habitation,
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cattle keeping, and agricultural activity resulted in changes soil urease activity that have been
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preserved to present day.
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The level of urease activity is considered as a novel feature of cultural layers and soils of
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ancient manured lands and may be applied for their characterization. We believe that increased
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values of urease activity in certain parts of a settlement may indicate the location of cattle pens.
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High urease activity in soils within large areas around ancient settlements is associated with
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additional income of urea via manure fertilization of arable plots. This is confirmed by the close
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relationship between the level of urease activity and the number of pottery fragments in soils.
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To date, archaeologists had no readily available and simple way to reveal the locations of
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cattle keeping at archaeological sites. Such locations were conditionally determined by specific
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form and size of buildings as well as peculiarities of artifacts distribution in soils. In other case
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fixation of cattle pens was impossible. In our work we offer simple and effective method for
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such fixation. It may be also valuable for revealing the infrastructure of settlements and
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determination of residential and cattle keeping zones. The application of the method is promising
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for indication ancient manured lands around settlements.
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Acknowledgments
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The work was supported by Russian Foundation for Basic Research (projects No 12-06-
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00272; 14-06-00200) and the Program for Fundamental Research of the Presidium of Russian
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Academy of Sciences.
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Fig. 1. Study area location.
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Fig. 2. Alanic settlement Podkumskoe-2 (A); cultural layer – section B-341 (В), background
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walls).
Fig. 5. Urease activity at the sites Podkumskoe-3 and Podkumskoe-7 (A – settlement, B – section).
Fig. 6. The amounts of pottery fragments and the urease activity in soils along the transect at the sites Podkumskoe-3 (A) and Podkumskoe-7 (B).
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Fig. 4. Urease activity at the site Podkumskoe-2 (A – section, B – small section, C – ruined
Fig. 7. Phosphorus content at the sites Podkumskoe-3 (A) and Podkumskoe-7 (B).
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Podkumskoe-3 - B-346 (B) and B-344 (C).
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Fig. 3. Alanic settlements Podkumskoe-3 and Podkumskoe-7 (A); soil sections at the site
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soil – section B-342 (C).
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Anthropogenic impact on soil results in an increase in soils urease activity. Elevated level of urease activity have been preserved to present day. High level of urease activity was observed in soils of ancient cattle pens. Urease activity may be used as diagnostic feature of ancient manured lands.
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