4 Daphnia magna ACUTE LETHALITY

4

Daphnia magna ACUTE LETHALITY

TOXICITY TEST PROTOCOL

by

D. G. Poirier, G. F. Westlake, and S. G. Abernethy

ONTARIO MINISTRY OF THE ENVIRONMENT

AQUATIC TOXICITY UNIT

AQUATIC BIOLOGY SECTION

WATER RESOURCES BRANCH

QH90.57.B5P7531988

APRIL 1988

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Daphnia magna ACUTE LETHALITY

TOXICITY TEST PROTOCOL

by

D.G. Poirier, G.F. Westlake and S.G. Abernethy

Ontario Ministry of the Environment

Aquatic Toxicity Unit

Aquatic Biology Section

Water Resources Branch

April 1988

Qc Queen's Printer for Ontario, 1988

ISBN 0-7729-3798-2

ABSTRACT

Daphnia magna (Straus) is a small planktonic crustacean which is widely distributed in ponds and lakes inOntario, and is representative of filter-feeding zooplankton communities. This organism has been used intoxicity testing for over 100 years. As a test organism Daphnia has the advantages of being small(therefore only small amounts of effluent are required for each test), they are generally very sensitive,respond quickly to toxicants, and they are easily reared in large numbers In the laboratory.

Proper culture conditions consist of moderately hard water (120 - 250 mg/L), with a pH between 6.5 and8.5, and maintained at 20°C. Light intensity should not exceed 800 Lux at the water surface, and shouldfollow a 16h:8h light:dark photoperiod.

The toxicity test Is a 48h static, acute test which uses only healthy neonate (1 st Instar) D. magna less than24h old. The test chambers can be any reasonable size, but must be constructed of glass and have asurface area to volume ratio (+ 10%) similar to that of a 25 x 150 mm test tube. Loading densities must notexceed 1 neonate per 15ml of test solution, and a minimum of ten Daphnia per concentration is required.Dissolved oxygen, pH and conductivity must be measured for each test concentration before and after thetest. Estimation of the LC50 values, 95% confidence intervals, and slope of the probit line can becalculated by any acceptable method with preference to probit analysis using the maximum likelihoodmethod, where data permits. All data must be supplied as per the "toxicity test summary report sheet' inappendix three

RESUME

Daphnia magna (Straus) est un petit crustac6 planctonique que I'on trouve abondamment dans la plupartdes lacs et 6tangs de I'Ontario et qui forma une partie importante de la communaut6 zooplanctonique senourrlssant par la m6thode de filtration. On se sert de cet organisme depuis plus de cent ans pour 6tudierla toxicity aquatique. II offre piusieurs avantages pour ce type d'6tude. Tout d'abord, sa petite tailledemande un volume d'effluent moindre pour chaque test. Ensulte, II est trbs sensible aux agents toxiques,auxquels 11 r6agit rapidement. D. magna pout enfin titre 6lev6 facilement en grand nombre en laboratoire.

Les conditions aquatiques pour la culture an laboratoire n6cessitent une duret6 modeste (entre 120 et 250mg/L), une acidit6 allant de 6.5 a 8.5 at une temp6rature constants de 20°C. L'6clalrage ne dolt jamaisd6passer une intensH de 800 Lux A la surface de I'eau et le cycle lumineux dolt suivre le r6gime 16h/8h6clairage/noirceur.

L'analyse de la toxlcitd aigub dure 48 heures en 6tat statique et on utilise seulment les nouveau-n6s(premier stade) de 1'espbce Daphnia magna, qui dolvent titre Ag6s de moins de 24 heures et titre en bonnesant6. La pibces d'essal dolt titre d'une grandeur raisonnable. Elle dolt titre de verre et le rapport entre lasurface et le volume dolt titre semblable, A plus ou moins 10 p. 100, A celul dune 6prouvette mesurant 25sur 150 mm. La population des crustac6s ne devrais jamais d6passer une densit6 d'un nouveau-n6 par 15ml de solution. On dolt se servir d'au moins 10 organismes par concentration de solution. L'oxygbnedissous, I'acldlt6 et la conductivity dolvent titre mesur6s daps chaque concentration avant et aprbsI'analyse. La CI-50, les intervalles de confiance de 95 p. 100 et la pente de la ligne des probits peuvent trecalcul6s au moyen de toute m6thode acceptable. Toutes les donn6es dolvent titre consign6es sur laformule "Toxicity test summary report sheet", que I'on trouvera A I'annexe trois.

