Mediterranean Seagrass Posidonia Oceanica Research Articles

Ontogenetic transition from specialized root hairs to specific root-fungus symbiosis in the dominant Mediterranean seagrass Posidonia oceanica

Terrestrial plants typically take up nutrients through roots or mycorrhizae while freshwater plants additionally utilize leaves. Their nutrient uptake may be enhanced by root hairs whose occurrence is often negatively correlated with mycorrhizal colonization. Seagrasses utilize both leaves and roots and often form root hairs, but seem to be devoid of mycorrhizae. The Mediterranean seagrass Posidonia oceanica is an exception: its adults commonly lack root hairs and regularly form a specific association with a single pleosporalean fungus. Here we show that at two sites in the southern Adriatic, all its seedlings possessed abundant root hairs with peculiar morphology (swollen terminal parts) and anatomy (spirally formed cell walls) as apparent adaptations for better attachment to the substrate and increase of breaking strain. Later on, their roots became colonized by dark septate mycelium while root hairs were reduced. In adults, most of terminal fine roots possessed the specific fungal association while root hairs were absent. These observations indicate for the first time that processes regulating transition from root hairs to root fungal colonization exist also in some seagrasses. This ontogenetic shift in root traits may suggests an involvement of the specific root symbiosis in the nutrient uptake by the dominant Mediterranean seagrass.

Experimental evidence of warming-induced flowering in the Mediterranean seagrass Posidonia oceanica

Sexual reproduction in predominantly clonal marine plants increases recombination favoring adaptation and enhancing species resilience to environmental change. Recent studies of the seagrass Posidonia oceanica suggest that flowering intensity and frequency are correlated with warming events associated with global climate change, but these studies have been observational without direct experimental support. We used controlled experiments to test if warming can effectively trigger flowering in P. oceanica. A six-week heat wave was simulated under laboratory mesocosm conditions. Heating negatively impacted leaf growth rates, but by the end of the experiment most of the heated plants flowered, while controls plants did not. Heated and control plants were not genetically distinct and flowering intensity was significantly correlated with allelic richness and heterozygosity. This is an unprecedented finding, showing that the response of seagrasses to warming will be more plastic, more complex and potentially more resilient than previously imagined.

Preservation protocol for dimethylsulfoniopropionate and dimethylsulfoxide analysis in plant material of the Mediterranean seagrass Posidonia oceanica, and re-evaluation of dimethylsulfoniopropionate leaf content

We tested three treatments to preserve Posidonia oceanica leaves for the analysis of dimethylsulfoniopropionate (DMSP) and dimethylsulfoxide (DMSO): oven dried at 60°C for 24h, frozen at −20°C, and frozen-in-ice and kept at −20°C. The DMSP content was analyzed by proxy as dimethylsulfide (DMS) by gas chromatography after alkaline cleavage at room-temperature. The DMSP leaf content of P. oceanica in samples that were oven dried at 60°C for 24h, then stored at room temperature decreased by 87% over 80days of storage and then remained stable for about 88 additional days compared to the control. The DMSO leaf content of P. oceanica in samples that were oven dried increased nine-fold after 198days of storage following drying compared to the control. Both the DMSP and DMSO leaf content of P. oceanica remained stable for 198days compared to the control with frozen and frozen-in-ice treatments, which we both recommend as adequate protocols to preserve P. oceanica tissues for DMS(P,O) analysis. The annual average DMSP leaf content of P. oceanica at 10m in the Bay of Calvi (Corsica, France) was 205±58μmolg−1 (fresh weight) based on samples preserved frozen, two orders of magnitude higher than the value we previously reported based on samples that were oven dried. The newly determined DMSP leaf content allows ranking P. oceanica as the highest DMSP producer reported to date among marine and inter-tidal autotrophs.

Low frequency acoustic properties of Posidonia oceanica seagrass leaf blades.

The acoustics of seagrass meadows impacts naval and oceanographic sonar applications. To study this environment, a one-dimensional resonator was used to assess the low-frequency (1-5 kHz) acoustic response of the leaf blades of the Mediterranean seagrass Posidonia oceanica in water. Three separate collections of plants from Crete, Greece, and Sicily, Italy were investigated. A high consistency in effective sound speed was observed within each collection while a strong variability was observed between different collections. Average size, mass, and epiphytic coverage within each collection were quantified, and discoloration and stiffness are discussed qualitatively with respect to the observed acoustic variability.

