Jorg Bollmann^*, Bernhard Brabec
Mara Y. Cortés®, Markus Geisen
" G eo lo g ica l Institute, E T Z -Z u r ic h , S on n egg stra sse 5, 8 0 9 2 Zurich, S w itze rla n d
* S w iss F ederal In stitu te f o r S n o w a n d A v a la n ch e R esearch, F liielastra sse 1 1, 7260 D a vo s D orf. S w itze rla n d '' The N a tu ra l H isto ry M useum , P a la eo n to lo g ica l D epa rtm en t, C ro m w ell Road, L on do n S W 7 5BD , G reat B rita in
Received 22 December 1998; accepted 15 June 1999
A bstract
A quick new method is described for the quantification o f absolute nannofossil proportions in deep-sea sediments. This method (SM S) is the combination o f Spiking a sample with A/icrobeads and Spraying it on a cover slide. It is suitable for scanning electron microscope (SEM) analyses and for light microscope (LM) analyses. Repeated preparation and counting o f the same sample (30 times) revealed a standard deviation o f ±10.5% . The application o f tracer microbeads with different diameters and densities revealed no statistically significant differences between counts. The SMS-method yielded coccolith numbers that are statistically not significantly different from values obtained from the filtration-method. However, coccolith counts obtained by the random settling method are three times higher than the values obtained by the SM S- and the filtration-method. © 1999 Elsevier Science B.V. All rights reserved.
K eywords: coccoliths; absolute abundance; microbeads; preparation technique
1. Introduction
There is an increasing need for absolute calcare ous nannofossil counts in order to calculate coccolith fluxes from the photic zone into sediment traps or to estimate the varying coccolith carbonate accumula tion rates in geological records. In the past, several different techniques were developed, although there are currently only two basic methods that are in use: (a) the random settling technique introduced by Beaufort (1991) and modified by different authors (Williams and Bralower, 1995; Su, 1996; Flores and
* Corresponding author. Tel.: -f4I 1632 3684; Fax: -1-41 1632 1080; E-mail: [email protected]
Sierro, 1998) and (b) a filtration technique described by Backman and Shackleton (1983) and modified by Andruleit (1996). The accuracy and reproducibility of both techniques depends on three factors: ( 1) the even distribution of the particles on a filter or on a cover slide, (2) the assumption that no size-depen dent fractionation o f coccoliths occurred during the preparation procedure, and (3) that there is no loss or bias due to splitting procedures, filter funnels or settling devices.
Here, we demonstrate the application o f the com bination of two techniques that have been already applied or suggested for nannofossil analyses: (a) a spraying technique used by McIntyre et al. (1967) and (b) the addition o f microbeads as tracer parti
0377-8398/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PIT: 8 0 3 7 7 - 8 3 9 8 ( 9 9 ) 0 0 0 3 2 - 8
5 A p p e n d ix 175
30 J. B o llm a n n e t al./ M a rin e M ic ro p a leo n to lo g y 38 (1999) 2 9 - 3 8
cles (suggested by Okada, 1992). Furthermore, we compare the results obtained by this new method with the counts obtained by the filtration- and the settling-method.
1.1. Background
McIntyre et al. (1967) used a spray gun in order to distribute coccoliths of various sizes homogeneously on a slide and Okada (1992) suggested adding a known weight o f microbeads (Potter Ballotini Inc., type MB 10) to a sediment sample in order to cal culate absolute abundances of nannofossils. Okada (1992) assumed a constant number of microbeads per unit weight without knowing the real number of microbeads per weight unit. Therefore, his method enables only the counting versus a relative standard but not the calculation of absolute nannofossil con tent per gram sediment. However, Okada (1992) also suggested calibrating the number of microbeads per weight unit using a suitable standard if this standard were available.
Adding tracer particles or tracer chemicals is a common method, especially in chemistry, to calcu late the unknown amount o f a component. Particle tracers have been used by micropaleontologists since the 1960’s (Benninghoff, 1962; Stockmarr, 1971). Benninghoff (1962) added a known number o f Ly- copodium spores to a sediment sample in order to calculate the absolute abundance of pollen per gram sediment. However, until now it was not possible to apply this simple concept to coccolith counts be cause (a) no uniform tracer particles were available in the size range of coccoliths and (b) it was difficult to calculate the number of tracer particles that had to be added to a sample with an acceptable standard deviation.
