1、Designation: E 1203 98 (Reapproved 2004)Standard Practice forUsing Brine Shrimp Nauplii as Food for Test Animals inAquatic Toxicology1This standard is issued under the fixed designation E 1203; the number immediately following the designation indicates the year oforiginal adoption or, in the case of
2、 revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice describes procedures for hatching, harvest-ing, and testing the acceptability of
3、 brine shrimp nauplii for useas a food for certain fish and invertebrate species that are usedin aquatic toxicity tests. The term “brine shrimp” refers to allspecies in the genus Artemia (1)2although this practicespecifically deals only with those species for which cysts(encysted embryos) are commer
4、cially available.1.2 These procedures are applicable to all brine shrimpnauplii that are obtained by incubating commercially availablecysts. With appropriate processing, cysts collected by noncom-mercial harvesters can be subjected to these same procedures.1.3 Modification of these procedures might
5、be justified byspecial needs or circumstances.1.4 This practice is organized as follows:SectionReferenced Documents 2Terminology 3Summary of Practice 4Significance and Use 5Hazards 6Obtaining Cysts 7Selecting a Source 7.1Storage 7.2Hatching Cysts 8Harvesting Nauplii 9Testing Nauplii 10Feeding Assay
6、10.1Chemical and Physical Measurements 10.2Using Nauplii 11Report 121.5 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the a
7、pplica-bility of regulatory limitations prior to use. Specific hazardstatements are given in Section 6.2. Referenced Documents2.1 ASTM Standards:3E 1191 Guide for Conducting Life-Cycle Toxicity Testswith Saltwater MysidsE 1241 Guide for Conducting Early Life-Stage ToxicityTests with Fishes3. Termino
8、logy3.1 The words “must,” “should,” “may,” “can,” and “might”have very specific meanings in this practice. “Must” is used toexpress an absolute requirement, that is, to state that the testought to be designed to satisfy the specified condition, unlessthe purpose of the test requires a different desi
9、gn. “Must” isonly used in connection with factors that directly relate to theacceptability of the test. “Should” is used to state that thespecified condition is recommended and ought to be met ifpossible. Although violation of one “should” is rarely a seriousmatter, violation of several will often r
10、ender the resultsquestionable. Terms such as “is desirable,” “is often desirable,”“might be desirable” are used in connection with less importantfactors. “May” is used to mean “is (are) allowed to,” “can” isused to mean “is (are) able to,” and “might” is used to mean“could possibly.” Thus the classi
11、c distinction between “may”and “can” is preserved, and “might” is never used as asynonym for either “may” or “can.”3.2 Descriptions of Terms Specific to This Standard:3.2.1 brine shrimp cysta gastrula-stage embryo that isenclosed in an envelope and cuticle for resistance to desicca-tion. Dried brine
12、 shrimp cysts are often incorrectly referred toas eggs. Upon hydration, embryonic development proceedsuntil a nauplius emerges from the encysting shell.3.2.2 brine shrimp naupliusa newly hatched, freely swim-ming, instar I stage larva. Nauplii are incapable of exogenousfeeding until the instar II st
13、age that occurs approximately 24 hafter hatch at 25C. The reddish-brown color of the nauplii isdue to the presence of yolk on which they rely for endogenousfood.1This practice is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fate and is the direct responsibility
14、 of SubcommitteeE47.01 on Aquatic Assessment and Toxicology.Current edition approved April 1, 2004. Published April 2004. Originallyapproved in 1987. Last previous edition approved in 1998 as E 1203 98.2The boldface numbers in parentheses refer to the list of references at the end ofthis practice.3F
15、or referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C7
16、00, West Conshohocken, PA 19428-2959, United States.3.2.3 feeding assaya test in which one or more life stagesof an aquatic animal species is raised on a diet to determine theadequacy of the diet for the life stage(s) of the species.3.2.4 Reference Artemia cystsa homogeneous collectionof cysts that
17、has been tested and declared a Reference sample.Formerly two repositories of reference cysts existed,4but bothsupplies have been exhausted without replacement.3.2.5 straina geographical population of brine shrimp thatis genetically distinct from other brine shrimp populations ofthe same species. Gen
18、etic distinction is determined electro-phoretically (2).3.2.6 time to 90 % hatchthe amount of time that expiresbetween the immersion of dried brine shrimp cysts in water andthe hatching of 90 % of the hatchable cysts. Time to 90 %hatch is usually abbreviated T90.4. Summary of Practice4.1 One can of
