The effects of environmental factors on biomass and microcystin production by the freshwater cyanobacterial genera Microcystis and Anabaena

Several cyanobacterial genera produce the hepatotoxins, microcystins. Microcystins are produced only in cells that have microcystin synthetase gene (mcy) clusters, which encode enzyme complexes involved in microcystin biosynthesis. Microcystin-producing and nonmicrocystin-producing genotypes of single cyanobacterial genus may occur simultaneously in situ. Previously, the effects of environmental factors on the growth and microcystin production of cyanobacteria have mainly been studied by means of isolated cyanobacteria cultures in the laboratory. Studies in the field have been difficult, owing to the lack of methods to identify and quantify the different genotypes. In this study, genus-specific microcystin synthetase E (mcyE) gene primers were designed and a method to identify and quantify the mcyE copy numbers was developed and used in situ. Microcystis and Anabaena mcyE genes were observed in two Finnish lakes. Microcystis appeared to be the most abundant microcystin producer in Lake Tuusulanjärvi and in one basin of Lake Hiidenvesi. Because the most potent microcystin-producing genus of a lake can be identified, it will be possible in the future to design genus-targeted strategies for lake restoration. Effects of P and N concentrations on the biomass of microcystin-producing and nonmicrocystin-producing Microcystis strains and an Anabaena strain were studied in cultures. P and N concentrations and their combined effect increased cyanobacterial biomass of all Microcystis strains. The biomass of microcystin-producing Microcystis was higher than that of nonmicrocystin-producing strains at high nutrient concentrations. The P concentration increased Anabaena biomass, but the effect of N concentration was statistically insignificant for growth yield, probably due to the ability of the genus to fix molecular N2. P and N concentrations and combined nutrients caused an increase in cellular microcystin concentrations of the Microcystis strain cultivated in chemostat cultures. Cyanobacteria are able to hydrolyse nutrients from organic matter through extracellular enzyme activities. Leucine aminopeptidase (LAP) activity was observed in an axenic N2-fixing Anabaena strain grown in batch cultures. The P concentration caused a statistically significant increase in LAP activity, whereas the effect of N concentration was insignificant. The highest LAP activities were observed in the most eutrophic basins of Lake Hiidenvesi. LAP activity probably originated mostly from attached heterotrophic bacteria and less from cyanobacteria.

Genetic and environmental influences on human responses to odors

Olfaction, the sense of smell, has many important functions in humans. Human responses to odors show substantial individual variation. Olfactory receptor genes have been identified and other genes may also influence olfaction. However, the proportion of phenotypic variation in odor response due to genetic variation remains largely unknown. Little is also known about which genes modify specific responses to odors. This study aimed to elucidate genetic and environmental influences on human responses to odors. Individuals from Finnish families (n=146) and Australian (n=413), British (n=163), Danish (n=336), and Finnish (n=399) twins rated intensity and pleasantness of a set of 12 (families) or 6 (twins) odors and tried to identify the odors. In addition, the participants rated their own sense of smell and annoyance experienced with different environmental odors. The odor stimuli of a commercial smell test (The Brief Smell Identification Test; banana, chocolate, cinnamon, gasoline, lemon, onion, paint thinner, pineapple, rose, smoke, soap, and turpentine) were presented in the family study. Based on the results of the family study and a literature survey, a new set of odor stimuli (androstenone, chocolate, cinnamon, isovaleric acid, lemon, and turpentine) was designed for the twin studies. In the family sample, heritabilities of the traits were estimated and underlying genomic regions were searched using a genome-wide linkage scan. In the pooled twin sample, variation in the measured traits was decomposed into genetic and environmental components using quantitative genetic modeling. In addition, associations between nongenetic factors (e.g., sex, age, and smoking) and olfactory-related traits were explored. Suggestive evidence for a genetic linkage for pleasantness of cinnamon at a locus on chromosome 4q32.3 emerged from the family sample. High heritability for the pleasantness of cinnamon was found in the family but not the twin study. Heritability of perceived intensity of androstenone odor was determined to be ~30% in the twin sample. A strong genetic correlation between perceived intensity and pleasantness of androstenone, in the absence of any environmental correlation, indicated that only the genetic correlation explained the phenotypic correlation between the traits (r=-0.27) and that the traits were influenced by an overlapping set of genes. Self-rated olfactory function appeared to reflect the odor annoyance experienced rather than actual olfactory acuity or genetic involvement. Results from nongenetic analyses supported the speculated superiority of females' olfactory abilities, the age-related diminishing of olfactory acuity, and the influences of experience-dependent factors on odor responses. This was the first study to estimate heritabilities and perform linkage screens for individual odors. A genetic effect was detected for only a few responses to specific odors, suggesting the predominance of environmental effects in odor perceptions.