2. Materials and methods
2.7. Microbeads
Microbeads o f uniform diameter have recently be come available commercially and thus allow a sim ple theoretical calculation of microbeads per solid weight (instead o f enumerating complete aliquots, see Stockmarr, 1971) with a standard deviation of
<2%. These microbeads are made of polystyrene, latex or borosilicate and they are available in many different sizes, accuracy o f diameter and physical and chemical properties. In order to use these mi crobeads for calcareous nannofossil counting they have to be stable in water suspensions with a pH of 8.5 or in alcohol. Furthermore, they have to be resis tant to ultrasonic treatment and they should not form large aggregates during the preparation procedure.
We used two different kinds o f microbeads that meet the above-mentioned requirements in our new method: (a) polystyrene microbeads (mean diameter 4.0 p,m ± 0.06 |xm standard deviation; density = 1.09 g/cm^) and (b) borosilicate microbeads (mean diameter 5.1 |xm ± 0.8 p.m, density = 2.5 g/cm^; for details see Table 1).
The number of microbeads per solid weight (e.g. 1 g) can be calculated as follows:
M-xot = -77T — - — — = ---z--- r ( 0 Pm X
where A / j o t = number of microbeads per gram; = weight o f one microbead; Pm = density o f one microbead; Fm = volume o f one microbead.
2.2. Sediment samples
In order to test the new method we used two dif ferent sediment samples: (a) the fine fraction (<38 p,m) o f a pure coccolith ooze from the mid-Pleis- tocene Gephyrocapsa dominance interval (Bollmann et al., 1998) of North Atlantic DSDP Hole 607 that is composed of 93% o f Gephyrocapsa placoliths (607, 2-2, 29-32 cm), and (b) a hemipelagic Holocene sample (bulk) with low carbonate content (Blagnac sample: off Cape Verde; W 18°15.1; N 21°19.76; 2002 m water depth). This sample was already used for an international intercalibration experiment be tween thirteen nannofossil experts. Until now, the results o f this experiment have not been published and therefore, we show only our own determinations (Table 2).
2.3. Spraying device
We used an Effa Spray Mounter and small glass capillaries (for details see Table 1). The distance between the target and the spray mounter was found
J. B o llm a n n e t a l . /M a r in e M ic ro p a leo n to lo g y 38 (1999) 2 9 - 3 8 31
Table 1
Materials used for SMS preparation
Item Ordering No. Amount Price Supplier
Polystyrene Microbeads; diameter: 4.0 ± 0.06 [im; density: 1.0® g/cm^
Product number: PS055N/001168; Inv. Nr. L970305E
I g $ 270,- Bangs Laboratories, Inc., 9025 Technology Drive,
Fishers, IN 46038-2886 USA, e-mail: [email protected]
Borosilicate Microbeads; diameter: 5.1 ± 0.8 |im; density: 2.5 g/cm^
Product number: 9005; LOT Nr. 19324
1 g ~ $ 150,- Duke Scientific Corp., 2463 Faber Pl., Palo Alto,
P.O. Box 50005, CA 94303, USA, e-mail: [email protected]
Spray gun 11805 or 11800 1 $ 9 5 ,- Ernest Fullam., 900 Albany Shaker Rd. Latham,
New York 12110, USA, e-mail: [email protected]
Small glass capillary tubes 11802 100 $ 13,- Ernest Fullam., 900 Albany Shaker Rd. Latham,
New York 12110, USA, e-mail [email protected]
Triton X 100 detergent XlOO 100 ml S 28,- Sigma Chemical Company, P.O. Box 14508, St.
Louis, Missouri 63178-9916, USA
Laser granulometer, GALAl CIS 1 L.O.T. - Oriel GmbH, D-64295 Darmstadt,
Germany
to be optimal at ca. 20 cm with respect to particle density and distribution.
2.4. Sample preparation 2.4.1. Spraying (Figs. 1 and 2)
Step 1. Microbeads and dry sediment are weighed with a high-precision balance (e.g. 1.0 mg with a Mettler balance AE 260 with a precision of 10~® g). We suggest adjusting the ratio between the weight of microbeads and the weight of sediment in order to avoid counting excessively large numbers o f mi crobeads with low numbers of coccoliths, or vice versa. For pure coccolith oozes a weight ratio of microbeads (polystyrene) to coccoliths (<38 |xm fraction) o f 2 to 1 is recommended and for samples with low carbonate content (e.g. 40%) we recom mend to use a ratio of 1 to 4. If bulk samples are used, we suggest increasing the amount of sediment to a minimum of 10 mg in order to avoid inhomo- geneous samples. However, if fine-fraction samples are used about 1 mg sediment is sufficient in order to guarantee homogeneous samples.
