Nanoleakage related to bond strength in RM-GIC and adhesive restorations.
Abstract: AIM: This was to investigate the nanoleakage of Resin Modified Glass Ionomer Cement (RMGIC) and composite resin (CR) restorations in sound and caries-affected primary dentine, submitted to load cycling and cariogenic challenge in vitro. METHOD: Occlusal cavities were prepared in 60 sound exfoliated primary second molars and 30 specimens were subjected to chemical induction of artificial caries lesions and the others were restored without caries induction. All prepared teeth were divided into 2 groups according to restorative materials. From each dentine condition 5 restored teeth and restorative material were subjected to microtensile bond strength and nanoleakage tests immediately or after load-cycling or submitted to the pH-cycling procedure before testing. RESULTS: The adhesive presented bigger areas of silver leakage at the interfaces on caries-affected dentine (2.46 [+ or -] 0.47) [mm.sup.2] than sound dentine (0.90 [+ or -] 0.19) [mm.sup.2]. RMGIC nanoleakage was not influenced by the sound (1.75 [+ or -] 0.11) [mm.sup.2] or caries-affected (2.08 [+ or -] 0.39) condition of the substrate. A significant moderate inverse correlation was revealed between the bond strength and silver leakage area at the interface, (r= -0.55, p<0.001). CONCLUSIONS: Nanoleakage is greater in caries-affected primary teeth dentine than sound dentine in adhesive restorations although at the interfaces of RMGIC does not differ. As nanoleakage increases, bond strength decreases significantly.

Key words: nanoleakage, primary dentine, adhesion
Article Type: Report
Subject: Dental adhesives (Health aspects)
Dental adhesives (Research)
Dental glass ionomer cements (Health aspects)
Dental glass ionomer cements (Research)
Dental caries (Care and treatment)
Dental caries (Research)
Authors: Marquezan, M.
Skupien, J.A.
da Silveira, B.L.
Ciamponi, A.L.
Pub Date: 02/01/2011
Publication: Name: European Archives of Paediatric Dentistry Publisher: European Academy of Paediatric Dentistry Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 European Academy of Paediatric Dentistry ISSN: 1818-6300
Issue: Date: Feb, 2011 Source Volume: 12 Source Issue: 1
Topic: Event Code: 310 Science & research
Product: Product Code: 3843075 Dental Adhesives NAICS Code: 339114 Dental Equipment and Supplies Manufacturing SIC Code: 3843 Dental equipment and supplies
Geographic: Geographic Scope: Brazil Geographic Code: 3BRAZ Brazil
Accession Number: 277106721
Full Text: Introduction

Different causes that interact synergistically contribute to restoration failures, with hydrolysis being the main event, because of water entering at resin/dentine interface. Hydrolysis breaks not only the links between collagen fibers, but those between the resinous polymers as well [Hashimoto et al., 2000]. This process can be accelerxated by the action of enzymes released by bacteria or dentine [Pashley et al., 2004]. This intrinsic degradation was described in partially demineralised dentine collagen and is attributed to the action of matrix metalloproteinases (MMPs) [Hebling et al., 2005; Carrilho et al., 2007].

The word nanoleakage was introduced to describe the occurrence of nanosized spaces inside the hybrid layer, even in the absence of gaps at the interface [Sano et al., 1994; Sano et al, 1995]. Nanoleakage can represent:

* incomplete collagen network impregnation by the adhesive;

* incomplete solvent evaporation;

* unpolymerized monomers;

* hydrolytic degradation of collagen;

* resin degradation.

Thus, silver deposition in specimens tested immediately (within 24 hours) represents residual water from the adhesive procedure, while in older specimens it represents water absorption and consequent degradation [Breschi et al., 2008].

As nanoleakage represents the degradation of interfaces, causing reduction in the adhesive bond strength, an inverse correlation between bond strength and nanoleakage is expected [Okuda et al., 2002]. Reis et al. [2007a] demonstrated an inverse and significant relationship between bond strength and nanoleakage.

Bond strength of caries-affected dentine is lower than that of sound dentine; there is greater MMP activity in affected dentine [Hebling et al., 2005], and the bond interface to caries affected dentine is more susceptible to degradation [Erhardt et al., 2008]. Thus, it is relevant to consider that when the affected dentine layer is kept on the pulpal wall of the cavity, chemical mechanism of bonding and fluoride release can be beneficial to resistance against degradation [De Munck et al., 2005], as well to as facing cariogenic challenge. In this sense, it seems plausible to investigate the behaviour of restorations in caries-affected dentine, made with Resin Modified Glass Ionomer Cement (RM-GIC) and composite restorations (CR) made with a bonding agent containing functional agents.