A matter of taste : genetic and environmental influences on responses to sweetness

Diet is a major player in the maintenance of health and onset of many diseases of public health importance. The food choice is known to be largely influenced by sensory preferences. However, in many cases it is unclear whether these preferences and dietary behaviors are innate or acquired. The aim of this thesis work was to study the extent to which the individual differences in dietary responses, especially in liking for sweet taste, are influenced by genetic factors. Several traits measuring the responses to sweetness and other dietary variables were applied in four studies: in British (TwinsUK) and Finnish (FinnTwin12 and FinnTwin16) twin studies and in a Finnish migraine family study. All the subjects were adults and they participated in chemosensory measurements (taste and smell tests) and filled in food behavior questionnaires. Further, it was studied, whether the correlations among the variables are mediated by genetic or environmental factors and where in the genome the genes influencing the heritable traits are located. A study of young adult Finnish twins (FinnTwin16, n=4388) revealed that around 40% of the food use is attributable to genetic factors and that the common, childhood environment does not affect the food use even shortly after moving from the parents home. Both the family study (n=146) and the twin studies (British twins, n=663) showed that around half of the variation in the liking for sweetness is inherited. The same result was obtained both by the chemosensory measurements (heritability 41-49%) and the questionnaire variables (heritability 31-54%). By contrast, the intensity perception of sweetness or the responses to saltiness were not influenced by genetic factors. Further, a locus influencing the use-frequency of sweet foods was identified on chromosome 16p. A closer examination of the relationships among the variables based on 663 British twins revealed that several genetic and environmental correlations exist among the different measures of liking for sweetness. However, these correlations were not very strong (range 0.06-0.55) implying that the instruments used measure slightly different aspects of the phenomenon. In addition, the assessment of the associations among responses to fatty foods, dieting behaviors, and body mass index in twin populations (TwinsUK n=1027 and FinnTwin12 n=299) showed that the dieting behaviors (cognitive restraint, uncontrolled eating, and emotional eating) mediate the relationship between obesity and diet. In conclusion, the work increased the understanding of the background variables of human eating behavior. Genetic effects were shown to underlie the variation of many dietary traits, such as liking for sweet taste, use of sweet foods, and dieting behaviors. However, the responses to salty taste were shown to be mainly determined by environmental factors and thus should more easily be modifiable by dietary education, exposure, and learning than sweet taste preferences. Although additional studies are needed to characterize the genetic element located on chromosome 16 that influences the use-frequency of sweet foods, the results underline the importance of inherited factors on human eating behavior.

Miniaturized mass spectrometric ionization techniques for environmental analysis and bioanalysis

Miniaturized mass spectrometric ionization techniques for environmental analysis and bioanalysis Novel miniaturized mass spectrometric ionization techniques based on atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) were studied and evaluated in the analysis of environmental samples and biosamples. The three analytical systems investigated here were gas chromatography-microchip atmospheric pressure chemical ionization-mass spectrometry (GC-µAPCI-MS) and gas chromatography-microchip atmospheric pressure photoionization-mass spectrometry (GC-µAPPI-MS), where sample pretreatment and chromatographic separation precede ionization, and desorption atmospheric pressure photoionization-mass spectrometry (DAPPI-MS), where the samples are analyzed either as such or after minimal pretreatment. The gas chromatography-microchip atmospheric pressure ionization-mass spectrometry (GC-µAPI-MS) instrumentations were used in the analysis of polychlorinated biphenyls (PCBs) in negative ion mode and 2-quinolinone-derived selective androgen receptor modulators (SARMs) in positive ion mode. The analytical characteristics (i.e., limits of detection, linear ranges, and repeatabilities) of the methods were evaluated with PCB standards and SARMs in urine. All methods showed good analytical characteristics and potential for quantitative environmental analysis or bioanalysis. Desorption and ionization mechanisms in DAPPI were studied. Desorption was found to be a thermal process, with the efficiency strongly depending on thermal conductivity of the sampling surface. Probably the size and polarity of the analyte also play a role. In positive ion mode, the ionization is dependent on the ionization energy and proton affinity of the analyte and the spray solvent, while in negative ion mode the ionization mechanism is determined by the electron affinity and gas-phase acidity of the analyte and the spray solvent. DAPPI-MS was tested in the fast screening analysis of environmental, food, and forensic samples, and the results demonstrated the feasibility of DAPPI-MS for rapid screening analysis of authentic samples.