Step 2. Polystyrene microbeads and sediment are suspended in 2 to 4 ml of denatured alcohol. We used alcohol in order to provide a homogeneous mixing between polystyrene microbeads and sedi ment because of the comparatively low density of
these microbeads. Borosilicate microbeads and sedi ment are suspended in 2 to 4 ml water. We buffered the water with NH3 to a pH of 8.5 in order to avoid dissolution or overgrowth o f coccoliths. In addition, a very small amount o f Triton X 100 detergent was added to avoid coagulation o f the particles.
Step 3. The suspension is ultrasonicated for about 30 s at 35 kHz.
Step 4. The suspension is sprayed onto a cover slip (5 to 10 times) using small glass capillaries (Figs. 1 and 2). However, for routine work we recommend using a 5 ml syringe in combination with a glass capillary (Fig. 2). The syringe is filled with the suspension and the suspension is sprayed onto the target. This offers the advantages o f (a) spraying a complete suspension onto a target without refilling capillaries and (b) a better control on how much material is sprayed onto the target.
Step 5. The dry cover slide is mounted on an aluminium stub for scanning electron microscope (SEM) analysis or on a microscope slide for light microscope (LM) analysis.
Detailed ordering information (Table 1) and a full description of the spraying setup is given on: http://www.geology.ethz.ch/m p/data/m icrobeads/ beads main.html
T a b l e 2 T e s t f o r r e p r o d u c i b i l i t y a n d p r e c i s i o n o f t h e S M S - m e t h o d S a m p l e M e t h o d M i c r o b e a d t y p e M i c r o b e a d s ( g X 1 0 - “ ) S e d i m e n t ( g X 1 0 - “ ) M i c r o b e a d c o u n t s C o c c o l i t h c o u n t s C o c c o l i t h s ( x l O ' V g ) E. huxleyi ( x l O ' O / g ) G ephyrocapsa s p p . ( x I O ' ° / g ) E p m fu n d a ( x l O ' V g ) O t h e r s ( X l O ' V g ) (%) D S D P 6 0 7 - I S M S P 7 . 6 8 2 6 . 1 5 2 0 3 1 2 2 8 4 . 8 7 4 . 5 3 0 . 2 0 0 . 1 3 8 . 0 1 D S D P 6 0 7 - 2 S M S P 5 . 4 4 1 5 . 0 3 1 8 0 9 3 7 5 . 1 6 - 4 . 8 9 0 . 1 4 0 . 1 3 8 . 5 4 D S D P 6 0 7 - 3 S M S P 5 . 4 4 1 5 . 0 3 2 0 1 1 0 5 4 5 . 2 0 - 4 . 7 9 0 . 1 9 0 . 2 2 8 . 1 3 D S D P 6 0 7 - 4 S M S P 3 . 7 8 1 1 . 1 9 2 1 2 1 0 4 5 4 . 5 6 - 4 . 2 0 0 . 2 1 0 . 1 4 7 . 9 7 D S D P 6 0 7 - 5 S M S P 8 . 1 4 2 5 . 7 2 2 0 1 1 1 2 3 4 . 8 4 - 4 . 5 6 0 . 1 3 0 . 1 6 8 . 0 9 D S D P 6 0 7 - 6 S M S P 6 . 0 4 1 3 . 2 4 2 0 1 8 1 2 5 . 0 5 - 4 . 8 4 0 . 0 9 0 . 1 2 8 . 3 0 D S D P 6 0 7 - 7 S M S P 9 . 5 3 4 . 2 2 1 5 3 1 0 2 5 5 . 0 9 - 4 . 7 3 0 . 1 6 0 . 2 0 9 . 0 5 D S D P 6 0 7 - 8 S M S P 4 . 