Daphnia magna Toxicity Test 2

1. INTRODUCTION

The Ontario Ministry of the Environment is responsible for ensuring the maintainanceand improvement of the quality of surface waters in the province. Fundamental to theMinistry's water management policy is the reduction of toxicity from discharges tosurface waters (M.O.E., 1978). In addition, regulations and legislation, bothprovincial (Ontario Water Resources Act, Environmental Protection Act) and federal(Canadian Fisheries Act), obligate the Ministry to monitor and control toxicity ineffluents. The Water Resources Branch of the M.O.E. is developing and evaluatingtoxicity tests to measure the lethal and sublethal biological impacts of effluents onaquatic organisms from different trophic levels. The Daphnia magna acute lethalitytoxicity test protocol describes one of these tests.

Daphnia, a small planktonic crustacean, has been used in toxicity testing for over100 years (APHA, 1985). It is widely distributed in ponds and lakes in Ontario, and isrepresentative of filter-feeding zooplankton communities. As a test organism, Daphniahas the advantage of being small (adult Daphnia are normally slightly larger than thehead of a pin). Because of the size of these animals, only small amounts of effluentare required for each test. Daphnia are generally more sensitive and respond morerapidly to toxicants than fish (Johnson and Finley, 1980). They can easily be reared inlarge numbers in the lab, so that animals can be available for testing throughout theyear. Large numbers of young are produced in cultures maintained with reasonablecare for the nutritional and environmental requirements of the animals. The skills andknowledge required to perform this toxicity test are modest. Training normally requiresonly one demonstration of the test and minimal subsequent supervision. Commonsense instructions like "Do not rip off the legs when pipeting.", are generally all that isrequired. Numerous protocols (ISO, 1982; U.S.EPA, 1982; APHA, 1985; ASTM, 1986)have been reviewed in designing the following procedure and the most usefulcomponents of each have been retained. We feel that this protocol provides an easyto use, standardized toxicity test which is not unduly restrictive. This protocol hasbeen used on a wide variety of industrial effluents samples for more than a year at theOntario Ministry of Environment laboratory in Rexdale and has proven to beconsistent, reliable and repeatable. It should be applicable to a wide variety of waterpollution problems.

2. DEFINITIONS

DEATH is defined to occur when no heartbeat is discernible. It is important to definedeath for Daphnia toxicity tests because the animals tend to die in "stages". That is,they may lose locomotory control first, or be rendered completely immobile (either ofwhich could spell death for the organism in the wild), before the heart actually stopsbeating.

IMMOBILIZATION is the condition in which the organism can no longer propel itselfthrough the water, yet still may have a heartbeat. It is important to differentiatebetween death and immobility because each response may provide information onthe mode of action of the toxicants being tested. Many chemicals, for example, act on

3 Daphnia magna Toxicity Test

the nervous system, immobilizing the organism without immediately killing, eventhough the animal may perish from starvation or from some other cause in the wild. Insome cases the organism may recover if it is transfered to "clean" water.

NEONATES are the first instar. Crustaceans have a hard outer shell or exoskeletonwhich does not stretch or grow as do the internal organs. Instead, the animal makesa new skeleton inside, sheds the old one, absorbs water to expand the new skeletonbefore it hardens and then grows inside this larger skeleton. Each time the organismsheds this outer shell and forms a new one it is called a "molt", and the new largerstage of the organism is called an "instar". Daphnia average between 20 and 30 moltsin their lifetime. A neonate is the first instar or youngest stage.

STATIC TESTS are ones in which the toxicant, or effluent is not replenished duringthe exposure period.

48 hr LC50 is the median lethal concentration of chemical or effluent (i.e. theconcentration which causes 50% mortality of the test organisms during 48 hr ofexposure).

An ACUTE response is usually a reaction occuring within a short period of time,whereas CHRONIC effects occur as a result of long term exposure, usually over atleast one generation of the test species. The distinction between acute and chronic isrelated to the life cycle of the test organism. For D. magna an acute test takes twodays to conduct, and a chronic test takes approximately seven days.