Development of analytical techniques for studies on dispersion of actinides in the environment and characterization of environmental radioactive particles

Radioactive particles from three locations were investigated for elemental composition, oxidation states of matrix elements, and origin. Instrumental techniques applied to the task were scanning electron microscopy, X-ray and gamma-ray spectrometry, secondary ion mass spectrometry, and synchrotron radiation based microanalytical techniques comprising X-ray fluorescence spectrometry, X-ray fluorescence tomography, and X-ray absorption near-edge structure spectroscopy. Uranium-containing low activity particles collected from Irish Sea sediments were characterized in terms of composition and distribution of matrix elements and the oxidation states of uranium. Indications of the origin were obtained from the intensity ratios and the presence of thorium, uranium, and plutonium. Uranium in the particles was found to exist mostly as U(IV). Studies on plutonium particles from Runit Island (Marshall Islands) soil indicated that the samples were weapon fuel fragments originating from two separate detonations: a safety test and a low-yield test. The plutonium in the particles was found to be of similar age. The distribution and oxidation states of uranium and plutonium in the matrix of weapon fuel particles from Thule (Greenland) sediments were investigated. The variations in intensity ratios observed with different techniques indicated more than one origin. Uranium in particle matrixes was mostly U(IV), but plutonium existed in some particles mainly as Pu(IV), and in others mainly as oxidized Pu(VI). The results demonstrated that the various techniques were effectively applied in the characterization of environmental radioactive particles. An on-line method was developed for separating americium from environmental samples. The procedure utilizes extraction chromatography to separate americium from light lanthanides, and cation exchange to concentrate americium before the final separation in an ion chromatography column. The separated radiochemically pure americium fraction is measured by alpha spectrometry. The method was tested with certified sediment and soil samples and found to be applicable for the analysis of environmental samples containing a wide range of Am-241 activity. Proceeding from the on-line method developed for americium, a method was also developed for separating plutonium and americium. Plutonium is reduced to Pu(III), and separated together with Am(III) throughout the procedure. Pu(III) and Am(III) are eluted from the ion chromatography column as anionic dipicolinate and oxalate complexes, respectively, and measured by alpha spectrometry.

Development of analytical methods for the separation of plutonium, americium, curium and neptunium from environmental samples

In this work, separation methods have been developed for the analysis of anthropogenic transuranium elements plutonium, americium, curium and neptunium from environmental samples contaminated by global nuclear weapons testing and the Chernobyl accident. The analytical methods utilized in this study are based on extraction chromatography. Highly varying atmospheric plutonium isotope concentrations and activity ratios were found at both Kurchatov (Kazakhstan), near the former Semipalatinsk test site, and Sodankylä (Finland). The origin of plutonium is almost impossible to identify at Kurchatov, since hundreds of nuclear tests were performed at the Semipalatinsk test site. In Sodankylä, plutonium in the surface air originated from nuclear weapons testing, conducted mostly by USSR and USA before the sampling year 1963. The variation in americium, curium and neptunium concentrations was great as well in peat samples collected in southern and central Finland in 1986 immediately after the Chernobyl accident. The main source of transuranium contamination in peats was from global nuclear test fallout, although there are wide regional differences in the fraction of Chernobyl-originated activity (of the total activity) for americium, curium and neptunium.

Eurasian Neogene large herbivorous mammals and climate

The development and changes in the distribution of herbivorous mammal communities during the Neogene is complex. The Eurasian scale environmental patterns reflect the large scale geographical and climatic patterns. The reorganization of these affect the biome distribution throughout the continent. The distribution of mammal taxa was closely associated with the distribution of biomes. In Eurasia the Neogene development of environments was twofold. The early and middle Miocene that seemed to have been advantageous for mammals was followed by drying of environments during the late Neogene. The mid-latitude drying was the main trend, and it is the combined result of the retreat of Paratethys, the uplift of Tibetan Plateau and changes in the ocean currents and temperatures. The common mammals were "driving" the evolution of mammalian communities. During the late Miocene we see the drying affecting more and more regions, and we see changes in the composition of mammalian communities.