19、cysts is purchased. Samples are hatched inaerated salt water and nauplii are collected on a small-meshscreen and rinsed. Nauplii are fed to a species of aquatic animalto determine if the nauplii are an acceptable complete food forthat life stage of that species. Chemical and physical measure-ments m
20、ight also be performed on the cysts or on the nauplii,or on both. If the nauplii are acceptable, a large number of cansfrom the same lot are purchased, appropriately stored, hatched,and fed to that life stage of that species. An alternative is tofeed algae in addition to the brine shrimp to the test
21、 organisms.5. Significance and Use5.1 In certain toxicity tests during which small aquaticanimals must be fed, the food of choice is often live brineshrimp nauplii because of their availability, ease of use, andpresumed good nutritional quality. In addition, many testspecies that are cultured or hel
22、d in the laboratory must be fedprior to toxicity testing, even if they are not fed during the test.5.2 Brine shrimp nauplii can be readily hatched in thelaboratory from cysts that are obtained naturally from manygeographical areas around the world. Cysts from a few of thoseareas are available commer
23、cially, but might represent two ormore species of brine shrimp (1).5.3 Nauplii of different strains of the same brine shrimpspecies from different geographical areas can differ substan-tially in their nutritional value, contaminants, and acceptabilityas a complete food for the life stage of the spec
24、ies to whichthey are fed (3-8).5.4 The results of a toxicity test can depend on the brineshrimp nauplii used as food (9). It is desirable to determineprior to use whether a particular batch of brine shrimp is ofadequate quality for the test organisms.5.5 The primary requirements of acceptable brine
25、shrimpare that they are an appropriate size for the test organisms, haveadequate nutritional value, and do not contain excessiveconcentrations of contaminants. Whether a given lot of brineshrimp meets these requirements for a given species can onlybe determined by a feeding assay.5.6 Standardization
26、 of the hatching, harvesting, testing, andusing of brine shrimp will probably increase the reproducibilityof results of toxicity tests with some test species by decreasingthe use of unhealthy organisms caused by feeding them poorquality brine shrimp.6. Hazards6.1 Because water is a good conductor of
27、 electricity, use ofground fault systems and leak detectors should be consideredto help avoid electrical shocks. Salt water is such a goodconductor that protective devices are strongly recommended.7. Obtaining Cysts7.1 Selecting a SourceSelection of a given lot of cysts fora feeding assay can be bas
28、ed on information concerning thegeographical origin of the cysts, the way the cysts were driedand stored, and possibly the size of the cysts and resultingnauplii. However, commercial suppliers might not divulge thisinformation, and information that is provided might not becorrect. The preferred proc
29、esses for drying and storing brineshrimp cysts are, respectively, fluidized bed drying and storageunder vacuum or nitrogen (10). Cysts processed and stored inother ways might be acceptable, but hatchability will probablybe reduced. Size of nauplii at hatch varies from strain to strainbut is relative
30、ly constant within a strain (11). Mortality of somelarval fish increases with increasing naupliar size (12) becausesome larvae simply cannot ingest the larger nauplii. Nutritionalvalue and levels of contaminants vary both among and withinstrains, and depend mostly on the quality of the water and the
31、algae in the water from which the cysts were harvested (13).The only way to determine the acceptability of cysts is toperform a feeding assay. Therefore, it is desirable to purchaseand test a small sample (for example, one can) of cysts fromone lot before purchasing a large quantity.7.2 StoragePrope
32、rly sealed cans of cysts can be stored forseveral years at room temperature, under refrigeration, orfrozen. If they are frozen, they should be removed from thefreezer at least 48 h, and preferably about 1 week, prior to useto ensure adequate hatching. After a can has been opened, theoptimal conditio
33、ns for long term storage are, of course, lost. Ifthe contents will be used up in 1 to 2 months, it is onlynecessary to close the can tightly (for example, with a plasticlid) after each use and store it in a cool (about 4C), dry place,such as in a refrigerator. If the contents will not be used upwith
34、in 2 months, it is best to distribute the cysts among smallercontainers that hold abouta1to2month supply of cysts andseal the containers under nitrogen or vacuum or freeze them.One simple method is to place the cysts in a sealable plasticfreezer bag and introduce nitrogen via a pipet inserted throug