The aims of this study were firstly to investigate the resistance to degradation of RMGIC and adhesive/CR restorations in sound and caries-affected primary dentine, submitted to load cycling and cariogenic challenge in vitro, by evaluating nanoleakage. The second aim was to verify the relationship between nanoleakage and microtensile bond strength. The null hypotheses tested were that the nanoleakage of the RMGIC and the adhesive/CR restorations does not differ when these materials are applied in sound or affected dentine; nanoleakage is not modified by the load and pH cycling; and nanoleakage is not related to bond strength.

Material and methods

In this study 60 sound exfoliated primary second molars were used. The human primary molars were obtained after the institutional informed consent from all donors. Teeth were cleaned with pumice/water slurry, rinsed and stored in a solution of distilled water and thymol in a refrigerator (4[degrees]C) until use. The pulp chambers of 60 crowns were filled with CR and their cusps) flattened with 220-grit abrasive paper. Occlusal Class I cavities (7mm x 5mm x 2mm deep) were prepared using a high-speed handpiece with a cylindrical medium-grit (100 [micro]m) diamond bur (#842) (Komet, Lemgo, Germany) under water irrigation. Each diamond bur was replaced every five preparations. A sub-set of 30 specimens was subjected to the induction of artificial caries lesions and the other 30 were restored without artificial caries induction.

Artificial caries induction. The entire surface of each specimen, except for the internal surfaces of the cavity, was painted with two layers of an acid-resistant red varnish (Colorama, Sao Paulo, SP, Brazil). Simulated dentine carious lesions were created by a pH-cycling procedure, according to the protocol described in a previous report [Mendes and Nicolau, 2004]. The demineralising solution contained 2.2 mM Ca[Cl.sub.2], 2.2 mM Na[H.sub.2]P[O.sub.4], and 50 mM acetic acid adjusted to a pH of 4.8. The remineralising solution contained 1.5 mM Ca[Cl.sub.2], 0.9 mM Na[H.sub.2]P[O.sub.4], and 0.15 M KCl adjusted to a pH of 7. Each specimen was cycled for 8 hrs in the demineralising solution (10 ml) and 16 hrs in the remineralising solution (10 ml). This procedure was performed for 14 days with solutions being renewed at each change, at 37[degrees]C, and without shaking. The depth of dentine demineralisation with this same method has been reported to be over 100 pm deep [De Munck et al., 2005]. After the induction period, a diamond bur with tapered safe end was used to clean the walls surrounding the cavities keeping the demineralised dentine layer at the bottom of the cavities.

Restoration procedures. The prepared teeth were randomly divided into 2 groups (Figure 1) according to the restorative materials: a RM-GIC (Vitremer[R]) (3M ESPE, St. Paul, MN, USA) and a total-etch adhesive system (Adper Single Bond 2[R]) (3M ESPE, St. Paul, MN, USA) followed by a CR (Filtek Z100[R]) (3M ESPE, St. Paul, MN, USA). The methods of application of these materials were in accordance with the manufacturer's instructions (Table 1). Filtek Z100 was inserted using an incremental technique, and each layer was light polymerized for 40 seconds with a Translux EC (Kulzer GmbH, Bereich Dental, Wehrheim, Germany) halogen light-curing unit. The output intensity was monitored with a Demetron Curing Radiometer (Model 100) (Demetron Research Corporation, Danbury, CT, USA). A minimal output intensity of 600mW/[cm.sup.2] was used for the experiments.


After storage in distilled water 37[degrees]C for 24 hrs, the occlusal surfaces of the restorations were ground in order to assure exposure of enamel-restorative material interfaces. Then 5 restored teeth from each dentine condition and restorative material were subjected to one of the following procedures: 1) Sectioned and tested for microtensile bond strength and nanoleakage (control 24 hrs); or 2) mounted in plastic rings using acrylic resin, for load-cycling (50,000 cycles, 90 N, 3 Hz) under water, with a compressive load applied to the centre of the restoration using a 5 mm diameter spherical stainless steel plunger, attached to a cyclic loading machine (S-MMT-250NB) (Shimadzu, Tokyo, Japan) before testing; or 3) submitted to the pH-cycling procedure, being alternately placed into containers with the demineralizing solution for 8 hrs and remineralizing solution for 16 hrs. Solutions were the same as those described above for carious lesion induction, but the cycling procedure was performed for 10 days, as proposed by Rocha et al. [2007].

