Phytolith analysis for the Patrok Aike Lake Drilling Proyect: Sample treatment protocols for the PASADO Microfossil manual

ALEJANDRO F. ZUCOL, ESTEBAN PASSEGGI, MARIANA BREA, NOELIA I. PATTERER, MA. GABRIELA FERNÁNDEZ PEPI AND MARIA DE LOS MILAGROS COLOBIG.

Patrok Aike Maar Lake Sediment Archive Drilling Project

Proyecto Editorial PIPA.

Año: 2010; p. 81 - 84

~ Drilling Projects serie (SALSA and PASADO). some of the researchers from the various iplines involved are coordinating their protocols. in order to help standardize the various hodologies used to achieve their research objectives (Schabitz and Wille, Unpub.). This ument has therefore been created to inform the PASADO microfossil community of the ´hodologies that are being applied in the phytolith analyses, as well as to discuss the entiaI types of paleobotanical and paleoenvironmental reconstructions being focused n as objectives. e phytoliths from the Potrok Aike Lake sedimentary sequence are being studied jointly by research groups: Diamante and Mar del Plata. This presentation is focused on mmunicating the details of the protocol that the Diamante group is implementing for the tment and study of these samples, with the eventual goal of coordinating these thodologies to develop one common protocol used by both groups. mple treatment for phytolith analysis -Sample storage. ed samples of undisturbed sediments are taken from the core sequence, and are stored in metic plastic tubes until the time of processing . -Sample treatment and classification. e set of samples are classified according to the different sections that are to be studied by ach research group. Each sample consists of approximately 5g of dry sediment. Sets of arnples are prepared and classified, then packaged to be sent to the various laboratories ere the treatment will take place. aboratory procedures can be summarized as involving two stages: 1) sample pre-treatment, luding removal of soluble salts , carbonate material, and organic matter, as well as eflocculation of the clastic material; and 2) sample treatment to separate the various ranometric components and to concentrate the biomorphic particles using heavy liquid tation . • -Sample pre-treatment. a Elimination of carbonates, humic components and concretions. The sediment sample a iquot (5g) is placed in a Pyrex beaker and dilute hydrochloric acid (10 %) is added, with oderate warming in a water bath. Reaction with carbonate and calcium cements is evident the release of gasses that produces bubbling in the sample, with this release being creasingly intense in accordance with the abundance of these compounds. The presence f humic components and iron oxides is demonstrated by yellowish coloration of the solution. his treatment is repeated until gaseous release is no longer observed and the solution ecomes translucent. " his solution is then left to settle for 30 minutes to prepare for decanting . The upper column of the solution is removed by pipette or siphoning and the presence of chloride ions in the wash water is tested using silver nitrate solution (1%). dditional stages 81 1: If it is necessary to preserve all particles sizes, the sample wash should be performed using a porous filter, or instead the sample can be centrifuged, in order to avoid loss of material . 2: If iron oxides persist in the sample, the treatment can be intensified by adding stannous chloride crystals to the hydrochloric acid solution, being careful to maintain the pH level between 4.5 and 8.5. b. Elimination of soluble salts. The remaining sediment from the previous stage is put in a new Pyrex beaker and 200 ml of distilled water is added, stirring with a glass rod. When the content becomes uniform the solution is allowed to settle for 30 minutes to allow decanting. The clean liquid above the deposit is eliminated. These steps are repeated two to three times with cold water and once more with immersion in a warm water bath (50-70°C), in order to eliminate all of the soluble sales that may be present. Additional stages 1: To test for the presence of chloride ions, transfer an aliquot of the solution to a glass test tube. Add a few drops of concentrated nitric acid and 4 drops of 4% silver nitrate. If the solution becomes white , it will be necessary to repeat the above elimination steps. To test for the presence of sulphate ions, transfer an aliquot of the solution to a glass test tube and first add a few drops of concentrated hydrochloric acid , then 5 drops of saturated barium chloride solution. If a precipitate forms then washing of the sample will have to be continued. c. Elimination of organic matter. The sediment remaining after the previous stage is put in a Pyrex beaker and 30% hydrogen peroxide is added for a period of 24 hours, stirring periodically with a glass rod. The solution is then warmed in a water bath (50-70°C) with further addition of hydrogen peroxide until gaseous release is no longer observed. At this point the solution is allowed to evaporate until the sample has become dry. Additional stages 1: If the organic content is especially high, the hydrogen peroxide reaction may cause the level of liquid to rise to the point of overflowing. In this case the reaction can be diminished by adding a few drops of common alcohol. The reaction produced by the organic matter is different than the one that is produced by the presence of manganese , which is indicated by violent bubbling with release of a dense white steam. 