Impact of Inorganic Pollution on the Wood Structure and Infradensity of Avicennia germinans Wood from the Gabon Estuary

Experimental sites and sampling

Wood from Avicennia germinans (A. germinans) was collected from two separate experimental sites located in the Gabon Estuary region. The Alénakiri site (00°18.6927 N; 009°31.3063’ E), located in the municipality of Owendo, was selected as the polluted site because it lies in the heart of an urban area and near an open-air landfill characterised by heavy metal pollution, among other contaminants [6]. The Ayémé maritime site (00°28.8516’ N; 009°30.9994’ E), located in a peri-urban area, is distant from neighbouring communities. At each site, three vigorous A. germinans trees of similar diameter (70 to 80 cm) were sampled (Figure 1). Trunk discs 5 centimetres thick were collected at a height of 1.3 m above the ground.

Figure 1: Location of the different sampling sites of the mangrove trees. Ayémé maritime site (a). Alénakiri site (b).

Salinity measurement

Salinity and pH were measured in situ at a depth of 15 cm in the soil using a multiparameter pH meter (a portable device capable of measuring 15 parameters).

Ash content

The ash content analyses were performed according to the protocol described by Sluiter et al. [7]. Two jars, one small and one large, were cleaned and then dried in a desiccator at 105 °C for 30 minutes. We then weighed the pots and recorded the mass as M0. Next, we placed 2 g of sawdust in the small pots and 5 g in the large pots, dried them in a desiccator at 105°C until a constant mass was reached, and recorded the mass as M1. Finally, we placed the jars in an oven at 550±25°C for 4 hours until light grey or white ash was obtained. At the end of this time, we let them cool for one (1) hour. We weighed the test specimens and recorded the mass M₂.

Calculation of the ash content

T(%) = (M2 – MO) / (M1-MO) X 100                                         (1)

With :

T(%) : Ash content

M0: Mass of the container

M1: Mass of the container + anhydrous sample

M2: Mass of the container + ash

Vessel density

The episcopic microscope coupled to a camera allows images to be obtained on wood samples (100 x to 700 x) using normal reflected light. The light is directed onto the sample by conical beams through the objective, providing an adjustable image of the structural details. To determine vessel density, about twenty blocks of wood from each sapwood and heartwood compartment (1cm³) were prepared in the heartwood compartment. To obtain good surface quality, the transverse face of the sample was sanded with 4000-grit sandpaper (5µm average grain size) and cleaned with compressed air. The wood blocks were then placed under a Leica DM2700M optical microscope equipped with a reflected light source and a digital camera for acquisition. A script was developed under FIJI software [8] for quantitative anatomy analysis. Some twenty wood samples were analysed for each sample type.

Analysis of infradensity

Infradensity analysis was assessed according to Bakour (2003). Twenty-two 1 cm³ wood blocks from the Oveng and Mamboumba wood samples were machined from each sapwood and heartwood compartment, then oven-dried for 48 h at 103°C. After vacuum saturation of the samples in a vacuum pump, they were weighed on a balance (Denver Instrument, max=400g, d=0.001 g) using Archimedes’ buoyancy. Infradensity is the ratio between the mass of wood in its anhydrous state and its water-saturated volume or “green volume”:

$\rho =\frac{M0}{Vs}\text{ (2)}$

where:

ρ= is the infradensity in g/cm³

M₀ = oven-dry mass of the wood,

Vs = saturated volume.

Data analysis

All data were analysed using a one-way analysis of variance (ANOVA), followed by Fisher’s test, with a significance level of α = 0.05, using the Rr643.0.2 software.

Measurement of salinity and pH

pH and salinity were measured at the contaminated site in Alénakiri and the uncontaminated site in Ayémé; the results are shown in Table 1. This table shows that the salinity at the polluted site in Alénakiri (39.75 g/l) is higher than that at the unpolluted site in Ayémé (38.5 g/l). Regarding pH, the pH at the polluted site is acidic (4.78), while the pH at the unpolluted site is slightly basic (8.5).

Ash content of A. germinans wood

Figure 2 shows the ash content of the sapwood and heartwood compartments of A. germinans wood collected from the contaminated site at Alénakiri and the uncontaminated site at Ayémé maritime. This figure reveals that ash content is significantly higher (p < 0.05) in the sapwood and heartwood compartments of wood from the contaminated Alénakiri site. This higher ash content in the contaminated wood from Alénakiri can be explained by the presence of heavy metals in the wood [6].