A six thousand‐year record of climate and land‐use change from Mediterranean seagrass mats

Summary The Mediterranean seagrass Posidonia oceanica maintains a biodiverse ecosystem and it is a world‐wide important carbon sink. It grows for millennia, accumulating organic‐rich soils (mats) beneath the meadows. This marine habitat is protected by the European Union; however, it is declining rapidly due to coastal development. Understanding its response to disturbances could inform habitat restoration, but many environmental impacts predate monitoring programs (<50 years). This research explores the palaeoecological potential of Posidonia mats to reconstruct six thousand years of environmental change that could have affected Posidonia meadows and, in turn, left an imprint on the mats. Palynological, microcharcoal, magnetic susceptibility and glomalin‐related soil protein (GRSP) analyses on Posidonia mats enabled us to detect climate‐ and human‐induced environmental processes impacting on the seagrass during the Late Holocene. The pollen and microcharcoal records reconstructed anthropogenic disturbances attributed to agriculture. The record of GRSP shows that agrarian activities affected continental soil quality. Changes in magnetic susceptibility reveal that enhanced soil erosion was caused by both climate (major flooding events in the NW Mediterranean) and humans (cultivation) which impacted on the Posidonia mat. Finally, increased human impact is linked to eutrophication of coastal waters since Roman‐Medieval times. Synthesis. This study shows that climate and land‐use changes in the western Mediterranean resulted in enhanced loadings of terrigenous material to the coastal zone since the Late Holocene, likely disturbing the Posidonia meadows and their mat carbon accumulation dynamics. Under the current global change scenario in which CO2 emissions are projected to increase, restoring carbon sinks is a priority. Seagrass habitat restoration should consider not only the coastal perturbations, but also the continental ones at a catchment scale to preserve the socio‐economic ecosystem services provided by seagrasses.

Trace element compartmentation in the seagrass Posidonia oceanica and biomonitoring applications

This study investigated the trace element bioaccumulation capacity of the Mediterranean seagrass Posidonia oceanica, and its suitability as a bioindicator of contamination in water and sediments. Results showed that P. oceanica leaves accumulate higher concentrations of Ni and Zn. Since P. oceanica regenerates its leaves periodically, the higher concentrations in aerial organs may suggest a “removal” strategy according to which P. oceanica accumulates greater concentrations of trace elements in its temporary organs. In turn, P. oceanica seems to adopt an exclusion strategy for toxic non-essential elements (As, Cr, Pb). Results showed also that P. oceanica organs are correlated with As, Cd, Cu, Ni, and Zn concentrations in sediments. No significant relationship was found between P. oceanica and water. This study showed that P. oceanica may adopt different tolerance strategies compared to mainland-rooted macrophytes, and its possible use as a bioindicator of trace elements in sediments should be considered.

Seagrass (Posidonia oceanica) seedlings in a high-CO2 world: from physiology to herbivory.

Under future increased CO2 concentrations, seagrasses are predicted to perform better as a result of increased photosynthesis, but the effects in carbon balance and growth are unclear and remain unexplored for early life stages such as seedlings, which allow plant dispersal and provide the potential for adaptation under changing environmental conditions. Furthermore, the outcome of the concomitant biochemical changes in plant-herbivore interactions has been poorly studied, yet may have important implications in plant communities. In this study we determined the effects of experimental exposure to current and future predicted CO2 concentrations on the physiology, size and defense strategies against herbivory in the earliest life stage of the Mediterranean seagrass Posidonia oceanica. The photosynthetic performance of seedlings, assessed by fluorescence, improved under increased pCO2 conditions after 60 days, although these differences disappeared after 90 days. Furthermore, these plants exhibited bigger seeds and higher carbon storage in belowground tissues, having thus more resources to tolerate and recover from stressors. Of the several herbivory resistance traits measured, plants under high pCO2 conditions had a lower leaf N content but higher sucrose. These seedlings were preferred by herbivorous sea urchins in feeding trials, which could potentially counteract some of the positive effects observed.