For the microtensile test, 5 teeth per group were sequentially sectioned with a water-cooled diamond disc (Isomet 4000) (Buehler, Lake Bluff, IL, USA), along the mesiodistal and buccolingual axis, in order to obtain beams with a square cross-sectional area of about 1 [mm.sup.2] for microtensile testing. 45 beams were obtained per group. Each beam was attached to a modified Bencor Multi-T testing apparatus (Danville Engineering Co., Danville, CA, USA) with cyanoacrylate adhesive (Zapit) (Dental Venture of America Inc., Corona, CA, USA) and stressed to failure under tension, in a universal testing machine (Instron 4411) (Instron Corporation, Canton, MA, USA) at a crosshead speed of 0.5 mm/min. Bond strength values were expressed in MPa.

Nanoleakage test. One section of each restoration, which was not cut for the microtensile test, was prepared for the nanoleakage test. The sections were waterproofed with the nail varnish, with the exception of the pulpal floor interface of the cavity, and immersed in aqueous ammoniacal silver nitrate for 24 hrs. The tracer was prepared by dissolution of 25g of silver nitrate crystals in 25 mL of distilled water. Concentrated ammonium hydroxide (28%) was used for titration of the black solution until it became white and the ammonium ions were converted from silver into silver diamine ions. This solution was diluted in 50 ml of distilled water to reach a concentration of 50% (pH=9.5). The specimens impregnated with silver were rinsed in distilled water and immersed in photo developing solution for 8 hrs under fluorescent light to reduce silver ions into metallic silver grains within voids along the interface.

The cosmetic varnish was removed by the application of 600-grit SiC paper, the sections were fixed and included in epoxy resin to be polished with a sequence of SiC papers (1,000, 1,550 and 4,000), followed by felt that was impregnated with 1 and 0.25 pm grit diamond slurry used in an automatic polishing machine. Between each granulation and at the end, the specimens were cleaned and immersed in water in an ultrasonic vat. After this, the slices were dehydrated in increasing concentrations of ethanol for five minutes in each concentration (30, 50, 70, 90, 96%).

The last bath was in absolute alcohol for 10 mins, repeated twice. The specimens were immersed in HMDS for 30 minutes and left to dry in laminar flow chapel equipped with an exhaust. After this, each polished slice was mounted on a metallic stub and covered with carbon. SEM was operated in the backscattered mode, and silver presented white in the image. So that the images would present a magnification suitable for verifying the areas of silver impregnation, a 1 mm wide area in the depth of the cavity was captured at 150x. White areas at the interfaces were checked by EDX sounding (energy dispersive x-ray detector) for determining the chemical elements presented and to discard the possibility of false-positives results. The captured images were analysed in Leica QWin program (Leica Microsystems, Heildelberg, Germany), which allowed leakage area to be measured by the tracer through the number of pixels in white colour (silver) converted into [micro][m.sup.2].

Statistical analysis. The silver nitrate leakage area values in [micro][m.sup.2] were analyzed by descriptive statistics using SPSS software. Normal distribution was verified with the Shapiro-Wilk test and homogeneity by Levene's test. The data were raised in logarithms in base 10 to equalize the variances. A multiple ANOVA was carried out followed by multiple comparisons Tukey's test. The mean bond strength value for each tooth was correlated to the silver leakage area at the interface by Spearman's correlation coefficient.


The mean areas of silver nanoleakage at the interface and their respective standard deviations for each experimental group are presented in Table 2. The factor dentine (F=37.87; p<0.001) significantly influenced silver nanoleakage at the interface, whereas the other factors: material (F=1.69; p=0.20) and challenge (F=0.51; p=0.60) did not affect leakage. The interaction between the factors dentine and material were significant (F=13.03, p<0.01).

The RM-GIC Vitremer presented no difference in the nanoleakage pattern as a function of sound or affected substrate; whereas, the adhesive Single Bond 2 presented larger areas of silver nanoleakage at the interfaces with caries-affected dentine in comparison with sound dentine. This was valid for baseline (24hrs) and after pH cycling.

