ESTUARINE ANIMALS: A MANUAL ON COLLECTION, PRESERVATION AND MAINTENANCE

All estuarine biologists either working at research or aca<iemic institutes should he aware uf atleast the simple collection procedures of estuarine aninlals and their uccurale identification for environmental studies. After knowing such procedures un uttenlpt should he made to build a reference collection centre covering the local fl(lra and fauna which will later assist in various studies connected wtih ecology, cUI111l1erciai exploitatil)n. conservation and environmental assessment studies. The present ,,,anual is a hlol towards such 'agoal which deals with the general collection, preservat ion and Illaintcnance procedures of estuarine animals.


I. INTRODUCTION
All estuarine biologists either working at research or aca<iemic institutes should he aware uf atleast the simple collection procedures of estuarine aninlals and their uccurale identification for environmental studies. After knowing such procedures un uttenlpt should he made to build a reference collection centre covering the local fl(lra and fauna which will later assist in various studies connected wtih ecology, cUI111l1erciai exploitatil)n. conservation and environmental assessment studies. The present ,,,anual is a hlol towards such 'agoal which deals with the general collection, preservat ion and Illaintcnance procedures of estuarine animals.

II. COLLECTION
Collection of intertidal, planktonic. nektonic and benthic animals are to be nlade by using suitable gear. The collection equipment recommended are very sinlple in design and can be obtained from manufacturers within the country (Appendix a). The nlain criteria for estuarine gear should be of light weight as these are to be uperateu in shallow waters of estuaries from indigenous craft anti fibre-glass boats operated with outboard motor engine.

u. Intertidal:
This area covers varied hahitats having different fauna and flora inhabiting mud flats to salt l11arshes and nlangroove S\Vanlps. Here the collections are mainly hand picked by using blunt forceps. chisels. hanlnlers, showels, spades. crowbars. brushes, s","uupnets, wide nlouth pipettes. sieves of different aperture sizes (2.Smm -O.Smln) anti aL')u by using fornlaldelyde. bleaching powder etc .• in pools exposed at low tide Records of tM ~ological SIUWY of India marks. One can visit this area frequently and make representative collections. Most of the animals (sessile or motile) are either buried in the mud, sand or associated with aquatic vegetation; attached to dead wood, rocks, within empty shells, oyster beds; under stones and other substrates. A tide table chart of the area is essential for collections CJs the animals will be exposed only during low tide and the area should be reached well before the low tide time. If one reached after the low tide, it is impossible to make representative collection as the area will be submerged during high tide water and one has to wait till the next low tide which may .take few hours and that time may not be suitable for making collections as it may be dark at that time. It is better to stay at the spot overnight to observe the tidal exposure of the coast and to collect the animals during both high and low tides for better representative collections.
b. Plankton: Planktonic animals range from few microns to few centimeters along with bigger forms viz., medusae, jellyfishes, salps. All these organisms are collected through plankton nets made of bolting silk cloth which are towed behind a boat at low speed. The most widely used plankton net ( Fig. 1) consists of a circular metal ring of 30 cm. in diameter, with a canvas collar of 10 cm. height followed by a concial net of bolting silk approximately 100 cm. height ending in a canvas cloth of 10 cm. height at the narrow end (Cod end) to which a tu bular metalic bucket is clamped. The bottom of the bucket is covered and clamped with the same bolting silk cloth used for the plankton net. Three bridles are tied to the eyes provided on the metal ring which in turn are connected to a small ring or shackle which is connectoo to a towing rope for towing the net behind the boat at a reasonable distance (5-10 ° mts) to avoid the disturbances caused by the Boat. The plankton collected in the bucket after towing for a period (15-30 min.) is transferred to th~ container after washing the sides of the bucket and the bloting silk cloth clamped to the bucket using 10'Yo neutral formalin for fixing and preservation. Do not use water for washing the sample after collecting plankton into the container. which will dilute the preservative thus spoiling the plankton sample in due course.
Different types (numbers) of bloting silk cloth should be used to collect different types of plankton-macro (0-4) micro (5-15) and nanoplankton (J 5-25 and they range from subtidal to the deeper parts of the estuary. The quipment used for the collection of benthic organisms are mostly a varie~y of grabs and corers wbich are designed to penetrate the bottom and grab the sediment sample with the associated fauna and folora. The retrived sediment sample is passed through sieves uf diffe.rent aperture sizes (2 mm -0.05 mm). The macrobenthos are those which are retained in the sieves of 0.5 mm while meiobenthos and microbenthos are those witich pass through 0.5 mm sieves and below.
The most common grab is the Peterson grab which consists of 2 jaws which are hinged together and are held open at the ti~e of lowering. As soon as the grab hluches the bottom. the clamp is released facilitating the 'grab to close its jaws and while doing so .it takes a bite of the floor containing a segment of sediment sample with its associated organisms. Here we recommend use of a light weight grab of less than 8 Kg weight which can be operated in shallow waters from country crafts ( Fig. 4). In addition to the above Peterson grab. a modified version of Van Veen hand grab (Fig. 5) is found to be highly suitable in estuaries of shallow depths. Detailed account of the various instruments used in the collection of benthos is given by Holme & McIntyre 1971. The benthic organisms are also collected by using dredges. beam trawl and Agassiz trawl (Figs. 6-8). They ~re used for collection of organisms crawling on the floor but not for burrowing and others living within the sediment. A light weight dredge \vith a square mouth of 30 cm which can be hand h~uled from a snlall boat. is suited for the collections of smaller epibenthic organisms from a limited area. For the collection, of bigger epibenthic organisms over a wide area a beanl trawl or Agassiz trawl are well suited for sampling. however these are difficult to operate fronl it country boat. A smaller version of these trawls with a mouth width of one meter can be operated from fibre glass boats with uutboard motors. The dredges and trawls should not be operated for more than 15-20 minutes as there is a chance of clogging its mouth with large sediments and making it difficult to lift them.