The end point of a LETHAL TEST is death of the test organism, whereas the endpoint of a SUBLETHAL TEST is not mortality, but may include changes in suchthings as locomotion, growth rate, reproduction, respiration, biochemical responses,etc.

3. BIOLOGY OF Daphnia magna

Daphnia magna is one of the larger members (adults < 5 mm) of the order Cladocera.D. magna is laterally flattened, and is covered by a transluscent, folded shell called acarapace (Figure 1). Internal organs and limbs are easily visible through thecarapace using a magnifying glass or light microscope.

D. magna is found in ponds and lakes in Asia, Europe and North America. They livein open water or amongst shoreline vegetation, moving in short hops using rapidstrokes of their powerful second antennae (Figure 1). Their flattened appearance andhopping motion has earned them the common name of "water fleas". They feedprimarily on algae, protozoans and bacteria using their setose ("hairy") thoracic legsto strain food particles from the water. In turn, Daphnia are a very important foodsource for fish and aquatic invertebrates.

Under favourable environmental conditions reproduction is usually parthenogenic, thatis female Daphnia produce more female Daphnia without requiring a male to fertilizethe eggs. Eggs are produced in the ovaries, and are released, via the oviduct, intothe brood chamber (Figure 1). In the brood chamber the eggs continue to develop,eventually hatching (within 1 to 3 days) into young which are genetically virtually


identical to the parent. The young neonates are then released to the outside at thenext molt. Usually one clutch of eggs is produced for each adult instar, with between2 and 100 (mean = 50) eggs per clutch.

Figure 1: Anatomy of Daphnia magna.

During adverse environmental conditions (eg. crowding, accumulation of excretoryproducts, decrease in available food, temperature changes, etc.) males and femalescapable of sexual reproduction are produced. The eggs, fertilized during copulation,are released to the brood chamber which then thickens and darkens to form anephipplum. This ephippium is then shed with the next molt. Ephippia usually mustundergo some type of environmental stress such as freezing or drying before theyhatch. This is the normal way that Daphnia survive adverse conditions.


Figure 2: Ephippium of D. magna.

Young Daphnia magna grow very quickly reaching reproductive maturity in about 10days, and will live over 35 days under good conditions. The optimal temperature forgrowth, reproduction and survival appears to be around 20°C. Daphnia magna canwithstand low oxygen concentrations, but are stressed at concentrations less than40% saturation. Most are found in water with pH between 6.5 and 8.5.

There are few known diseases of Daphnia. On rare occasions they may get a fungalor bacterial infection in culture situations (Unestam, 1973).

4. TEST ORGANISM

The test organism required by this protocol is Daphnia magna Straus. The sameprocedures would probably work with D. pulex de Geer, but this Daphnid is poorlytaxonomically defined. Both species proved to be similarly susceptible to manychemical toxicants (Canton and Adema, 1978), are widely distributed and fill similartrophic niches in nature. D. magna is more easily handled because of its larger size,and is more suitable for the range in water hardness found throughout Ontario.

5. EQUIPMENT AND SUPPLIES

temperature control device (incubator, temperature controlled room, water bath,or submersible aquarium heater.)

. widemouth glass jars (250 ml to 4000 ml)

10 L or larger glass aquaria or cylindrical tanks

screw top glass test tubes and test tube racks (e.g. 25 x 150 mm)

graduated cylinders (glass)

small aquarium net (mesh size approx. 0.1 x 0.1 mm)


. stainless steel or plastic screen (mesh 1 x 2mm)

. air supply (oil free) - use laboratory supply or small aquarium pump

fluorescent light supply and photoperiod timer - light intensity at water surface<800 lux, squewed towards blue end of the spectrum (colour rendering index90) spectrum.

. thermometer ( 0.50C gradations)

The test lab must have the facilities to test water quality parameters (using testingmeters below or accepted chemical methods), or send water samples out to aqualified laboratory to be tested.

oxygen meter

pH meter

conductivity meter

hardness meter

microcomputer

statistical analysis program capable of calculating LC50s

light table (to aid in sorting and observing neonates)

dissecting microscope - capable of >40X magnification

light intensity meter

Fk#W"C" Ugnl

Main Cuttwa Tank

I

Figure 3: Daphnia culturing and toxicity testing apparatus.


