In order to obtain good-quality slides, all samples used in this study were prepared using a settling technique. Approximately 1000 samples were processed and at least one sample was examined from each section of core. Different settling techniques have been described in published literature mainly to prepare samples used for scanning electron microscope (SEM) studies (e.g., Hay 1977; Knüttel 1986; Backman 1980). The settling techniques are used to eliminate particles sized below 2 μm and above 30 µm. In this study, a special settling technique has been developed to prepare slides for the light microscope (LM).
A constant volume of 25 mm3 of sediment was put into a graduated 15 mL test tube, which was filled with distilled water to 8 mL. The sample was shaken several times within an hour and then held still for 30 s to let the larger particles sink to the bottom. A capillary tube was used to remove the bottom solution down to 3 mL, and water was added to 15 mL. Next, the sample was shaken several times for 5 min and then held still for 90 s. A graduated capillary tube was used to place 8 mL of solution from the top of the tube on a cover slide. Up to 30 samples could be processed at regular intervals during an hour.
The same settling technique was used to prepare a gridded circular cover slide for transferring and observing the same specimen in both LM and SEM. Eight drops on a gridded cover slide usually provided a good concentration of nannofossils for both LM and SEM. Twenty-two transferred specimens are illustrated on the 14 plates. The specimen transfer technique used herein was developed by Dr. Frank H. Wind at the Florida State University.
This settling technique, using a constant volume of sediment and solution, has two important advantages: first, it is a relatively quick method, and, second, the coccolith dispersion is uniform over the slides, which is very important for a quantitative analysis. Also, occurrences of markers or rare species are found more quickly on a settled slide than on an ordinary smear slide. This has helped in this study to improve the biostratigraphic ranges of many species while at the same time improving the resolution of the Oligocene-Miocene zonation.
Abundances of individual species in each sample were counted for each sample using a Zeiss Photomicroscope III under x1250 magnification and by using cross-polarized and phase-contrast light. With computer assistance using BugWare (BugWare, Inc.), between 300 and 500 specimens were counted on each slide and their frequency converted to the number of specimens per mm2. Every specimen of the nannofossil assemblage was counted at the species level. Next, two long traverses of the slide were observed for rare taxa excluded from the initial counts; these were added to the counts with a value of one. Backman and Shackleton (1983) detailed different methods to obtain quantitative abundance. They also indicated that similar information is obtained by counting specimens per mm2 or by counting the number of microfossils per gram of sediment. Following their comments we chose to use a constant volume of sediment instead of sediment weight and to express the abundance relative to the area observed. This approach has the advantage of speed and easy application by other biostratigraphers.
For each site, a table with the absolute number of specimens per mm2 was generated. These large numeric tables (numeric range charts are not presented in this paper) were transferred into Cricket-Graph to draw the abundance curves (Fig. 3, Fig. 5, Fig. 8, Fig. 10, Fig. 11, Fig. 12, Fig. 13, Fig. 14, Fig. 15, Fig. 16, Fig. 17).
The range-charts presented herein in Table 3, Table 5, Table 7, and Table 9 were obtained directly from the numeric tables with the absolute numbers of specimens per mm2 translated in seven levels of single species abundance according to the following definitions:
P (present) = 1 to 4 specimens/ mm2;
R (rare) = 5 to 10 specimens/ mm2;
F (few) = 11 to 50 specimens/ mm2;
C (common) = 51 to 150 specimens/ mm2;
A (abundant) = 151 to 1000 specimens/ mm2;
V (very abundant) = 1001 to 5000 specimens/ mm2;
O (ooze) = 5001 to 25000 specimens/ mm2.
The same definitions are used for estimations of total abundance of each sample, with the additional definition of B (barren of nannofossils).
In addition to the abundance of individual species, special attention was focused on the occurrence of reworked Mesozoic and Paleogene (mostly Eocene) species. Except for the early Oligocene Zone NP21, the presence of reworked material did not interfere with the biostratigraphic results. Both groups of reworked specimens were counted separately and presented in the first two columns of the range charts. The absolute number of specimens per mm2 of Mesozoic species is presented for Hole 900A and correlated with the sequence chronostratigraphy.
Different estimations of calcareous nannofossil preservation have been used and discussed in previous studies. The following estimation are based on the comments of Roth and Thierstein (1972). The qualitative evaluation of calcareous nannofossils preservation was denoted as follows:
P (poor) = severe dissolution, fragmentation and/or overgrowth has occurred; most primary features have been destroyed and many specimens cannot be identified at the species level;
M (moderate) = Dissolution and/or overgrowth are evident; a significant proportion (up to 25%) of the specimens cannot be identified to species level with absolute certainty;
X (mixed) = Dissolution and/or overgrowth are evident among certain taxa, but nearly all specimens (up to 95%) can be identified with certainty;
G (good) = Little dissolution and/or overgrowth is observed; diagnostic characteristics are preserved and all specimens can be identified;
E (excellent) = No dissolution is seen; all specimens can be identified.