III. COLLECTION TREATMENT IN THE FIELD
l. Sorting: The intertidal. planktonic. ne'ktonic and benthic animals are to be sorted immediately after collection. The pJanktonic samples are to be fixed in 5-10-ro n.eutral formalin. The nekton samples are sorted out group wise (appendix -B & C), fixed and preserved in 10'10 neutral formalin. Bigger organisms need a slit in the belly through anal opening. Benthic organisms retrieved after sieving through a set of sieves of 2 mm down to 0.5 mm are weighed. fixed and preserved in 10' Yo neutral formalin. For groups like sponges. coelenterates. annelids. moll~sca and echinoderms special treatment of anaesthetization and fixing is needed and the details of such procedure are givne in Table I. b. Anaesthetization: Wherever anaesthetization is necessary. crystals of menthoL megnesium sulphate. magnesium chloride, chloral hydrate are sprinkled over the clean estuarine waters containing the animals allowing them to expand gradually ( Table I).

Records of the Zoological Survey o/India c. Fixation:
Formalin is the best field fixative which is available commercially as 38" aqueous solution of formaldehyde gas. For dilution purposes tbe 38% solution is treated as 100% formalin and animals are fixed in 5-10% neutral formalin sollution generally. The IO' Yo solu flon .is prepared by adding 9 parts of water to t pan of commercial formalin. (Before dilution, 1 litre of commercial formalin should be neutralized either with 20 gms of Borax or 200 gms of Hexamine).

d. Preservation:
After fixation (Killing in formalin) the liquid fixative generally becomes diluted and dirty. Further some groups are not to be preserved in formalin even for short time. Therefore, suitable preservative should be used before packing them in containers as it may not be possible to unpack the collections immediately at the lahoratory (Table I).