Sexual reproduction in chydorid cladocerans (Anomopoda, Chydoridae) in southern Finland - implications for paleolimnology

The relationship between sexual reproduction of littoral chydorid cladocerans (Anomopoda, Chydoridae) and environmental factors in aquatic ecosystems has been rarely studied, although the sexual behavior of some planktonic cladocerans is well documented. Ecological monitoring was used to study the relationship between climate-related and non-climatic environmental factors and chydorid sexual reproduction patterns in nine environmentally different lakes that were closely situated to each other in southern Finland. Furthermore, paleolimnological ephippium analysis was used to clarify how current sexual reproduction is reflected in surface sediments of the same nine lakes. Additionally, short sediment cores from two of the lakes were studied with ephippium analysis to examine how recent climate-related and non-climatic environmental changes were reflected in chydorid sexual reproduction. Ephippium analysis uses the subfossil shells of asexual individuals to represent asexual reproduction and the shells of sexual females, i.e. ephippia, to represent sexual reproduction. The relative proportion of ephippia of all chydorid species, i.e. total chydorid ephippia (TCE) indicates the relative proportion of sexual reproduction during the open-water season. This thesis is part of the EPHIPPIUM-project which aims to develop ephippium analysis towards a quantitative climate reconstruction tool. To be able to develop a valid climate model, the influence of the environmental stressors other than climate on contemporary sexual reproduction and its reflection in sediment assemblages must be clarified so they can be eliminated from the model. During contemporary monitoring a few sexual individuals were observed during summer, apparently forced to sexual reproduction by non-climatic local environmental factors, such as crowding or invertebrate predation. Monitoring also revealed that the autumnal chydorid sexual reproduction period was consistent between the different lakes and climate-related factors appeared to act as the main inducers and regulators of autumnal sexual reproduction. However, during autumn, chydorid species and populations among the lakes exhibited a wide variation in the intensity, induction time, and length of autumnal sexual reproduction. These variations apparently act as mechanisms for local adaptations due to the genetic variability provided by sexual reproduction that enhance the ecological flexibility of chydorid species, allowing them to inhabit a wide range of environments. A large variation was also detected in the abundance of parthenogenetic and gamogenetic individuals during the open-water season among the lakes. On the basis of surface sediment samples, the general level of the TCE is ca. 3-4% in southern Finland, reflecting an average proportion of sexual reproduction in this specific climate. The variation in the TCE was much lower than could be expected on the basis of the monitoring results. This suggests that some of the variation detected by monitoring may derive from differences between sampling sites and years smoothed out in the sediment samples, providing an average of the entire lake area and several years. The TCE is always connected to various ecological interactions in lake ecosystems and therefore is always lake-specific. Hypothetically, deterioration of climate conditions can be detected in the TCE as an increase in ephippia of all chydorid species, since a shortening open-water season is reflected in the relative proportions of the two reproduction modes. Such an increase was clearly detected for the time period of the Little Ice Age in a sediment core. The paleolimnological results also indicated that TCE can suddenly increase due to ephippia of one or two species, which suggests that at least some chydorids can somehow increase the production of resting eggs under local environmental stress. Thus, some environmental factors may act as species-specific environmental stressors. The actual mechanism of the increased sexual reproduction seen in sediments has been unknown but the present study suggests that the mechanism is probably the increased intensity of gamogenesis, i.e. that a larger proportion of individuals in autumnal populations reproduce sexually, which results in a larger proportion of ephippia in sediments and a higher TCE. The results of this thesis demonstrate the utility of ephippium analysis as a paleoclimatological method which may also detect paleolimnological changes by identifying species-specific environmental stressors. For a quantitative TCE-based climate reconstruction model, the natural variation in the TCE of surface sediments in different climates must be clarified with more extensive studies. In addition, it is important to recognize the lakes where the TCE is not only a reflection of the length of the open-water season, but is also non-climatically forced. The results of ephippium analysis should always be interpreted in a lake-specific manner and in the context of other paleoecological proxies.

