Results

3.1. Enzymatic activities in grasslands and croplands

The mean values of enzymatic activities measured at the three dates are shown in Table III.2. The enzymatic activities were generally 2 to 3-fold higher in grassland than crop soils. Only urease activity presented the same activity in both land use types for the two sites.

At the Saint-Georges sur Fontaine site, dehydrogenase activity was not significantly different for the two studied cultural practices and alkaline phosphatase showed greater activity in cropland than grassland. The variation coefficients showed that enzymatic activities were highly variable in grassland and cropland of both sites including at the lowest level of activity.

Table III.2. Average values of enzymatic activities in crop and grassland soils Saint Georges sur Fontaine Yvetot

Grassland Crop Grassland Crop

Acid Phosphatase(nmol PNP g^-1 h^-1) 1201.6 457.3 1509.7 464.8

Bold number is the average value including spatiotemporal variability (soils sampled in April, June and October 2005; n = 20 for each activity). Italic number corresponds to the standard deviation. Coefficient of variation (%) is in italic and bold type.

The variation range of enzymatic activities is presented in Figure III.2.

Figure III.2. Spatiotemporal variability in grassland and cropland at Yvetot (a) and at Saint-Georges sur Fontaine (b). For each enzyme, boxes represent the average activity;

open circles represent minimal and maximal values.

The enzymes which showed the greatest variation were acid and alkaline phosphatases, and to a lesser extent β-glucosidase. The variation was greater in grassland than cropland. For example at Yvetot (Fig. III.2a), acid and alkaline phosphatases and β-glucosidase activities varied respectively from 135 to 2501, 7 to 3022and 426 to 1960 nmol PNP g^-1h^-1 in grassland soils. For these same activities the range was lower in cropland soils:

from 210 to 1842, 24 to 1291 and 136 to 641 nmol PNP g^-1h^-1. N-acetyl-β-glucosaminidase exhibited less variability in two cultivation practices. The values for grassland were ranged from 86 to 552 nmol PNP g^-1h^-1 and for cropland, 20 to 263 nmol PNP g^-1h^-1. The variations

of dehydrogenase and urease activities do not appear clearly in the graph because of the weak activity measured. In fact, the levels of activity were recorded between 3 and 80 nmol TPF g

-1h^-1 for dehydrogenase and 5 to 25 nmol N-NH₄⁺ g^-1h^-1 for urease in grassland and crop together.

For the site at Saint-Georges sur Fontaine (Fig. III.2b) the enzymatic activities presented less variability than for the site of Yvetot. Only the acid phosphatase showed a greater range of variability in particular in grassland which varied from 137 to 2808 nmol PNP g^-1h^-1. In the soil under crops, alkaline phosphatase showed the greatest variation from 34 to 1376 nmol PNP g^-1h^-1. On this site, activities were greater in grassland than in crops except for alkaline phosphatase.

A variation analysis was carried out in order to identify the most important factor affecting the soil enzymatic activities: the cultivation practice, the site or the sampling date (Table III.3). The statistical analysis demonstrated clearly an effect of the cultivation practice only on acid phosphatase and N-acetyl-β-glucosaminidase activities. Urease activity was mainly influenced by the sampling date. None of the three factors can explain the variation for the other enzyme activities.

Table III.3. Influence of natural and anthropic factors on soil enzymatic activities in plots as affected by variance analysis (%)

Enzymatic activities Cultural practices

Site Date Residual

variability

Acid phosphatase 65.6 0.0 14.7 19.7

Alkaline phosphatase 0.0 0.0 43.4 56.6

β-glucosidase 21.6 31.7 36.7 10.0

N-acetyl-β-glucosaminidase 41.8 0.0 28.3 29.9

Dehydrogenase 14.7 3.2 26.5 55.6

Urease 0.0 0.0 93.7 6.3

(2005-campaign; n = 200)

3.2. Validity of TMEs in relation to the studied plots

Figure III.3 represents the enzymatic activities measured both in the Yvetot plots and the TMEs at the same times: at the beginning and the end of TME incubation in outside conditions. The results showed that no matter which enzymes are considered or cultivation system used, the natural variation of activities was found both in the plots and in the TMEs.

For the grassland, no significant difference was observed between the measurements recorded in the TMEs and the plots. For the cropland, significant differences existed for acid and alkaline phosphatase activities. In these cases, the activity measured in the TMEs was higher than that measured in the plot but it remains in the range of variability previously observed for these activities.

Figure III.3. Comparison of soil enzymatic activities measured both in uncontaminated cosmes incubated for 70 days in outside conditions and in Yvetot field plots (a, grassland; b, crop). Symbols * and + indicate significant differences between cosmes and plots (p<0.05).

3.3. Impact of copper on enzymatic activities

Control and contaminated TMEs were incubated for 70 days in outside conditions.

The results of TMEs realized from grassland are shown in Figure III.4 and from cropland in Figure III.5. The activities varied over time for both the control and the contaminated TMEs.

In the case of grassland TMEs, copper did not have a significant effect on soil activities compared to the control TMEs, except for acid phosphatase. A significant effect on this activity can be observed in grassland at 7 days for the two levels of copper contamination: 2 and 200 ppm. This effect disappeared for the 2 ppm level of copper whereas it persisted in the TMEs treated with 200 ppm of copper during the incubation period. In the case of cropland

*

TMEs, two enzymatic activities were affected significantly by the presence of 200 ppm of copper after 70 days of incubation: dehydrogenase and N-acetyl-β-glucosaminidase.

Dehydrogenase activity was inhibited by the metal whereas that of N-acetyl-β-glucosaminidase was increased. It is important to note that the significant variations observed for enzymatic activities remains in the range of spatiotemporal variability already established.

Figure III.4. Soil enzymatic activities in grassland cosmes uncontaminated (□) or contaminated to 2 (■) or 200 (■) ppm of copper. *, indicate a significant difference between the control and contaminated cosmes (p < 0.05)

*

Figure III.5. Soil enzymatic activities in cropland cosmes uncontaminated (□) or contaminated to 2 (■) or 200 (■) ppm of copper. *, indicate a significant difference between the control and contaminated cosmes (p < 0.05)

0

Analysis of variance was carried out in order to identify the most important factor affecting the soil enzymatic activities: the cultivation practice, the presence of copper, the incubation time and the TMEs establishment (Table III.4). The results confirmed the absence of effect due to the TMEs establishment on the enzymatic activities. They also showed the lack of effect of copper on enzymatic activities: 0% to 19.2% of the variation can be explained by the metal for the six enzymatic activities. The highest percentage was obtained for alkaline phosphatase. Therefore the variation can not be fully explained by any of the factors in this analysis.

Table III.4: Influence of factors on soil enzymatic activities in undisturbed soil cosmes by variance analysis (%)

Enzymatic activities Cultural practices

Incubation time

Cosme Copper Residual variability

Acid phosphatase 0.0 47.0 0.0 10.8 42.2

Alkaline phosphatase 12.0 9.6 14.2 19.2 45.0

β-glucosidase 17.6 39.3 0.0 0.0 43.1

N-acetyl-β-glucosaminidase 49.4 13.1 9.1 2.0 26.4

Dehydrogenase 1.2 27.9 ND 5.6 65.3

Urease 0.0 89.2 ND 2.8 8.0

(2006-campaign; n=114, except dehydrogenase and urease n=90) ND means not determined