e. Containers:
It is always preferable to use screw capped plastic tubes and wide mouth plastic bot ties. jars and drums with screw caps for keeping the collected material in the field. The bigger animals -crabs, squids and certain fishes etc., should b~ wrapped in cheese cloth (gauze cloth) a.nd packed in bigger screw capped, wide mouJ.b plastic drums. The collections contained in the smaller tu bes are to be packed in 1-5 litre screw capped wide mouth plastic jars with cotton padding a~l around.
f. Labelling: Every sample either kept in the tubes or wrapped in cheese cloth should contain invariably an internal label printed on a tracing paper. Avoid paper labels as they become soft and disintegrate. Labels should be written with water proof black I ndian Ink or with soft lead pencil ( Fig. 9).

g. Documentation:
As it is not possible to write all the details on the field label. it is important to keep collection data in a field note book. The sample number given on the label should be same as mentioned in the field note book/station book so that the detailed field data can be transferred on to the permanent label in the laboratory. Data sheets are to be filled while collecting the plankton, nekton and benthos and proforma data sheets are given for guidance (Appendix -d-O. h. Packing: The final field packing of the collections kept in different small plastic containers are packed in steel trunks with sufficient soft padding material all around. The big plastic drums c~n be carried after covering with gunny bags. All these packages should contain a destination label on the outside as well as inside.

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Photographic presentation of the collection spots as well as the s~ens in live condition. preferably in situ and also fixed is necessary fer better record of the collections and also for future publication purposes.

IV. COLLECTION TREATMENT IN THE. LABORATORY
The collections brought from the field will be unpacked and transferred to suitable containers with final preservatives (Table I).
8. Sorting: E ventho~gh the samples were well cleaned and preserved in the field. it needs further attention as it is not possible to tdtve thoro\lgh cleaning of the sample· in the field. As there is every chance of some specimens being damaged during transit. only the specimens in good condition should be finally preserved. After that. the collections are sorted out finally groupwise (Appendix -C). For subsorting the collection samples of lower groups a good binocular stereo microscope with wide field eyepieces and good illumination is necessary.

b. Preservation:
After subsorting. either the collections were transfered to 5-10'Yo neutral formalin or to 75% ethyl alcohol (i.e. 4 parts of rectified spirit and one part of water). Soft bodied animals are to be transfered to 75'Yo alcohol by passing them through 30% and 50% alcohol for few hours to avoid strinkage. The bigger animals like fishes, crusta<reans are to be washed before transfering them to 75'Yo·alcohol ( Table I). It is advisable to preserve bigger animals in formalin as they reql}ire large quantity of ethyal alcohol which is expensive as well as difficult to procure and maintain. while the formalin preservative is cheaper and need less attention. t~()~gh it is unpleasent to work.

c. Containers:
Wi~e mouth glass jars and bottles of different sizes provided with plastic scre~ caps or glass stoppers are ideally suited for long. ternl preservation Qf anImal collections. While preserving the smaller specimens. it. is hetter to use small specimen tubes (glass/plastic) which will he plugged with cutton and kept immersed in the preservative using the above mentioned glassjars/bottleswith ColLon padding at the bottom and top. The large specimens which cahnot be kept ib· glass jars should be stored in plastic tanks fitted with trolly frame'\ and tight lids. After the transfer of the materials to tie suitable ·containers. a 'suitabfe label filled with all field data written in water proof Indian Ink should be kept inside (Figs. 10-13). These labels are very important for future study of the col'1eetions as it may take even a long. time to look into thorn.
Records 01 the Zoological Survey of IIIflia e. Registration (unnamed register) : The unnamed material thus collected and preserved must be registered serially in a good bound ruled ledger register using water proof Indian Ink only. The format of the unnamed register is given in the (appendix-g).

f. Documentation:
It is useful to prepare a list of collections, group wise present in the unnamed collection for a better inventory as the unnamed register will not give a ready information of all the unnamed collections of a particular group.
g. Holding of the unnamed collections: (unnamed collection room) : A seperate collection room with shelves capacity is necessary for keeping and arranging the unsorted. sorted and registered collections group-wise. The plan of arrangement of collections should be displayed at the entrance of the collection rooms.
h. Upkeep: The future of the collection depends upon the upkeep and mainlenance i.e. routine check up, including the filling of preservative whenever necessary and dusting of the containers as if may take even years to work out the unnamed collections. I t is better to evolve a routine procedure for checking the collectiuns as the collections increase through years.