The pH or load cycling challenges did not promote difference in silver nanoleakage in comparison with baseline (24 hrs) for any of the materials and substrates. Figures 2 and 3 represent the nanoleakage pattern found in the experimental groups. Figure 4 presents the dispersion graph between the bond strength values (MPa) and measurements of the silver nitrate nanoleakage area at the interface ([micro][m.sup.2]). Spearman's correlation coefficient revealed a significant moderate inverse correlation between the bond strength and silver nanoleakage area at the interface, which means that as nanoleakage increases, bond strength decreases significantly (r= -0.55, p<0.001).





As the hybrid layer is a mix of dentine organic matrix, hydroxyapatite crystals, resin monomers and solvents, ageing can affect each one of these components individually or in combination [Breschi et al., 2008]. Hydrolysis is the main mechanism of resin degradation in the hybrid layer. Water is also responsible for the collagen degradation. The combined effect of degradation of the resinous portion and collagen increases the presence of water at the interface, leading to an additional deleterious effect on the longevity of the bond [Breschi et al., 2008]. As hydrolytic degradation only occurs in the presence of water, the hydrophilicity of the bonding agent, its water sorption, and the subsequent degradation are related [Tay et al., 2002a; Tay et al., 2003]. This means that irrespective of the adhesive strategy (total-etching or self-etching), the presence of a hydrophilic monomer in the adhesive leads to the formation of hybrid layers that behave as permeable membranes that allow the water to flow, even after adhesive polymerization [Tay et al., 2002b].

Adhesives with a higher percentage of hydrophilic monomers (simplified) show a higher degree of permeability after polymerization, and consequently, more nanoleakage expression [Tay et al., 2002b]. The hydrophilic monomer HEMA is a common component of adhesive agents to improve their infiltration into moist substrates. However, HEMA decreases the water vapour pressure, making it difficult to remove, and then retaining water at the interface. This makes the hybrid layer act as a hydrogel, absorbing and releasing water, contributing to nanoleakage [Tay et al., 2002c]. The Vitremer primer is a light polymerizable liquid with high concentration of HEMA (46%) [Pereira et al., 2000]. The presence of a high concentration of this hydrophilic monomer can produce incomplete polymerization and water permeability, causing dissolution and degradation of resinous components, as also occurs with the simplified adhesive systems [Breschi et al., 2008]. Navarra et al. [2009] found a relationship between a low degree of conversion and the presence of nanoleakage, and this could affect the quality and durability of the adhesive interface over the course of time.

In seeking a resistant and lasting bonding, the addition of functional agents to adhesive systems has been suggested. Thus, the composition of the bonding agent also influences the effectiveness of bonding. The Adper Single Bond 2 contains a functional monomer, the copolymer of polyalkenoic acid, which has some degree of interaction with the calcium ions of dentine through acid-based reactions. Preventing calcium loss from dentine matrix can contribute to stability of the adhesive interface over the course of time [Osorio et al., 2002].

However, silver deposits were detected throughout the hybrid layer and also in the adhesive layer of Adper Single Bond [Osorio et al., 2002; Reis et al, 2007b], as an amorphous phase that was identified as the copolymer of polyalkenoic acid, which does not infiltrate into the collagen network, due to its high molecular weight [Osorio et al., 2002]. After ageing for 6 months in water, Erhardt et al. [2008] and Reis et al. [2007b] found an increase in silver leakage into the hybrid and adhesive layers, suggesting that water comes from an external source oriented to the copolymer mass by self propagable water channels. Although these results are similar to the present study for Single Bond 2 in affected dentine, the interaction between dentine-material seems to explain better the occurrence of nanoleakage, as nanoleakage at the baseline represents residual water retained at the interface, and the challenges used did not have any effect on the amount of silver nitrate leakage. However, in sound dentine it was not possible to observe the details described by Reis et al. [2007b] because of the lower resolution and the use of scanning electron microscopy instead of transmission electron microscopy.

The presence of fluorides in the bonding agent has also been reported as a component to improve the durability of bonding. Sodium fluoride crystals were observed in the adhesive layer of Clearfil Protect Bond. These fluoride crystals can be extracted from the adhesive and inhibit the enzyme action in the interface, being responsible for its better long-term behaviour in vitro [Reis et al., 2007b]. GIC by nature liberates fluoride, which, in accordance with the hypothesis previously cited, would contribute the longevity of the restoration. Although, in our study, the pattern of silver leakage observed for sound dentine specimens restored with Vitremer was higher than that for adhesive restorations. Thus, the first null hypothesis was rejected. Two reasons can be related to this fact: 1) GIC are materials prone to hydric imbalances, which possibly contributed for a high water presence in the interface; and 2) the presence of great amount of HEMA in the Vitremer can have contributed for a higher retention and permeability to water.