6 8 1 4 . 9 0 2 0 3 1 1 8 0 5 . 0 0 - 4 . 6 9 0 . 2 1 0 . 1 0 8 . 0 3 D S D P 6 0 7 - 9 S M S P 8 . 1 1 6 . 3 5 2 0 5 8 1 0 5 . 3 6 - 5 . 0 7 0 . 1 7 0 . 1 3 8 . 2 4 D S D P 6 0 7 - I 0 S M S P 5 . 9 4 5 . 9 3 3 0 7 6 8 1 6 . 0 9 - 5 . 6 4 0 . 2 5 0 . 2 0 7 . 3 5 D S D P 6 0 7 - I I S M S P 5 . 4 9 4 . 4 6 3 0 9 5 2 5 5 . 7 3 - 5 . 2 8 0 . 2 9 0 . 1 5 7 . 6 3 D S D P 6 0 7 - I 2 S M S P 1 0 . 7 8 1 8 . 1 9 2 0 9 5 9 0 4 . 9 9 - 4 . 7 1 0 . 1 4 0 . 1 4 8 . 4 6 D S D P 6 0 7 - I 3 S M S P 1 5 . 9 4 1 7 . 4 4 3 0 3 7 4 4 6 . 1 5 - 5 . 7 2 0 . 2 4 0 . 1 9 7 . 2 9 D S D P 6 0 7 - 1 4 S M S P 1 1 . 5 5 1 4 . 5 2 3 0 1 7 7 7 5 . 6 2 - 5 . 2 4 0 . 2 4 0 . 1 4 7 . 2 7 D S D P 6 0 7 - I 5 S M S P 1 2 . 6 2 1 4 . 7 6 2 0 3 5 3 3 6 . 1 5 - 5 . 6 5 0 . 3 3 0 . 1 6 8 . 6 5 D S D P 6 0 7 - 1 6 S M S P 1 3 . 1 4 2 0 . 8 8 2 0 4 6 2 2 5 . 2 6 - 4 . 8 3 0 . 2 5 0 . 1 7 8 . 4 8 D S D P 6 0 7 - 1 7 S M S P 2 1 . 0 6 3 1 . 0 6 2 7 4 8 4 2 5 . 7 1 - 5 . 2 9 0 . 2 4 0 . 1 7 7 . 4 2 D S D P 6 0 7 - 1 8 S M S P 9 . 6 1 1 2 . 2 1 2 6 4 7 6 1 6 . 2 1 - 5 . 7 1 0 . 2 9 0 . 2 1 7 . 6 0 D S D P 6 0 7 - I 9 S M S P 8 . 4 9 7 . 9 4 2 0 4 4 3 2 6 . 2 0 - 5 . 6 9 0 . 3 0 0 . 2 2 8 . 8 8 D S D P 6 0 7 - 2 0 S M S P 8 . 5 3 6 . 2 9 3 0 6 5 0 1 6 . 0 8 - 5 . 4 9 0 . 3 9 0 . 2 1 7 . 7 1 D S D P 6 0 7 - 2 I S M S P 9 . 5 2 1 9 . 0 8 2 0 6 9 1 8 6 . 0 9 - 5 . 6 5 0 . 3 3 0 . 1 1 8 . 1 4 D S D P 6 0 7 - 2 2 S M S P 6 . 9 1 2 . 4 8 2 0 4 9 0 4 6 . 7 1 - 6 . 3 2 0 . 2 0 0 . 1 9 8 . 1 8 D S D P 6 0 7 - 2 3 S M S P 7 . 9 8 1 2 . 7 4 2 1 2 7 4 1 6 . 0 0 - 5 . 5 8 0 . 2 3 0 . 1 9 8 . 2 1 D S D P 6 0 7 - 2 4 S M S P 1 8 . 5 1 3 . 6 8 3 1 5 5 0 0 5 . 8 8 - 5 . 4 1 0 . 1 5 0 . 3 2 7 . 6 5 D S D P 6 0 7 - 2 5 S M S P 6 . 1 9 1 1 . 7 7 2 0 1 7 1 4 5 . 1 2 - 4 . 7 4 0 . 1 7 0 . 2 0 8 . 4 0 D S D P 6 0 7 - 2 6 S M S P 1 3 . 9 1 6 . 6 9 3 0 9 8 3 0 6 . 1 3 - 5 . 7 9 0 . 1 3 0 . 2 1 7 . 1 5 D S D P 6 0 7 - 2 7 S M S P 1 0 . 4 1 3 . 1 6 3 0 1 6 1 9 4 . 4 5 - 4 . 2 0 0 . 1 4 0 . 1 2 7 . 4 9 D S D P 6 0 7 - 2 8 S M S P 8 . 6 5 1 5 . 8 6 3 0 2 9 8 1 4 . 8 5 - 4 . 5 4 0 . 1 6 0 . 1 5 7 . 0 8 D S D P 6 0 7 - 2 9 S M S P 1 0 . 1 7 1 0 . 1 0 3 0 7 6 0 7 5 . 4 5 - 5 . 0 4 0 . 2 5 0 . 1 6 7 . 4 7 D S D P 6 0 7 - 3 0 S M S P 7 . 1 8 9 . 4 1 2 0 9 5 6 7 5 . 6 7 - 4 . 9 8 0 . 2 7 0 . 4 2 8 . 5 0 A v e r a g e 5 . 5 2 7 . 9 8 S t a n d a r d d e v i a t i o n 0 . 5 8 % S t a n d a r d d e v i a t i o n 1 0 . 5 3 D S D P 6 0 7 - 3 1 S M S B 7 7 . 4 6 3 3 . 6 5 2 0 0 8 4 4 5 . 6 0 - 4 . 9 7 0 . 3 1 0 . 3 1 0 . 2 8 D S D P 6 0 7 - 3 2 F - t - M B B 5 9 . 6 8 2 5 . 6 8 2 8 3 1 0 9 3 5 . 1 7 - 4 . 9 0 0 . 1 2 0 . 1 5 0 . 