35、ha small hole. After about 1 min, the pipet is removed and thehole is sealed.8. Hatching Cysts8.1 Brine shrimp cysts may be hatched in either reusable ordisposable containers, such as conical plastic bags. Separatoryfunnels are probably the most convenient reusable containers,4Artemia Reference Cent
36、er, State University of Ghent, Ghent, Belgium, orQuality Assurance Research Division, U.S. Environmental Protection Agency,Cincinnati, OH 45268.E 1203 98 (2004)2because the stopcock allows harvesting of newly hatchednauplii from the bottom of the container. Substances used toclean a reusable contain
37、er should be nontoxic and the containermust be rinsed completely after cleaning so that the brineshrimp are not exposed to substances such as detergents.8.2 Salt water used for hatching should have a salinity of 25to 35 g/kg salinity.8.3 Aeration is necessary for hatching and survival of brineshrimp
38、 nauplii. Introduction of air into the bottom of a funnelshaped container provides best continuous circulation of cysts.Containers can be aerated using a glass tube or pipet attachedto a flexible tube leading to an air supply. Air used for aerationshould be free of fumes, oil, and water; filters to
39、remove oil andwater are desirable. Although brine shrimp cysts will hatchunder a wide range of temperatures, standard hatching tests areconducted at 25C (14), and all discussion of hatching in thispractice assumes a temperature of 25C.8.4 Brine shrimp cysts should be subjected to light intensityof a
40、t least 1000 lx for the first 3 h after immersion in salt water,because they need a light trigger to initiate hatching (15-17).8.5 Brine shrimp cysts should be added to the water in theproportion of 5 cm3of cysts/L.8.6 Cysts can be decapsulated by soaking in water for 2 h,dissolving the chorion with
41、 a hypochlorite solution withappropriate control of temperature, soaking in thiosulfate, andrinsing before incubation (18, 19). Although decapsulationdisinfects the cysts and increases hatchability and the energycontent of the nauplii, it is not recommended because of thelabor involved and because o
42、f the possibility of contaminationby hypochlorite, thiosulfate, and chlorinated materials.9. Harvesting Nauplii9.1 Brine shrimp hatch as instar I nauplii, which is anonfeeding stage. They diminish substantially in weight, ca-loric value (20), and biochemical composition (21) until theybegin feeding,
43、 and these changes might reduce their value asfood for test organisms (22). Therefore it is important toharvest nauplii shortly after they hatch. However, because theydo not all hatch at once, they should be harvested about 2 to 6h after T90, that is, about 2 to 6 h after 90 % have hatched. IfT90is
44、not known, it can be determined by periodicallysampling the aerated hatching solution and counting thehatched nauplii, unhatched cysts, and empty shells (10).At25C, T90for most brine shrimp is between 20 and 32 h (seeTable 1) (14). Therefore, brine shrimp should be hatched in aconstant temperature r
45、oom or water bath so T90is as similar aspossible from day to day and from season to season. After thefeeding schedule for the test organisms has been selected,hatching chambers should be set up at appropriate times sonauplii can be harvested when needed. The setup schedule caneither be planned on th
46、e basis of the T90of the cysts andtemperature that are available or one can try to select cysts anda temperature that fit into a desirable setup and harvestingschedule.9.2 At the end of the incubation period, aeration is stopped.Hatched nauplii and unhatched cysts will sink to the bottom,whereas emp
47、ty shells will float to the top. There might also besome heavy debris that sinks very rapidly and can be drawn offimmediately, before the nauplii and unhatched cysts reach thebottom. Because nauplii are phototactic, shading the top of thecontainer with black plastic and shining a light on the bottom
48、of the container can hasten the settling of nauplii. To avoidextreme packing of nauplii at the bottom of the container, it isbest to harvest nauplii 5 min after removal of aeration, wait 5min more and harvest again, repeating the procedure as oftenas necessary. After the nauplii have been harvested,
49、 they shouldbe washed onto a 150-m screen with clean salt water toremove small extraneous matter and substances such as glyc-erol that are present in the hatching water. If the nauplii aremixed with excessive amounts of debris, phototaxis can againbe used for separation, for example, in a separator box (23).Nauplii can also be separated by reimmersing them in cleansalt water, shining a light on one side of the container, andcollecting nauplii with a siphon or pipet as they move towardthe light. In certain circumstances, some brine shrimp emergefrom the cysts but do not