Postglacial climate changes and vegetation responses in northern Europe

Postglacial climate changes and vegetation responses were studied using a combination of biological and physical indicators preserved in lake sediments. Low-frequency trends, high-frequency events and rapid shifts in temperature and moisture balance were probed using pollen-based quantitative temperature reconstructions and oxygen-isotopes from authigenic carbonate and aquatic cellulose, respectively. Pollen and plant macrofossils were employed to shed light on the presence and response rates of plant populations in response to climate changes, particularly focusing on common boreal and temperate tree species. Additional geochemical and isotopic tracers facilitated the interpretation of pollen- and oxygen-isotope data. The results show that the common boreal trees were present in the Baltic region (~55°N) during the Lateglacial, which contrasts with the traditional view of species refuge locations in the south-European peninsulas during the glacial/interglacial cycles. The findings of this work are in agreement with recent paleoecological and genetic evidence suggesting that scattered populations of tree species persisted at higher latitudes, and that these taxa were likely limited to boreal trees. Moreover, the results demonstrate that stepwise changes in plant communities took place in concert with major climate fluctuations of the glacial/interglacial transition. Postglacial climate trends in northern Europe were characterized by rise, maxima and fall in temperatures and related changes in moisture balance. Following the deglaciation of the Northern Hemisphere and the early Holocene reorganization of the ice-ocean-atmosphere system, the long-term temperature trends followed gradually decreasing summer insolation. The early Holocene (~11,700-8000 cal yr BP) was overall cool, moist and oceanic, although the earliest Holocene effective humidity may have been low particularly in the eastern part of northern Europe. The gradual warming trend was interrupted by a cold event ~8200 cal yr BP. The maximum temperatures, ~1.5-3.0°C above modern values, were attained ~8000-4000 cal yr BP. This mid-Holocene peak warmth was coupled with low lake levels, low effective humidity and summertime drought. The late Holocene (~4000 cal yr BP-present) was characterized by gradually decreasing temperatures, higher lake levels and higher effective humidity. Moreover, the gradual trends of the late Holocene were probably superimposed by higher-frequency variability. The spatial variability of the Holocene temperature and moisture balance patterns were tentatively attributed to the differing heat capacities of continents and oceans, changes in atmospheric circulation modes and position of sites and subregions with respect to large water bodies and topographic barriers. The combination of physical and biological proxy archives is a pivotal aspect of this work, because non-climatic factors, such as postglacial migration, disturbances and competitive interactions, can influence reshuffling of vegetation and hence, pollen-based climate reconstructions. The oxygen-isotope records and other physical proxies presented in this work manifest that postglacial climate changes were the main driver of the establishment and expansion of temperate and boreal tree populations, and hence, large-scale and long-term vegetation patterns were in dynamic equilibrium with climate. A notable exception to this pattern may be the postglacial invasion of Norway spruce and the related suppression of mid-Holocene temperate forest. This salient step in north-European vegetation history, the development of the modern boreal ecosystem, cannot be unambiguously explained by current evidence of postglacial climate changes. The results of this work highlight that plant populations, including long-lived trees, may be able to respond strikingly rapidly to changes in climate. Moreover, interannual and seasonal variation and extreme events can exert an important influence on vegetation reshuffling. Importantly, the studies imply that the presence of diffuse refuge populations or local stands among the prevailing vegetation may have provided the means for extraordinarily rapid vegetation responses. Hence, if scattered populations are not provided and tree populations are to migrate long distances, their capacity to keep up with predicted rates of future climate change may be lower than previously thought.

Environmental factors affecting the occurrence of periglacial landforms in Finnish Lapland: a numerical approach

