Coordination and participants
The inter-laboratory trial was organized and coordinated by the staff of the platform Biochem-Env (Biochem-Env, 2019), involved in the ISO/TC 190/SC 4/WG 4 "Soil quality – Biological methods – Effects on soil micro-organisms" working group.
Eight laboratories from four countries were participating in the validation study (one for Czech Republic, one from Portugal, two from Spain, and four from France), and all belonged to research institutions.
The participating laboratory quantified the ten enzymatic activities in six soil samples, in triplicate for each sample. Laboratories were requested to follow the protocol updated after the first voting stage (committee draft ISO/CD 20130).
A kick-off meeting has been held in Versailles in spring 2016 to present the interlaboratory trial, provide main documents and describe the protocol to be fulfilled. Electronic formatted spreadsheets (Excel™) have also been sent to the participants to facilitate the data collection and analysis.
Soil samples
Meadow and arable soils were sampled from French experimental sites in France (Pierre Plate, INRAE Versailles; La Cage, INRAE Versailles; Lycée Agricole et Agroalimentaire Yvetot (provided by UniLaSalle, Rouen); Qualiagro, SOERE PRO, INRAE-Veolia Recherche & Innovation partnership, Feucherolles; Efele, SOERE PRO, INRAE Le Rheu; PROspective, SOERE PRO, INRAE Colmar). For the purpose of the ring-test and in order to minimize the influence of external factors, six soils were sampled in autumn 2015 by the platform staff from the surface soil layer (0–20 cm), immediately sieved through a 2-mm sieve and air dried for 8 days at room temperature. Aliquots (100 g dry weight) were sent by the platform staff to each participant, at room temperature. The selected soils covered a wide range of agropedoclimatic context in France. Their main properties (texture, pH, organic carbon, Cation Exchange Capacity) and land use are reported in Table 1. The range of variation of these properties was consistent with cropped soils.
Table 1
Properties of the soils used in the inter-laboratory trial
Soil
|
Site
|
Soil
Nomenclature
(WRB 2015)
|
Silt
%
|
Sand
%
|
Clay
%
|
pH
|
Organic C
%
|
Total N
%
|
CEC
cmol+/kg
|
Land use
|
1
|
Pierre Plate
|
Luvic cambisol/cambisol
|
13.0
|
76.2
|
10.8
|
5.6
|
2.19
|
0.12
|
6.6
|
meadow
|
2
|
La cage
|
Luvisol
|
56.2
|
27.1
|
16.7
|
7.4
|
1.00
|
0.10
|
11.5
|
arable soil
|
3
|
Yvetot
|
Neoluvisol-Luvisol
|
63.5
|
19.7
|
16.8
|
5.5
|
2.57
|
0.25
|
8.1
|
meadow
|
4
|
Qualiagro
|
Luvisol
|
78.3
|
6.70
|
15.0
|
6.6
|
1.05
|
0.10
|
7.9
|
arable soil
|
5
|
Efele
|
Luvisol-Redoxisol
|
79.3
|
16,1
|
14.6
|
6.0
|
1.15
|
0.12
|
6.1
|
arable soil
|
6
|
PROspective
|
Calcasol
|
66.5
|
9.6
|
23.4
|
8.5
|
1.43
|
0.12
|
16.9
|
arable soil
|
CEC : cation exchange capacity |
Chemicals
All products were purchased from Sigma-Aldrich, subdivided by the platform staff and provided to the participants. Solutions for use were prepared by each laboratory.
Measurement of enzymatic activities
Ten enzyme activities were measured in soil samples, most of them according to protocols modified from older ones (e.g. Tabatabai and Bremner 1970; Dick et al. 1996; Sinsabaugh et al. 2000): α-glucosidase (αGLU, E.C. 3.2.1.20), β-glucosidase (βGLU, E.C. 3.2.1.21), β-galactosidase (βGAL, E.C. 3.2.1.23), acid phosphatase (ACP, E.C. 3.1.3.1), alkaline phosphatase (ALP, E.C. 3.1.3.2), phosphatase (PHOS, acid or alkaline according to the pH of soil solution), arylsulfatase (ARS, E.C. 3.1.6.1), N-acetyl-glucosaminidase (NAG, E.C. 3.2.1.52), arylamidase (ARN, E.C. 3.4.11.2) and urease (URE, E.C. 3.5.1.5).
The analyses were carried out, in triplicate. Soil suspensions were prepared by mixing 4 g (dry weight) of soil with 25 mL of deionized water (for ARS, αGLU, βGLU, βGAL, NAG, PHOS and URE), 50 mM Tris base pH 7.5 (for ARN); 50 mM Tris HCl pH 5.5 (for ACP) or 50 mM Tris base pH 11 (for ALP) into flat-bottom plastic flasks (30–60 mL). They were then homogenized for 10 min on an orbital agitator (250 min− 1) and maintained under stirring during suspension pipetting into 96-well microplates.
