Supplementary Material
Organic carbon hidden in urban ecosystems
J.L. Edmondson, Z.G. Davies, N. McHugh, K.J. Gaston, J.R. Leake
Soil sample preperation validation
Soil sampling methodology
Four replicate soil profiles were taken, from 0 – 100 cm depth, from a representative urban park in Leicester and a further four soil profiles were sampled from an arable field in the agricultural hinterland surrounding the study city. At each site soil profiles were taken from the four corners of a 1 x 1 m quadrat. Soils were sampled using a bulk density corer as detailed in the manuscript.
Soil sample preparation and analysis
It was not possible to split the soil cores in half lengthwise and still obtain reliable bulk density estimates for both halves as the distribution of stones would need to be identical for both halves. Thus, the eight soil profiles were allocated alternately to one or other of the sample preparation treatments (the conventional method using a 2 mm sieve to remove stones, followed by oven drying at 105 o
C and ball milling and analysis of the <2 mm fraction; and our method which involved oven drying at 105 o
C before ball milling, followed by 1 mm sieving to remove stones, and analysis of the <1 mm fraction). In both cases the inorganic carbon was removed from the ball milled soil using HCl, and samples were analysed for organic carbon, using a CN analyser, following the methodology outlined in the manuscript.
Statistical analysis
The proportion of soil total weight removed by the 1 mm or 2 mm seive (%) and the stone-free (>2mm before ball milling, >1mm after ball milling) soil bulk density (g cm
-3
), OC concentration (mg g
-1 total soil weight), and soil OC density (mg cm
-3
) were all compared across the two methods using analysis of covariance (ANCOVA). The factors in the
ANCOVA’s were method (1mm or 2 mm) and land-use (urban park or arable) and the covariate was soil depth. Per cent and concentration data were arcsine transformed prior to analysis.

Results
Descriptive statistics for the proportion of soil mass removed by the seiving process
(% dry weight), soil fine earth bulk density (g cm
-3
), soil OC concentration (mg g
-1 of total soil weight), and soil OC density (mg cm
-3
), show no difference between the two soil sample preparation methodologies (Table 1). The mean soil OC concentration determined across the soil profiles was 12.2 mg C g -1 ± 1.7 for the 1 mm methodology and 12.5 mg C g -1 ± 2.0 for the 2 mm methodology.
The soil sample analysis methodology had no significant effect on the proportion of material removed by sieving, despite the differences in sieve sizes (Table 2).
Furthermore there was no effect of sample analysis methodology on the soil fine earth bulk density (g cm
-3
), soil OC concentration (mg g
-1
) (Fig. 1), or soil OC density (mg cm
-3
) (Table
2).
Conclusion
In conclusion the two methods, as expected, are shown to be equivalent. The 1 mm method used for sample preparation and analysis did not cause a systematic over- or under-estimation of OC stocks when compared to the conventional 2 mm method. Furthermore the 1 mm method maximised the efficiency of sample preparation and analysis, and tended to give more accurate measurements of soil bulk density and OC concentration as reflected by slightly lower amounts of soil mass discarded and lower standard errors of the means.

Fig. 1: The relationship between soil OC concentration (mg g
-1
total soil weight) and soil depth in arable and urban soils treated with the 1 mm sieve method (brown symbols and dashed line of best fit; y = 43.963(+2.313 S.E.)e - 0.031 (+ 0.002 S.E.)x ) and 2 mm sieve method
(green data points and a continuous line of best fit; y = 48.909(+2.378 S.E.)e
- 0.035 (+ 0.002
S.E.)x
).

Table 1: Analysis of covariance of the effect soil sample preparation method, land use and soil depth on the proportion of soil weight removed by seive, soil fine earth bulk density (<2mm and <1mm), soil OC concentration, and soil OC density.
Proportion of soil weight removed by seive (%)
Soil fine earth bulk density (g cm -3 )
Soil OC concentration (mg g -1 total soil weight)
Soil OC density (mg cm -3 )
1 mm method 2 mm method
Mean S.E. Median Range Interquartile range Mean S.E. Median Range Interquartile range
1.8 0.6 0.6 0.0-15.1 2.3 3.3 1.3 0.4 0.0-35.0 2.2
1.4
12.2
14.7
0.1
1.7
1.9
1.5
6.5
9.1
0.9-1.7
2.5-35.9
4.1-33.6
0.4
15.2
16.8
1.3
12.5
15.2
0.1
2.0
2.3
1.3
6.2
9.0
0.9-1.7
2.9-39.1
3.6-37.3
0.5
15.4
23.7

Table 2: Analysis of covariance of the effect soil sample preparatin method, land use and soil depth on the proportion of soil weight removed by seive, soil fine earth bulk density, soil OC concentration, and soil OC density. d.f. F P value
Proportion of soil weight removed by seive (%)
Factor: Method (1 mm or 2 mm) 1,61 1.195
Factor: Land use (urban park or arable) 1,61 1.125
Covariate: Soil depth
Factor interaction: Method x land use
Soil fine earth bulk density (g cm -3 )
Factor: Method (1 mm or 2 mm)
Factor: Land use (urban park or arable)
Covariate: Soil depth
1,61 3.472
1,61 0.173
1,48 2.728
0.279
0.293
0.067
0.679
0.105
1,48 3.972 0.052
1,48 96.418 <0.001
1,48 0.018 0.894 Factor interaction: Method x land use
Soil OC concentration (mg g -1 )
Factor: Method (1 mm or 2 mm)
Factor: Land use (urban park or arable)
Covariate: Soil depth
1,63
1,63
1,63
0.005
5.126
276.826
1,63 0.002
0.942
0.027
<0.001
0.888 Factor interaction: method x land use
Soil OC density (mg cm -3 )
Factor: Method (1 mm or 2 mm)
Factor: Land use (urban park or arable)
Covariate: Soil depth
Factor interaction: Method x land use
1,48 0.032 0.859
1,48 1.430 0.238
1,48 187.039 <0.001
1,48 0.047 0.816