Photographs:
As it is not possible to photograph all the specimens in the field . . efforts shuultJ be made to photograph them in the laboratory with a view to prepare line dnl\\'ings at a later stage.
V IDENTIFICATION a. Labelling (named) : All identified material should be properly labelled on a good qu~lity drawing paper using water proof Indian Ink. The size of the label should be proportionate to the size of the container (Figs. 14-16).
b. Registration (named) : All the identified collections are to be entered in a bound ruled register made of good quality ledger paper. The entries of the named collections should be made with water proof Indian Ink. The format of the named registar t~ shown in the appendix -h. . This being the final arrangement of the collection. containers of suitable size should be selected keeping in view the size of the specimen to be kept inside. There should be at least 25% extra space around the specimens in the container so that they will be kept always immersed in the preservative. The containers should be of clear glass with plastic screw caps preferably with a liner inside to reduce the evaporation of the preservative. For tube collections (smaller specimens) and tank collections (very big specimens) procedure mentioned under (IV. c) can be followed. The named collections pertaining to smalVbig specimens of the same species from different localities are to be kept either in big glass jar/tank withQut mixing their localities. The label as written in (V. a) should be inserted in the container.

d. Documentation (catalogue cards) :
For ready information of the named collections. data belonging to each sp'ecies available in the collections are to be entered in the species catalogue cards (Fig. 17) and are to be kept in the index cabinets as per the suggested classified list (appendix-c ).

e. Preservation:
Proper upkeep of the named collections is necessary. Regular checking of the eollections and filling of the preservative should be done at intervals without fail.
f. Holding of named collections (named collection roon., : A seperate room for keeping the named collection is necessary and the room should be pruvided with shelves capacity so that the collections can be arranged as per the classified list (appendix -c). Special thought should be given in a·rranging and making them exhibited clearly as these collections itself should act as an aid to the identification for the future study of unnamed material.
The collection room should be protected from sun light and better lighting arrangement should be made so that the specimens can be seen clearly. All the id~ntiried collections should be handled (removed or replaced) by the qualified staff only.

Preparation of list:
All the. specimens (named! unnamed) should be sent out only after filling in the proforma in quadruplicate with all details and duly checked and signed by the concerned. The proforma for sending the loan collection is given of (appendix-i).
h. Packing and Forwarding: Proper care shou ld he taken in· selecting the packing material. They stiou Id be Records of the Zoological SIUWY of 111dia either light weight and unbreakable corrugated boxes, screw cape~ tins or thennocole boxes so as to withstand the transit through raiVroadl air. Good padding around the specimens with light cotton wool is very essential. otherwise the specimens will be pressed from all sides. Before closure of the packing. 3rd copy of the loan form duly filled should be kept inside the package. After closing the packet with a brown paper and sealing with cellotape, a despatch lable containing from and to addresses should be pasted ( fig. 18). It is better to insure/register the collection parcel while sending it with in the country/abroad. c. Despatct! & Receipt : Proper record of the specimens sent out and rec~ived ~hou ld be maintained in a register for further correspondence (appendix -J & K).

VII. LITERATURE
a. References group-wise: Literature references cards (Fig. 19) are to be maintained group-wise/specieswise/area-wise for preparation of reports. faunal lists and for other purposes. These cards should be kept in the inde" cabinets of SIr X 3" size for early reference. It is necessary to keep species cards and photography cards (Fig. 17. 20 & 21) also in the index cabinets of sizes 6" X 4" and 8" X 5" respectively~ h. Reference library: A reference library covering with books. journals and reprints dealing. mainly with taxonomic work (group-wise) is essential for the study of the animals so collected. preserved and maintaIned in the reference collection centre.