2: If the presence of organic matter persists, it is possible to try an alternative, more volatile oxidation treatment of the sample using 5% permanganate of potassium (KMn04) and addit ion of some drops of SUlphuric acid . This reaction is allowed to continue for 24 hours without heating and with periodic agitation , and then with warming to 100°C until all free oxygen has been released. d. Dispersion. Sample dispersion is performed using a 1N solution of sodium hexametaphosphate (a.k.a. Calgon or Sodium Beta), diluted in the sample to 0.1N. The sample is placed in this solution and is stirred with a mixer for 5 minutes, then left to settle for 24 hours . 4.-Granometric class separation . The granometric separation procedures will have to be adapted to the specific purposes and material types , although in all cases the various granometric classes are separated by sieving and/or by use of their differential sedimentation rates , with the steps adjusted according to which particle sizes need to be obtained. In our case, three main fractions will be first be separated : fine (particles with diameter from 5-53 urn). medium (from 53-250 IJm), and coarse (greater than 250 IJm). These three fractions can first be separated from each other bysieving usinga#50sieve (250 IJmmesh)anda#270sieve (53 IJmmesh). Separation of the fine fraction, which passes through both sieves, into fine and medium-fine sub-fractions can be performed using differential sedimentation if required. Separation using differential sedimentation is based upon the application 0 Stokes´ Law , which states that at a given temperature, the sedimentation velocity of a pant e depends upon its size. After 82 rticular lengths of settling time. I: ss e to calculate the particle sizes that will remain spended in the solution, as well 8_´ se a ill have settled to the bottom of the burette . -Heavy-liquid flotation. ´ter separation into different size casses e size fractions from 5 -250 urn in diameter is includes the fine and medium fracti s 8 e sed for flotation . .,ytoliths have a specific gravity ranging ´rom 1.5 to 2.3 g/cm3 (Piperno, 2006 and terences therein). There are numerous heavy liquids that can be used for densimetric paration, but we prefer sodium polytungstate. si ce densimetric adjustments can be made ing distilled water, the solution can be recycled. and this chemical possesses minimal vironmental toxicity. The phytolith flotation is performed by adding sodium polytungstate lution (with its specific gravity adjusted to 2.345 g/cm 3) to the processed sample, followed centrifugation at 1200 rpm for 5-10 minutes. nitoring of solution density is performed using a densimeter for precise measurement (a estphal balance or picnometer can also be used) , or by means of Kranz indicators that also ovide a measurement of density ranges. e supernatant liquid containing the floating material is removed with a pipette and overed by filtering in a funnel with a paper filter, then the material is washed with distilled ter. In a separate funnel, the same process is performed for the fraction of heavier terial that sinks during centrifugation. Both filters are washed with an abundance of stilled water until traces of the heavy liquid can no longer be observed in the wash water. e different subsamples obtained during these two steps (4 and 5) are then labeled and ded to the collection of sediment samples. -Microscope slide mounting. NO types of slide mounting can be used for the phytolith microscope observation: liquid and lid, and both have their advantages. Liquid mounting can preserve mobility of the material be observed , while solid mounting is more durable and allows better preservation in eference collections. th types of slides are made following standard procedures, but with specific types of unting media used that are most appropriate for the refractive index possessed by ytoliths (see Zucol and Osterrieth, 2002). Thus for the liquid slides, immersion oil is used , nile the solid slides are made using Canada balsam. t least one slide is made from each of the granometric fractions created in step 4 (more an one if needed for counting purposes), and these are stored in the microscopic slide Ilection of the Paleobotanical Laboratory for later reference.