Figure 2: Ash content in A. germinans wood. A: Sapwood. B: Heartwood. E; error bars represent the 95% confidence interval. Different letters indicate groups that show a significant difference (p < 0.05)

Effect of Salinity on the Density of Sapwood Vessels in A. germinans

Figure 3 shows that the vessel density in the sapwood of Alénakiri (62.62 ± 11.36 mm⁻²) is higher than that of Ayémé maritime (55.32 ± 15.4 mm⁻²), which is related to the salinity values at the different sites. The results showed that vessel density and sediment salinity were correlated. However, this difference is not statistically significant (p ⸖ 0.05). This non-significant difference can be explained by the fact that, in addition to the high soil salinity at these two sites, the sapwood compartment plays an important role. Indeed, sapwood is the living part of the wood, as it is through the sapwood vessels that water and mineral salts circulate and are transported to the leaves. Some of these vessels lack thylli and therefore have cavities that can facilitate the transport of these substances to the leaves [9,10]. Thus, salinity influences vessel density in the sapwood. Robert et al. [11] reported that vessel density in mangrove forests increased under conditions of high salinity.

Figure 3: Effect of salinity on the vessel density of the sapwood of A. germinans. The error bar represents the 95% confidence interval. The same letters indicate that the groups show no significant difference (p ⸖ 0.05).

Furthermore, one of the physiological mechanisms by which Avicennia adapts to this fragile environment is the accumulation of salts in the leaf vacuoles. High concentrations of salt accumulate in the leaf vacuoles. To maintain osmotic balance, the leaves then become succulent. The salt concentration in the sap can also be reduced by transferring salt to senescent leaves, the bark, and the wood [12]. Thus, if the sapwood of Alenakiri wood has a higher salt concentration, a significant portion of the salt will be transferred to the heartwood or other organs, which could increase the density of the vessels in the heartwood of its wood.

Effect of salinity on the vessel density of the heartwood of A. germinans

Salinity is one of the abiotic factors that influence mangrove growth. This salinity affects vessel density. Figure 4 shows the vessel density in the heartwood of A. germinans from two sites: Alénakiri and Ayemé maritime.

Figure 4: Effect of salinity on the density of vessels in the heartwood of A. germinans. E: Error bars represent the 95% confidence interval. Different letters indicate groups that show a significant difference (p < 0.05).

This figure shows that the vessel density in the heartwood of Alénakiri (50.82 ± 12.91 mm⁻²) is significantly higher (p < 0.05) than that in the heartwood of Ayémé maritime (40.01 ± 12.34 mm⁻²), which is related to the salinity values at the different sites. The Ayémé and Alénakiri sites have salinity levels of 38.5 g/l and 39.75 g/l, respectively. Indeed, vessel density is linked to salinity levels. In areas with high salinity, there is an increase in vessel density; conversely, under low-salinity conditions, mangroves of the species A. marina, R. mucronata, and L. racemosa exhibit low vessel density [13,14]. It is important to note that the Alénakiri site is contaminated with heavy metals, among other pollutants [6]; therefore, despite heavy metal contamination, salinity takes precedence over pollution, thereby influencing the vessel density in the heartwood of A. germinans.

Impact of pollution on the wood structure

Wood is a material whose internal structure comprises several tissues, including vessels, parenchyma, and growth rings. Figure 5 shows cross-sections of the sapwood and heartwood of A. germinans from the Alénakiri (polluted site) and Ayémé maritime (unpolluted site) locations.

Figure 5: Cross-sections of A. germinans wood showing differences in width between two growth rings. C: heartwood from Alénakiri; D: heartwood from Ayémé maritime. E: sapwood from Alénakiri; F: sapwood from Ayémé

This figure shows that there is a difference in colour between the wood (sapwood and heartwood) of A. germinans collected in Alénakiri and that (sapwood and heartwood) collected in Ayémé maritime. Generally speaking, heartwood is darker than sapwood. However, upon comparison, the heartwood from Alénakiri is darker than that from Ayémé maritime. However, the sapwood from Ayémé is darker than that from Alénakiri. This colour difference can be explained by the nature of the substrate, among other factors. Indeed, edaphic conditions influence the phenotype of the wood, both macroscopically and microscopically [19].

Furthermore, the spacing between the growth rings in A. germinans wood from the two sites is not the same. Cross-sections of the contaminated wood from Alénakiri show wider growth rings than those of the uncontaminated wood from Ayémé maritime. Despite the high soil salinity at the Alénakiri site, the spacing between two growth rings is greater there. However, salinity does indeed influence the distance between two growth rings. Mabicka Iwangou et al. [16] demonstrated that areas with low salinity increased the distance between growth rings of A. germinans growing at a site contaminated with hydrocarbons. In this case, heavy metal pollution appears to affect the distance between two growth rings; this is an area that warrants further  study. Excessive heavy metal pollution causes a series of physiological and biochemical changes affecting plant growth, metabolism, and cellular structure [17].  The diameter of the vessels in the polluted wood from Alénakiri appears to be larger than that in the unpolluted wood from Ayémé; furthermore, the polluted wood from Alénakiri contains fewer clustered vessels. However, given its high salinity, it should have small vessels and appressed vessels. According to [18] and [19], who report, respectively, that high salinity conditions result in small vessels and adjacent vessels. This suggests that heavy metal pollution affects the diameter and arrangement of the vessels.