Soil suspensions pipetted from each flask (125 µL for all activities and 50 µL for URE) were separately distributed in four replicate wells. One of the wells was used as a control to reveal the influence of chemical interactions between soil compounds in the spectrometric readings.
According to the enzyme, the substrates added to the corresponding wells were: 25 µL of 8 mM L-leucine βnaphthylamide hydrochloride for ARN, 25 µL of 25 mM potassium p-nitrophenyl sulphate for ARS, 25 µL of 50 mM p-nitrophenyl β-D-galactopyranoside for βGAL, 25 µL of 25 mM p-nitrophenyl α-D-glucopyranoside for αGLU, 25 µL of 10 mM p-nitrophenyl N-acetyl β-D glucopyranoside for βGLU, 25 µL of 50 mM p-nitro-phenylphosphate disodium salt hexahydrate for PHOS, ACP and ALP, and 40 µL of 400 mM urea for URE. Deionized water (150 µL in assay wells and 190 µL in control wells) was also added for URE.
The incubation conditions were: 30 min at 37 ºC for PHOS, PAC and PAK; 60 min at 37°C for αGLU and βGLU; 120 min at 37°C for NAG, ARN and βGAL; 180 min at 25°C for URE; 240 min at 37°C for ARS.
Reactions were stopped for ARS, βGAL, αGLU, βGLU, NAG, PHOS, ACP and ALP by adding 25 µL of 500 mM calcium chloride in the assay wells; and 100 µL of 100 mM Tris base buffer pH 12 plus 25 µL of the appropriate substrate in the respective enzyme control well. The plates were afterwards centrifuged for 5 minutes at 1,500 g and 20°C, and 200 µL of the supernatant was transferred into a new plate. The absorbance of the reaction product, p-nitrophenol, was measured in a microplate spectrophotometer UV/visible at λ = 405 nm, being its concentration determined from a p-nitrophenol calibration curve (cf. supporting information for further details).
For ARN, 150 µL ethanol 96% was added to each well including controls, and 25 µL of substrate solution was added into control wells. The plates were centrifuged 5 minutes at 1,500 g and 100 µL of supernatant was transferred into a new plate. To reveal the quantity of βnaphthylamine produced, 100 µL of acidic ethanol and 100 µL of 3.5 mM p-dimethylaminocinnamaldehyde were added in all wells. After 20 min, the reading of absorbance was performed in a microplate spectrophotometer UV/visible at λ = 540 nm, and the βnaphthylamine concentration was determined from a β-naphthylamine calibration curve.
For URE, 40 µL of salicylate reagent was added to each well, including controls. Salicylate reagent was prepared just before analysis by dissolving 865 mg of sodium salicylate, 853 mg of trisodium citrate, 276 mg of disodium tartrate and 12 mg of sodium nitroferricyanide in 20 mL of deionized water. After a 3 min period, 40 µL of cyanurate reagent was dispensed into each well, including controls. Cyanurate reagent was prepared just before analysis by dissolving 3.4 g of trisodium citrate, 414 mg of disodium tartrate, 134 mg of lithium hydroxide and 51 mg of dichloroisocyanurate in 20 mL of deionized water. Colorimetric reaction was achieved after 30 min, being stable for two hours. The plates were then centrifuged 5 minutes at 1,500 g and 20°C, and 200 µL of the supernatant was transferred into a new plate. The reading of absorbance was performed with a microplate spectrophotometer UV/visible at λ = 650 nm, and concentration determined from an ammonium chloride calibration curve.
Enzyme activities were expressed in mU g− 1 dry soil, corresponding to nmole of p-nitrophenol, β-naphthylamine or ammonium chloride released per minute and per g of dry soil.
Statistical Analysis
All analyses were performed using R-project software (The R Development Core Team, Ri386 3.5.0). Results are expressed as mean ± SD (standard deviation) and CV (coefficient of variation). Coherence check was ensured by identifying outlier data using the Grubbs test (P < 0.05) package “outliers” (Komsta 2011).
The intra-laboratory coefficient of variation (repeatability, CVr) and inter-laboratory coefficient of variation (reproducibility, CVR) were calculated as described below from enzyme activities. They were considered acceptable by the ISO TC190/SC4 when the CV values were below 30%.
CVr is the repeatability coefficient of variation; Si is the repeatability standard deviation calculated from intra-laboratory data; Xi is the mean of values calculated from intra-laboratory data; L is the number of the participating laboratory.
CVR is the reproducibility coefficient of variation; SR is the reproducibility standard deviation calculated from inter-laboratory data; X is the mean of values calculated from inter-laboratory data.
The comparison of the data provided by the laboratories for each soil were compared with PCA using the package ADE-4 (http://pbil.univ-lyon1.fr/ADE-4/home.php).