None of the challenges used was able to intervene in the pattern of silver infiltration. Li et al. [2002] also verified that the pattern of infiltration was not affected by load cycling, when applied over flat interfaces. Although porosities existed in the adhesive interfaces, it did not increase with simulated occlusal stress. Those authors emphasise that the load-cycling machine reproduces axial loads, while the chewing movements have a three-dimensional pattern. This condition of specificity of the simulated challenge in vitro must be considered for interpretation of results.

Reis et al. [2004] demonstrated an increase of silver particles into the hybrid layer formed by Single Bond[R] after six months in water. Literature is unanimous in pointing out that specimen storage in water is the best method to simulate ageing that restorations suffer in an oral environment [De Munck et al., 2005]. The pH cycling, however, aimed to simulate the cariogenic challenge that the restorations are subjected to in an environment characterized by caries activity. The deleterious effect of pH cycling over bond strength values of Adper Single Bond 2[R] and not over nanoleakage could be explained by the loss of minerals that weakened the union, without however, having perceivable effect on hydrolytic degradation visualized by the penetration of silver. Perhaps it would occur if pH cycling were extended to a longer period of time, a determinate factor for nanoleakage increase. Meanwhile, Vitremer[R] presented a silver leakage pattern coherent with its bond strength values, which did not vary as result of challenges. Thus, the second null hypothesis was accepted.

As nanoleakage is the expression of areas with potential depletion of the interface and the degradation per se, it is reasonable that there is an inverse association between the leakage pattern and bond strength [Okuda et al., 2002]. However existence of this relationship is not unanimously recognised in the literature, probably due to technical difficulties of obtaining data referring to leakage that is possible to measure, and which is representative of the interface; and at the same time, has morphologic details of the bond. Thus most studies make only qualitative analyses of nanoleakage [Erhardt et al., 2008]. Reis et al. [2007a] demonstrated an inverse and significant correlation between bond strength and nanoleakage after ageing of specimens for two years. Those authors believed that the results were able to express its inverse correlation because of ageing, which allowed the expression of degradation by nanoleakage and reduction in bond strength.

Otherwise the study of Hashimoto et al. [2004], which did not evaluate these properties in the long-term, did not find a correlation; and Ding et al. [2009] also found no statistically relevant relationship between nanoleakage and bond strength. In the present study, although the challenges did not influence the area of silver leakage, they did influence the bond strength, and a moderate negative correlation between the two properties was found, which denotes the influence of substrate and material characteristics on the bond effectiveness. Thus, the third null hypothesis was rejected.

The final aim of the adhesive procedures is for bonding agent to completely envelop the collagen fibers, to protect the interface from degradation. As the effectiveness and durability of the bond seems to depend on the hybrid layer quality, some strategies have been suggested to improve monomer infiltration; reduce the degree of water absorption and reduce collagen degradation, such as: 1) use of systems in which the primer and bond are separate; 2) increase polymerization time; 3) improve of impregnation by a longer application time and friction, and 4) use a MMP inhibitor [Breschi et al., 2008; Ricci et al., 2010]. Furthermore, with the intention of protecting restorations from degradation, it seems coherent to use bonding agents with an additional chemical mechanism, such as GIC. Neelakantan et al. [2009] found a lower amount of nanoleakage for glass ionomer based bonding agents compared with dentine adhesives.

The method of using of bonding agents is appropriate to verify interface degradation and the results herein demonstrated an inverse correlation between bond strength and nanoleakage. The use of restorative material with chemical mechanism of bonding is encouraged in patients with caries activity, because these materials are resistant to cariogenic challenge.


Nanoleakage is greater in caries-affected dentine than sound dentine in adhesive restorations although at the interfaces of RMGIC does not differ. As nanoleakage increases, bond strength decreases significantly. Load cycling and cariogenic challenge do not modify the pattern of silver nitrate leakage at the interfaces of both RMGIC or adhesive/composite.


The research was approved by the Research Ethics Commission of University of Sao Paulo. We thank Dra. Celia Rodrigues, who passed away during the preparation of this research, for all her knowledge.


