Samples processing
All samples are processed in the laboratory of the company using soft technics preserving the fossil material. Some special processings such as cleaning, sieving and picking of microfossils for carbon and oxygen isotope measurements may be performed.
Calcareous nannofossils
For examining the calcareous nannoplankton, smear-slides are prepared for each sample. The processing starts with the creation of the freshened surface of the sample, using a razor-blade, and a small amount of sediment is scraped from this surface and deposited onto a glass cover slip and diluted with distilled water. The suspension is smeared with a flat-side toothpick along the cover slip and then dried on a hotplate. The cover slip is fixed to a glass slide using Norland Optical Adhesive.
All slides are examined with a LM (light microscope) Olympus a x1,200 magnification in polarized light.
Concerning the condition of preservation of the calcareous nannofossils, the following classes are distinguished:
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good : > 75 % of the identified specimens are ascribed to genus and species level;
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moderate : 50 – 75 % of the identified specimens are ascribed to genus and species level, the rest of specimens may show overgrowths and/or signs of dissolution;
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poor : < 50 % of the identified specimens are ascribed to genus and species level, the rest of specimens may show overgrowths and/or signs of dissolution.
Biostratigraphy of the calcareous nannofossils refers to Martini (1971) and Perch-Nielsen (1985). Absolute age of Tertiary calcareous nannofossil events is indicated according to BugCam (2002) and Gradstein et al., 2012.
Pollen grains and spores
For the analysis of pollen grains and dinoflagellate cysts, each sample is processed using a standard method: acid digestion (HCl, HF, HCl), concentration using ZnCl2(at density 2.0), and sieving at 10 μm. A 50-μl volume of residue is mounted between cover slip and microscope slide using Norland Optical Adhesive. Each sample is examined with a LM (light microscope) Zeiss at x 400, x 600 and x 1,000 magnifications. Pollen grains are counted at x250 magnification, and identified at x1,000, using a AX10 Lab.A1 ZEISS microscope. Their botanical identification is based on detailed morphological analysis supported by the comparison with modern pollen from (1) our reference pollen collection and bank of photographs, (2) atlases and specialised literature. The pollen grains of trees are generally identified at the genus level, sometimes at the species level. Herb pollen grains are usually identified at the family level, sometimes at the genus level, seldom at the species level. Counting is performed until a minimum pollen sum (excluding spores, reworked and unidentified pollen grains) is reached. Taxa are grouped according to the ecological requirements of their present-day representatives. The results are displayed in detailed pollen tables. Percentages of each group are calculated with respect to the total pollen sum (reworked pollen excluded). The percentage of reworked pollen is calculated with respect to the total pollen sum (reworked pollen included). A synthetic pollen diagram displays the variations of the main vegetation groups stacked up to 100%. Identification and interpretation of pollen grains is based on the specialised literature and databases of Geobiostratdata.
Dinoflagellates Cysts
Dinoflagellate cysts are extracted from ca. 20 grams (dry weight) of sediment after the following process: (1) 200 ml of cold HCl (35%) for 2 hours, (2) 100 ml of cold HF (75%) for 24 hours, and (3) 200 ml of cold HCl (35%) for 6 hours. Between each acid treatment, sediments are washed three times. Residues are concentrated using ZnCl2 (density 2.0) and sieved at 10 μm before mounting within glycerol. Dinoflagellate cysts are counted at x250 magnification, and identified at x1,000 or x630, using a AX10 Lab.A1 ZEISS microscope (equiped of specific light source for fluorescence analysis). Their identification is based on detailed morphological analysis supported by comparison with modern and fossil dinoflagellate cyst databases from (1) our reference dinoflagellate cyst collection and bank of photographs, (2) atlases and specialised literature. Taxa are grouped into four groups according to their ecological significance: (1) brackish endemic species, including all morphotypes; (2) marine stenohaline species (i.e., oceanic species); (3) marine euryhaline species; and (4) freshwater algae. The reworked taxa are also counted. Identification for dinoflagellate cysts is based on scientific literature and database of Geobiostratdata.