Ultrasonic characterization of seagrass leaf blades (Posidonia oceanica)

It has been shown that simple mixture theory models are inadequate to describe low-frequency sound propagation through seagrass meadows and further understanding of the acoustic properties of seagrass tissue is necessary. To that end, we present two ultrasonic sound speed measurements on the Mediterranean seagrass Posidonia oceanica. First, a transit time measurement through a stack of 120 leaf blades at 2.25 MHz are compared to similar measurements made on macroalgae Ecklonia radiata. Sound speed differences are related to the gas content within the aerenchyma of seagrass and other tissue characteristics. Second, ultrasonic sound speed measurements through a suspension of finely divided P. oceanica leaf blades at frequencies between 1 and 4 MHz are compared to low-frequency (1-8 kHz) sound speed obtained on the same blade suspension by means of a resonance chamber.

High rates of microbial dinitrogen fixation and sulfate reduction associated with the Mediterranean seagrass Posidonia oceanica

Seagrass meadows of Posidonia oceanica represent hotspots of productivity in the oligotrophic Mediterranean Sea. The lack of dissolved inorganic nitrogen (DIN) in the seawater suggests that the N-demand of these meadows might be in part supported by microbial dinitrogen (N2) fixation. However, currently there are no direct N2 fixation measurements available for this productive marine macrophyte. Here we investigated N2 fixation activity associated with P. oceanica leaf, rhizome and root pieces. In 15N2 incubations, the roots exhibited highest rates of N2 fixation. The rates varied considerably between replicates, presumably due to a patchy microbial colonization of the roots. Additions of organic carbon compounds (acetate, glucose, sucrose or algal lysate) did not enhance the N2 fixation rates. Sulfate reduction rates measured alongside were also highest in root incubations. Correspondingly, sequences of the nifH gene (a marker gene for the iron protein of the N2-fixing enzyme nitrogenase) related to known sulfate-reducing bacteria were retrieved from P. oceanica roots. Other nifH sequences clustered with known heterotrophic diazotrophs previously identified in other marine macrophytes. In particular, many sequences obtained from P. oceanica roots were similar (>94%) to a saltmarsh rhizosphere-associated heterotrophic diazotroph, indicating that heterotrophic lifestyle might be common among marine macrophyte-associated diazotrophs.

Population ecology of Gibbula umbilicaris and Gibbula ardens (Gastropoda: Trochidae) in a Posidonia oceanica seagrass bed

Population structures of the gastropods Gibbula umbilicaris and Gibbula ardens (Trochidae), the two most common species of the genus Gibbula living in shallow beds of the Mediterranean seagrass Posidonia oceanica, were investigated in the seagrass bed off Lacco Ameno of Ischia (Gulf of Naples), at six stations along a depth gradient (1, 3, 5, 10, 15 and 25 m). Sampling was performed on a monthly basis for 1 year. A total of 853 individuals were counted and measured. The two gastropod populations analysed show a rather similar distributional trend in all seasons and depths. Recruitment occurred at the shallowest stations (1–3 m depth), where the populations were characterised by higher values of abundance and biomass, confirming that G. umbilicaris and G. ardens are typical species of the shallow community of P. oceanica seagrass beds. Juveniles were exclusively found in 1 month of the whole year: G. umbilicaris in April, G. ardens in July. The survivorship curves of both Gibbula species, derived from the size-frequency distribution of shells, showed a very low mortality rate in the first months after settlement. Demographic analysis of G. umbilicaris suggests a rather slow growth rate and a polymorphic maturation strategy with a lifespan of 1 year for most individuals, but lifespan may also reach about 2 years in a few specimens. Gibbula ardens has a lifespan of about 1 year and a fast growth rate, especially during the first couple of months after settlement. Therefore, on the basis of lifespan and recruitment time, both species seem to be semelparous.

Communities of Cultivable Root Mycobionts of the Seagrass Posidonia oceanica in the Northwest Mediterranean Sea Are Dominated by a Hitherto Undescribed Pleosporalean Dark Septate Endophyte.