Impact of pollution on the infradensity of A. germinans sapwood exposed to high salinity

Figure 6 shows the lower density of the sapwood of A. germinans from different sites. The infradensity of the sapwood of A. germinans from the Ayémé maritime site (0.7±0.02 g/cm³) is significantly higher (P < 0.05) than that of A. germinans wood from the polluted Alénakiri site (0.66 ± 0.01 g/cm³). The decrease in the underdensity of the sapwood of A. germinans from the Alénakiri site can be explained by the increase in voids within the wood, which is closely linked to environmental conditions, particularly pollution. Indeed, as mentioned above, A. germinans wood from Alénakiri has wide growth rings and wider, non-cohesive vessels. This demonstrates that pollution has a greater impact than salinity. In fact, in areas of high salinity, the growth rings of A. germinans wood are narrow, the vessels are mostly clustered, and the vessel diameter is small [11]. This leads to an increase in the underdensity and density of A. germinans wood. Areas of high salinity result in narrow growth rings and, consequently, an increase in both the underdensity and density of A. germinans wood [16,19].

Figure 6: Effect of pollution on the infradensity of A. germinans sapwood exposed to high salinity. Different letters indicate groups with significant differences (p < 0.05).

Salinity also influences the density of mangrove wood, which is related to fibre wall thickness and a reduction in the lumen diameter of the vessels. Both factors (diameter and fibre wall thickness) depend on salinity levels. Yáñez Espinosa et al. [20] reported that in low-salinity areas, A. germinans has thin fibre walls; and wood collected from sites with salinities of 30.09 and 12.57 g/l exhibited fibre wall thicknesses of approximately 4.8 µm (with a vascular lumen diameter of 3.2 µm) and 3.8 µm (with a vascular lumen diameter of 5.5 µm), respectively.

Impact of pollution on the infradensity of A. germinans heartwood exposed to high salinity

Unlike Figure 6, which showed that the sapwood of A. germinans from the Ayémé maritime site had significantly lower density than that of the sapwood from the Alénakiri site (p < 0.05), Figure 7, on the other hand, shows that there is no significant difference in the infradensity of the heartwood of A. germinans from the different sites (p < 0.05). The infradensity of the heartwood of A. germinans from the Ayémé maritime site is 0.84 ± 0.046 g/cm³, while that of the heartwood of A. germinans from the Alénakiri site is 0.85 ± 0.032 g/cm³. We observe that the standard deviation of the heartwood underdensity at the Ayémé maritime site is higher, indicating greater variation. This can be explained by physiological conditions, particularly high concentrations of salts that accumulate in the vacuoles. In fact, to cope with high salinity and maintain osmotic balance, the leaves become succulent. The salt concentration in the sap can also be reduced by the transfer of salt to senescent leaves, the bark, and the wood [12]. Thus, it is possible that a significant amount of salt was transferred to other parts of the aforementioned plant, leading to a decrease in salt content in the heartwood and wider growth rings (Figure 8), and consequently a reduction in infradensity.

Figure 7: Effect of pollution on the infradensity of A. germinans heartwood exposed to high salinity. Identical letters indicate that the difference between groups is not statistically significant (p ⸖ 0.05).

Figure 8: Cross-section of the heartwood of A. germinans from the Ayémé maritime site showing wide growth rings.

Thus, the objective of this study was to highlight the impact of inorganic pollution on the woody tissue and the reduced density of Avicennia germinans wood from the Gabon Estuary.

Two abiotic factors, namely salinity and heavy metal pollution, were studied to determine their impact on the properties of A. germinans wood. This study shows that the sapwood and heartwood of polluted wood from the Alénakiri site have a higher ash content than those from the unpolluted Ayémé maritime site. Salinity has a more pronounced effect than pollution, but only on the increase in vessel density in the sapwood and heartwood of A. germinans wood from the Alénakiri site. In contrast, the polluted wood from Alénakiri exhibited changes in its internal structure, notably: wide growth rings, non-adjoining vessels, and large vessels in the heartwood. This led to a decrease in the wood’s infradensity, particularly in the sapwood. Thus, inorganic pollution had a greater impact than salinity.

We plan to expand this study to understand the physiological process that led to wide growth rings and non-adjoining vessels in the polluted wood of A. germinans despite the high soil salinity.