Foraminifera and other marine organisms
Soft lithologies (clay, marl, silt) are disintegrated in warm solution of sodium carbonate. Harder lithologies (siltstone, claystone, limestone) are crushed by hydraulic crusher and then mechanically treated by rubber stopper on rubber plate. Disintegrated rock is at last washed and sieved at 63 μm and 100 μm. The size fractions 63-100 μm and >100 μm are analysed. Microfossils are manually picked under low-power stereomicroscope. The benthic foraminifer density is calculated from weights of rock, residue and picked residue. When applicable, the % of planktonic foraminifera in foraminifer taphocoenosis is calculated. In very rich samples the count is made planimetrically from picking tray and the result is estimated.
Semi-quantitative evaluation of microfossils distinguishes the following classes:
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*markers supporting interpretation of the sample;
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dominant: the most abundant element of assemblage, 30 % and more;
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abundant: approximately 10-30 %;
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frequent: approximately 5 – 10 %;
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rare: approximately < 5 %;
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reworked.
Taxonomic concept of determined planktonic and benthic foraminifera follows the modern planktonic atlases and manuals.
Biostratigraphy interpretation is based on the regional and global stratigraphic charts.
Diatoms
For selecting diatom material, two complementary methods are used. First, in order to select great diatom frustules that are often dissoluted by chemical treatment, samples are washed with distilled water and great fractions (50 µm and 150 µm) are separated. Picked diatoms from the great fraction are gently applied on a slide and definitely mounted with Eukitt (Mounting medium for microscope preparation). Secondly, in order to obtain the entire diatom association, material is cleaned, mounted and then prepared for permanent slides using the method of Schrader and Gersonde (1978).
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Chemical treatment. The original samples are subject to chemical treatment as follows: 10 g of sediment is placed in a 400 ml beaker with 25 ml of 30% hydrogen peroxide solution (H2O2) and boiled during 30 minutes for oxidation of organic matter. After boiling, the beaker is filled with distilled water and samples are shaken. After 10 seconds, the lighter fraction (in suspension) is moved into another beaker. The coarser material left at the bottom is removed. The beaker with the lighter fraction has to stand for 24 hours. Then the samples are shaken. Floating liquid is decanted and distilled water is added. This process is repeated several times at one-hour interval. The cleaning with Hydrochloric acid (HCl) follows in order to remove the calcareous particles: 25 ml of 30% of HCl are added and boiled during 30 mn. After boiling the samples need to stand for 24 hours. Then, decantation is performed several times at one-hour interval.
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Preparation of permanent slides. The 10 ml diluted sample is shaken carefully and about 0.5 ml is taken in the middle of the beaker using a micropippet; the drop is deposed on a slide and then dried; after drying, a drop of Eukitt is deposed on the slide and a cover glass is applied.
The microsopic slides are analysed under light microspore Zeiss Axioskop 40 with 60X and 100X objectives. A scanning electron microscope GEOL 4600 is also used for fine diatom frustules. For SEM observation, one drop of diluted suspension is placed on circular plot; after drying, ion spattering of gold is carried out for 5 mn.
Charophytes
Recorded since the Late Silurian, Characeae constitute a group of aquatic plants (algae), living in brackish and lacustrine environments. This group is represented by the calcified fructifications (gyrogonites), called charophytes that are preserved in fossil record. Taxonomy is based on the morphological characters of gyrogonites, the high rate of evolution of which since the Mesozoic indicates their sensitivity to geological events. Charophytes are used in biostratigraphy for freshwater and brackish environments. Charophyte fossil remains can be extracted from 2-4 kg of sediment per sample after crumbling in water oxygen peroxide and Na2CO3 solution before sieving at 1 cm, 0.5 mm and 0.2mm. Gyrogonites are picked out under a light microscope, measured at 40x magnification and identified (hundred gyrogonits per species). The biostratigraphic interpretation of a charophytes assemblage is done according to the international scientific bibliography .