Seagrasses, a small group of submerged marine macrophytes, were reported to lack mycorrhizae, i.e., the root-fungus symbioses most terrestrial plants use for nutrient uptake. On the other hand, several authors detected fungal endophytes in seagrass leaves, shoots, rhizomes, and roots, and an anatomically and morphologically unique dark septate endophytic (DSE) association has been recently described in the roots of the Mediterranean seagrass Posidonia oceanica. Nevertheless, the global diversity of seagrass mycobionts is not well understood, and it remains unclear what fungus forms the DSE association in P. oceanica roots. We isolated and determined P. oceanica root mycobionts from 11 localities in the northwest Mediterranean Sea with documented presence of the DSE association and compared our results with recent literature. The mycobiont communities were low in diversity (only three species), were dominated by a single yet unreported marine fungal species (ca. 90 % of the total 177 isolates), and lacked common terrestrial and freshwater root mycobionts. Our phylogenetic analysis suggests that the dominating species represents a new monotypic lineage within the recently described Aigialaceae family (Pleosporales, Ascomycota), probably representing a new genus. Most of its examined colonies developed from intracellular microsclerotia occupying host hypodermis and resembling microsclerotia of terrestrial DSE fungi. Biological significance of this hitherto overlooked seagrass root mycobiont remains obscure, but its presence across the NW Mediterranean Sea and apparent root intracellular lifestyle indicate an intriguing symbiotic relationship with the dominant Mediterranean seagrass. Our microscopic observations suggest that it may form the DSE association recently described in P. oceanica roots.

Seasonal and spatial variability of dimethylsulfoniopropionate (DMSP) in the Mediterranean seagrass Posidonia oceanica

We investigated for the first time the occurrence of dimethylsulfoniopropionate (DMSP) in the leaves of Posidonia oceanica and we report its variability during 4 sampling periods covering the seasonal cycle (February, June, August and November) and along a gradient from 10m to 30m depth. The P. oceanica leaf DMSP content expressed per mass of dry weight (dw) ranged from 0.1 to 33.9μmolgdw−1 and averaged 5.0μmolgdw−1. It was higher than the DMSP content of roots and rhizomes that averaged ∼0.5μmolgdw−1. The leaf DMSP content showed seasonal variations, being highest in summer when primary production and biomass of P. oceanica were also highest. In August, the leaf DMSP content showed variations with depth, increasing from 30m to 10m depth. In summer, the leaf DMSP content was highest in the youngest sections of leaves (closest to base) than in the older ones (closest to apex). The seasonal and depth distribution suggest that the DMSP leaf content is positively related to irradiance, hence, we hypothesize that DMSP in P. oceanica plays a role as an antioxidant against reactive oxygen species, although we cannot unambiguously exclude other potential roles such as grazer deterrent. The average leaf DMSP content of P. oceanica is modest compared to high DMSP producing macroalgae and phytoplankton. Yet, the integrated DMSP stock associated to the meadows of P. oceanica is very large due to its enormous biomass, and at the community level it is 2 orders of magnitude higher than the potential integrated DMSP stock related to phytoplankton in the same area.

Evidences of adaptive traits to rocky substrates undermine paradigm of habitat preference of the Mediterranean seagrass Posidonia oceanica.

Posidonia oceanica meadows are acknowledged as one of the most valuable ecosystems of the Mediterranean Sea. P. oceanica has been historically described as a species typically growing on mobile substrates whose development requires precursor communities. Here we document for the first time the extensive presence of sticky hairs covering P. oceanica seedling roots. Adhesive root hairs allow the seedlings to firmly anchor to rocky substrates with anchorage strength values up to 5.23 N, regardless of the presence of algal cover and to colonise bare rock without the need for precursor assemblages to facilitate settlement. Adhesive root hairs are a morphological trait common on plants living on rocks in high-energy habitats, such as the riverweed Podostemaceae and the seagrass Phyllospadix scouleri. The presence of adhesive root hairs in P. oceanica juveniles suggests a preference of this species for hard substrates. Such an adaptation leads to hypothesize a new microsite driven bottleneck in P. oceanica seedling survival linked to substrate features. The mechanism described can favour plant establishment on rocky substrates, in contrast with traditional paradigms. This feature may have strongly influenced P. oceanica pattern of colonisation through sexual propagules in both the past and present.

How do harpacticoid copepods colonize detrital seagrass leaves?