Determination of the environmental factors controlling earth surface processes and landform patterns is one of the central themes in physical geography. However, the identification of the main drivers of the geomorphological phenomena is often challenging. Novel spatial analysis and modelling methods could provide new insights into the process-environment relationships. The objective of this research was to map and quantitatively analyse the occurrence of cryogenic phenomena in subarctic Finland. More precisely, utilising a grid-based approach the distribution and abundance of periglacial landforms were modelled to identify important landscape scale environmental factors. The study was performed using a comprehensive empirical data set of periglacial landforms from an area of 600 km2 at a 25-ha resolution. The utilised statistical methods were generalized linear modelling (GLM) and hierarchical partitioning (HP). GLMs were used to produce distribution and abundance models and HP to reveal independently the most likely causal variables. The GLM models were assessed utilising statistical evaluation measures, prediction maps, field observations and the results of HP analyses. A total of 40 different landform types and subtypes were identified. Topographical, soil property and vegetation variables were the primary correlates for the occurrence and cover of active periglacial landforms on the landscape scale. In the model evaluation, most of the GLMs were shown to be robust although the explanation power, prediction ability as well as the selected explanatory variables varied between the models. The great potential of the combination of a spatial grid system, terrain data and novel statistical techniques to map the occurrence of periglacial landforms was demonstrated in this study. GLM proved to be a useful modelling framework for testing the shapes of the response functions and significances of the environmental variables and the HP method helped to make better deductions of the important factors of earth surface processes. Hence, the numerical approach presented in this study can be a useful addition to the current range of techniques available to researchers to map and monitor different geographical phenomena.

Topographical, structural and geophysical characterization of fracture zones: implications for groundwater flow and vulnerability

The main objective of this study is to evaluate selected geophysical, structural and topographic methods on regional, local, and tunnel and borehole scales, as indicators of the properties of fracture zones or fractures relevant to groundwater flow. Such information serves, for example, groundwater exploration and prediction of the risk of groundwater inflow in underground construction. This study aims to address how the features detected by these methods link to groundwater flow in qualitative and semi-quantitative terms and how well the methods reveal properties of fracturing affecting groundwater flow in the studied sites. The investigated areas are: (1) the Päijänne Tunnel for water-conveyance whose study serves as a verification of structures identified on regional and local scales; (2) the Oitti fuel spill site, to telescope across scales and compare geometries of structural assessment; and (3) Leppävirta, where fracturing and hydrogeological environment have been studied on the scale of a drilled well. The methods applied in this study include: the interpretation of lineaments from topographic data and their comparison with aeromagnetic data; the analysis of geological structures mapped in the Päijänne Tunnel; borehole video surveying; groundwater inflow measurements; groundwater level observations; and information on the tunnel s deterioration as demonstrated by block falls. The study combined geological and geotechnical information on relevant factors governing groundwater inflow into a tunnel and indicators of fracturing, as well as environmental datasets as overlays for spatial analysis using GIS. Geophysical borehole logging and fluid logging were used in Leppävirta to compare the responses of different methods to fracturing and other geological features on the scale of a drilled well. Results from some of the geophysical measurements of boreholes were affected by the large diameter (gamma radiation) or uneven surface (caliper) of these structures. However, different anomalies indicating more fractured upper part of the bedrock traversed by well HN4 in Leppävirta suggest that several methods can be used for detecting fracturing. Fracture trends appear to align similarly on different scales in the zone of the Päijänne Tunnel. For example, similarities of patterns were found between the regional magnetic trends, correlating with orientations of topographic lineaments interpreted as expressions of fracture zones. The same structural orientations as those of the larger structures on local or regional scales were observed in the tunnel, even though a match could not be made in every case. The size and orientation of the observation space (patch of terrain at the surface, tunnel section, or borehole), the characterization method, with its typical sensitivity, and the characteristics of the location, influence the identification of the fracture pattern. Through due consideration of the influence of the sampling geometry and by utilizing complementary fracture characterization methods in tandem, some of the complexities of the relationship between fracturing and groundwater flow can be addressed. The flow connections demonstrated by the response of the groundwater level in monitoring wells to pressure decrease in the tunnel and the transport of MTBE through fractures in bedrock in Oitti, highlight the importance of protecting the tunnel water from a risk of contamination. In general, the largest values of drawdown occurred in monitoring wells closest to the tunnel and/or close to the topographically interpreted fracture zones. It seems that, to some degree, the rate of inflow shows a positive correlation with the level of reinforcement, as both are connected with the fracturing in the bedrock. The following geological features increased the vulnerability of tunnel sections to pollution, especially when several factors affected the same locations: (1) fractured bedrock, particularly with associated groundwater inflow; (2) thin or permeable overburden above fractured rock; (3) a hydraulically conductive layer underneath the surface soil; and (4) a relatively thin bedrock roof above the tunnel. The observed anisotropy of the geological media should ideally be taken into account in the assessment of vulnerability of tunnel sections and eventually for directing protective measures.