2 8 D S D P 6 0 7 - 3 2 F B 0 2 5 . 6 8 1 0 9 3 4 . 5 0 - 4 . 2 7 0 . 1 0 0 . 1 3 D S D P 6 0 7 - 3 3 SE 4 -M B B 3 5 . 5 1 2 6 . 3 5 2 2 0 1 1 3 3 4 . 0 0 - 3 . 8 7 0 . 0 5 0 . 0 8 0 . 2 8 D S D P 6 0 7 - 3 3 S E B 0 2 6 . 3 5 1 1 3 3 1 2 . 3 4 - 1 1 . 9 5 0 . 1 5 0 . 2 4 B l a g n a c - 1 F B 0 1 0 2 . 0 0 1 2 7 6 0 . 2 7 0 . 0 8 3 0 . 0 8 0 . 0 4 0 . 0 6 B l a g n a c - 2 S M S B 7 2 . 0 3 2 9 3 . 6 3 2 1 0 4 3 8 0 . 2 9 0 . 0 9 6 0 . 1 1 0 . 0 5 0 . 0 4 0 . 2 8 T h e r e s u l t s o f t h e r e p e a t e d p r e p a r a t i o n s a n d c o u n t i n g o f D S D P s a m p l e 6 0 7 - 2 - 2 , 2 9 - 3 2 c m a n d t h e B l a g n a c s a m p l e a r e s h o w n . I n a d d i t i o n , t h e c o u n t s o f t h e s a m e s a m p l e s o b t a i n e d b y t h e f i l t r a t i o n a n d s e t t l i n g m e t h o d a r e g i v e n . T h e a b b r e v i a t i o n s i n c o l u m n ‘ M e t h o d ’ a r e : S M S = e s t i m a t e s o b t a i n e d b y s p i k i n g w i t h m i c r o b e a d s a n d s p r a y i n g ; F = e s t i m a t e s o b t a i n e d b y f i l t r a t i o n b a s e d o n p a r t i c l e s p e r a r e a ; F + M B = e s t i m a t e s o b t a i n e d b y m i c r o b e a d s a d d e d t o f i l t e r e d s a m p l e ( r a t i o m i c r o b e a d s / c o c c o l i t h s ) ; S E = e s t i m a t e s o b t a i n e d b y s e t t l i n g b a s e d o n p a r t i c l e s p e r a r e a ( a f t e r W i l l i a m s a n d B r a l o w e r , 1 9 9 5 ) . S E + M B = e s t i m a t e s o b t a i n e d b y m i c r o b e a d s a d d e d t o a s e t t l e d s a m p l e ( r a t i o m i c r o b e a d s / c o c c o l i t h s ) . T h e t y p e o f m i c r o b e a d i s i n d i c a t e d b y P = p o l y s t y r e n e m i c r o b e a d s , a n d B = b o r o s i l i c a t e m i c r o b e a d s . N o t e t h a t t h e n u m b e r s o f c o c c o l i t h s p e r g r a m s e d i m e n t h a v e t o b e m u l t i p l i e d b y I 0 ' ° . T h e e r r o r v a l u e i s c a l c u l a t e d f r o m t h e n u m b e r o f m i c r o b e a d s a n d c o c c o l i t h s c o i m t e d a n d t h e v a r i a n c e i n m i c r o b e a d s i z e s . S e e t e x t f o r d e t a i l s .
J. Bollm ann et al. /M a r in e M icropalcontology 3B (1999) 29-3 H 33
air gun (cross-section)
cover slide capillary tube with 'suspended preparation
20 cm working distance
Fig. 1. Sketch oF the spraying set-up. A distance o f 20 cm between gun and target was found to be optimal with respect to particle distribution. Cover slides and SEM stubs can be mounted with a small strip o f double-sided tape or a small strip o f spray glue.