An experiment was carried out investigating the colonization ability and specific pattern of copepods towards a provisional benthic habitat. Since copepods are known to disperse passively and actively, the experiment aimed to investigate the pool of colonizers of macrophytodetritus and the species-specific active colonization pathways. The experiment was performed in a Mediterranean seagrass Posidonia oceanica meadow on defaunated macrophytodetritus accumulations (mainly dead seagrass leaves) for two time intervals (24 and 96 h). Active colonization by copepods, independently of their adjacent potential source pool habitat (bare sandy sediments, P. oceanica canopy, water column and macrophytodetritus) occurred within 24 h. Natural densities (as in the control treatments) were only reached by active colonization through the water column. Both neither diversities nor species composition of natural macrophytodetritus were ever reached by one single migratory pathway, therefore only a combination of interstitial migration and water column migration can explain the species occurrence under natural condition. Moreover, every potential adjacent source pool habitat contributed species to the newly colonized macrophytodetritus. However, the main colonizers were mostly species with good swimming capabilities. The diverse pool of species present in the newly colonized macrophytodetritus underlines the complex communities and dispersion capabilities of copepods. Hence, macrophytodetritus possesses the potential ability to be a colonizer source pool for every adjacent habitat and thus behaves as a copepod hub for the entire seagrass ecosystem.

Photoacclimation of Caulerpa cylindracea: Light as a limiting factor in the invasion of native Mediterranean seagrass meadows

Reduction in light availability caused by the canopy of the Mediterranean seagrass Posidonia oceanica has been suggested as a critical mechanism to resist the invasion of the exotic macroalga Caulerpa cylindracea. We experimentally evaluated the role of light as a limiting factor on the capacity of colonization and spread of this invasive seaweed in P. oceanica meadows by assessing photoacclimation responses and productivity and growth capacity of C. cylindracea in mesocosm and in situ light manipulation experiments. Despite the high photoacclimative plasticity developed by the alga, the light regime within the seagrass meadow during the study period was close to the minimum light requirements for growth, restricting the development capacity of this species. In addition, while increases in light availability resulting from canopy alteration also enhanced the productive capacity of the invasive seaweed in the field, such increase was not followed by gains in biomass production. Our results thus support the hypothesis that light availability has a major role in the underlying resistance of seagrass meadows to the invasion by C. cylindracea, but also indicate that there are additional factors related to the canopy of P. oceanica that further hinder the growth and colonization capacity of the alga.

The exergy of a phase shift: Ecosystem functioning loss in seagrass meadows of the Mediterranean Sea

Sustained functioning of ecosystems is predicted to depend upon the maintenance of their biodiversity, structure and integrity. The large consensus achieved in this regard, however, faces to the objective difficulty of finding appropriate metrics to measure ecosystem functioning. Here, we aim at evaluating functional consequence of the phase shift occurring in meadows of the Mediterranean seagrass Posidonia oceanica, a priority habitat that is undergoing regression in many coastal areas due to multiple human pressures. Structural degradation of the P. oceanica ecosystem, consequent to increasing coastal exploitation and climate change, may result in the progressive replacement of this seagrass by opportunistic macrophytes, either native or alien. Reviewing published information and our personal records, we measured changes in biological habitat provisioning, species richness and biomass associated to each of the alternative states characterizing the phase shift. Then, ecosystem functioning was assessed by computing the exergy associated to each state, exergy being a state variable that measures the ecosystem capacity to produce work. Phase shift was consistently shown to imply loss in habitat provision, species richness, and biomass; structural and compositional loss was parallelled by a reduction of exergy content, thus providing for the first time an objective and integrative measure of the loss of ecosystem functioning following the degradation of healthy seagrass meadows.

An ecosystem-based approach to assess the status of a Mediterranean ecosystem, the Posidonia oceanica seagrass meadow.

Biotic indices, which reflect the quality of the environment, are widely used in the marine realm. Sometimes, key species or ecosystem engineers are selected for this purpose. This is the case of the Mediterranean seagrass Posidonia oceanica, widely used as a biological quality element in the context of the European Union Water Framework Directive (WFD). The good quality of a water body and the apparent health of a species, whether or not an ecosystem engineer such as P. oceanica, is not always indicative of the good structure and functioning of the whole ecosystem. A key point of the recent Marine Strategy Framework Directive (MSFD) is the ecosystem-based approach. Here, on the basis of a simplified conceptual model of the P. oceanica ecosystem, we have proposed an ecosystem-based index of the quality of its functioning, compliant with the MSFD requirements. This index (EBQI) is based upon a set of representative functional compartments, the weighting of these compartments and the assessment of the quality of each compartment by comparison of a supposed baseline. The index well discriminated 17 sites in the north-western Mediterranean (French Riviera, Provence, Corsica, Catalonia and Balearic Islands) covering a wide range of human pressure levels. The strong points of the EBQI are that it is easy to implement, non-destructive, relatively robust, according to the selection of the compartments and to their weighting, and associated with confidence indices that indicate possible weakness and biases and therefore the need for further field data acquisition.