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M. Marquezan, J.A. Skupien, B.L. da Silveira, A.L. Ciamponi

Dept. of Paediatric Dentistry, University of Sao Paulo, Sao Paulo, SP, Brazil.

Postal address: Dr. J.A. Skupien. Federal University of Santa Maria, RS, Santa Maria, RS, 97015-372, Brazil.

Table 1. Materials used in the experimental groups to investigate
the nanoleakage of RM-GIC and composite resin restorations.

Product/                Components              Mode of application
Manufacturer/ Batch #

Vitremer[R]             Primer: methacrylate    1) Apply Primer
                                                   for 30s, using a
3M ESPE, St Paul,       functional copolymer       light scrubbing
MN, USA                 of polyacrylic and         motion. Mild air
                        polyitaconic acids,        stream for 15s.
Primer: #6BJ            HEMA, ethanol and
                        photoinitiators.        2) Light polymerize
Powder: #6EJ                                       for 20s.
Liquid: #6FN            fluoroaluminosilicate   3) Hand-mixed
                        glass,                     manipulation
                        potassium persulfate    4) Insertion into
                        and ascorbic acid,         a cavity using a
                        small amounts of           syringe injector
                        pigments.                  in a single
                        Liquid: aqueous
                        solution of a           5) Light polymerize
                        polycarboxylic acid        for 40s.
                        modified with pendant
                        methacrylate groups,    6) Apply light-
                        methacrylate               polymerizing
                        functional copolymer       finishing gloss
                        of polyacrylic and         and light-
                        polyitaconic acids,        polymerize for
                        water, HEMA and            20s.

Adper Single            HEMA, water, ethanol,   1) Etch with
Bond 2[R]               Bis-GMA,                   phosphoric acid
                        dimethacrylates,           37% for 15 s.
(Adper Scotchbond       amines, methacrylate
1 XT in Europe)         functional copolymer    2) Rinse with
                        of polyacrylic and         water spray for
3M ESPE, St Paul,       polyitaconic acids,        15s, leaving tooth
MN, USA                 10% by weight of 5         moist.
#5CW                    spherical silica        3) Active
                        particles.                 application of two
                                                   consecutive coats
                                                   of the adhesive
                                                   with a fully
                                                   saturated brush
                                                   tip. Dry gently
                                                   for 2-5 s.

                                                4) Light polymerize
                                                   for 10 s.

Abbreviations: HEMA: 2-hydroxyethylmethacrylate; Bis-GMA: bis-phenol
A diglycidyl methacrylate

Table 2. Mean (SD) and [Log.sub.10] mean ([Log.sub.10]SD) values
for silver nanoleakage at the interface of Vitremer[R] and Adper
Single Bond 2[R] submitted to loading or pH cycling on sound or
affected primary dentine.

Challenge      Material                       Vitremer[R]

               Dentine              Sound Dentine     Affected Dentine

24 hours       Mean (SD)            57.90 (15.35)      151.50 (90.46)
               [Log.sub.10]Mean   1.75 (0.11) (Bab)   2.08 (0.39) (Bca)

Load Cycling   Mean (SD)            25.92 (20.66)       79.60 (57.10)
               [Log.sub.10]Mean   1.29 (0.37) (Aa)    1.77 (0.47) (Aa)

pH Cycling     Mean (SD)            88.40 (60.62)      152.40 (99.08)
               [Log.sub.10]Mean   1.88 (0.27) (Bb)    2.13 (0.23) (Ba)

Challenge      Material                 Adper Single Bond 2[R] +
                                             Filtek Z100[R]

               Dentine             Sound Dentine     Affected Dentine

24 hours       Mean (SD)            8.55 (3.77)      345.12 (193.26)
               [Log.sub.10]Mean   0.90 (0.19) (Aa)   2.46 (0.47) (Ca)

Load Cycling   Mean (SD)           60.26 (58.30)     214.32 (185.88)
               [Log.sub.10]Mean   1.08 (1.32) (Aa)   2.19 (0.39) (Aa)

pH Cycling     Mean (SD)            7.70 (9.21)      397.20 (220.81)
               [Log.sub.10]Mean   0.44 (0.89) (Aa)   2.55 (0.24) (Ba)

Means are shown in [micro][m.sup.2]. Means followed by the same
small letters in columns and capital letters in rows did not differ
significantly in Tukey's test (p<0,05).
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