Experimental in situ exposure of the seagrass Posidonia oceanica (L.) Delile to 15 trace elements

The Mediterranean seagrass Posidonia oceanica (L.) Delile has been used for trace element (TE) biomonitoring since decades ago. However, present informations for this bioindicator are limited mainly to plant TE levels, while virtually nothing is known about their fluxes through P. oceanica meadows. We therefore contaminated seagrass bed portions in situ at two experimental TE levels with a mix of 15 TEs (Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Mo, Ag, Cd, Pb and Bi) to study their uptake and loss kinetics in P. oceanica. Shoots immediately accumulated pollutants from the beginning of exposures. Once contaminations ended, TE concentrations came back to their original levels within two weeks, or at least showed a clear decrease. P. oceanica leaves exhibited different uptake kinetics depending on elements and leaf age: the younger growing leaves forming new tissues incorporated TEs more rapidly than the older senescent leaves. Leaf epiphytes also exhibited a net uptake of most TEs, partly similar to that of P. oceanica shoots. The principal route of TE uptake was through the water column, as no contamination of superficial sediments was observed. However, rhizomes indirectly accumulated many TEs during the overall experiments through leaf to rhizome translocation processes. This study thus experimentally confirmed that P. oceanica shoots are undoubtedly an excellent short-term bioindicator and that long-term accumulations could be recorded in P. oceanica rhizomes.

Effects of salinity on seed germination and early seedling growth of the Mediterranean seagrass Posidonia oceanica (L.) Delile

Previous research has demonstrated the low tolerance of Posidonia oceanica mature shoots to salinity variability but there is no information about the response of its seeds and seedlings to this impact. In the present study, two independent experiments were conducted to investigate the effects of salinity variation on germination, development and survival of P. oceanica seeds and seedlings under laboratory-controlled conditions. Maximum P. oceanica seed germination occurred at the control salinity (37), while mortality was lowest for this treatment. However, no significant differences were detected with other salinity levels (39–49). In contrast, salinity appears to play an important role in seedling development. The number of blades, the length of the primary root and the maximum length of leaves of seedlings were significantly reduced with increased salinities. The results observed in the present study are consistent with those obtained for mature P. oceanica shoots, and suggest that hypersalinities, such as those associated with brine discharge of a desalination plant, may limit P. oceanica seedling recruitment, affecting the expansion and recovery of Posidonia meadows.

Responses of the Mediterranean seagrass Posidonia oceanica to hypersaline stress duration and recovery

We studied the hypersaline stress responses of the Mediterranean seagrass Posidonia oceanica to determine if the species was tolerant to salinity increases that occur in coastal waters by the desalination industry. Water relations, amino acids, carbohydrates, ions, photosynthesis, respiration, chlorophyll a fluorescence, leaf growth and morphology, and plant mortality were analysed after exposing the mesocosm P. oceanica to a salinity level of 43 for one and three months followed by a month for recovery. One-month saline-stressed plants exhibited sub-lethal effects, including a leaf cell turgor pressure reduction, loss of ionic equilibrium and decreased leaf growth. There were also changes in photoprotective mechanisms, increased concentrations of organic osmolytes in leaves and reduced leaf ageing. All these dysfunctions recovered after removing the stress. After the longer exposure of three months, stress symptoms were much more acute and plants showed an excessive ionic exclusion capacity, increased leaf cell turgor, reduced plant carbon balance, increased leaf aging and leaf decay and increased plant mortality, which indicated that the plant had entered a stage of severe physiological stress. In addition, the long-term saline-stressed plants were not able to recover, still showing sustained injury after the one-month recovery period as reflected by unbalanced leaf ionic content, persistently impaired photosynthesis, decline in internal carbon resources and decreased leaf growth that resulted in undersized plants. In conclusion, P. oceanica was not able to acclimate to the saline conditions tested since it could not reach a new physiological equilibrium or recover after a chronic exposure of 3 months.