6. MAINTAINING CULTURES AND OBTAINING TEST ORGANISMS

Maintain the stock cultures of Daphnia in clean all glass or nalgene tanks >4L insize. These cultures will be the main supply of newly maturing adults for futurebreeding colonies.

Initiate the main cultures by combining 20% mixed (or 10% Selenastrum and 10%Chlorella) algal food stock (Appendix 1) with 80% dilution water. Dilution water mustbe of sufficient and consistent quality to maintain healthy, growing, reproducingstocks of D. magna. If the only available supply of water is chlorinated tap water, itmust be dechlorinated using an activated carbon filter column and vigorous aerationfor at least 24-h. Chlorine measurements are made using amperimetric titration andmust be non-detectable in dilution water. Water in the D. magna cultures must have atotal hardness of between 120 and 250 mg/L, and a pH between 6.5 and 8.5.

Feed all the cultures at a rate of 10 ml of mixed algal food stock per litre of culturesolution twice weekly (or 5ml Selenastrum culture and 5ml Chlorella culture). Everythird week feed only Selenastrum for two successive feedings, then return to the mainfeeding schedule. This feeding regime reduces the chances of nutritional deficienciesoccuring in the cultures (Taub and Dollar, 1968). Change the main culture solutionevery month. Use distilled water to replace any evaporated water weekly, and/orreplace 1/4 volume of the stock culture water weekly.

The cultures should be aerated gently and continuously while maintaining a 16h:8hlight:dark cycle. An 8-h light period stimulates sexual reproduction and a 16-h lightperiod stimulates asexual reproduction (Buikema et al., 1980).

Light intensity should not exceed 800 lux at the water surface, and should besquewed towards the blue end of the spectrum (colour rendering index > 90). Coolwhite fluorescent lights may be used for this purpose. Red and green wavelengthshave been shown to increase Daphnia sensitivity to toxicants, and ultraviolet andgreen wavelengths increase mortality in many aquatic organisms (Buikema, 1973).

Water temperature in the cultures should be 20°C ± 1 °C. Use an incubator, waterbath, controlled temperature room, or an aquarium heater to prevent watertemperature fluctuations from exceeding 1 °C per day.

A culture health test should be conducted in triplicate once per month (Appendix 6).

The toxicity experiments use only vigorously swimming neonate Daphnia. To obtainthese young organisms it is first necessary to establish several small containers(approximately 4-L) of adult Daphnia only. Initiate these breeding colonies bycombining dilution water with algal food stocks as previously described for initiatingstock cultures of Daphnia. Adult Daphnia are then separated from the main culturesby siphoning them out and straining them through a screen with 2 x 1 mm mesh, orby hand picking with a large diameter glass tube. Tip the screen quickly into thebrood stock containers to prevent dessication of the adults. This screening processmust be done gently to prevent damaging the adults. Stocking density should beapproximately 10 adults/L in these containers. Do not transfer animals to water which


differs from the main culture water by more than ±2°C, ±0.2 pH units or 10% totalhardness. Capture the screened culture water and return it, with any Daphnia whichmay have passed through the screen, to the main cultures. These brood culturesolutions and adults should be replaced once per month.

The following procedure must be conducted <24-h immediately prior to testing.Neonate Daphnia for toxicity testing are obtained from the brood cultures. Place ascreen (mesh size 2 x 1 mm) over the mouth of a small aquarium net (mesh size 0.1 x0.1 mm) and screen the contents of the brood culture jars through the pairedmeshes, while retaining the culture solution. Adult Daphnia will be captured on thelarger screen and the smaller ones on the finer screen. Return the large Daphnia tothe brood culture container, and the small Daphnia to the main cultures. The youngDaphnia captured by this screening method must not be used for toxicity testingbecause their age is unknown, and they may be damaged during the procedure. Byconducting this procedure 24-h before a test it can be assured that all the youngDaphniasubsequently produced in the brood cultures are < 24-h old.

Prior to testing, separate the neonates from the brood colonies using a large diameterglass tube (eg. a 10 ml pipette with the tip broken off and with the edges smoothedto remove sharp projections). The large bore of this tube reduces the risk ofdamaging the neonates while pipetting. Damaged animals are not used in these tests.

7. TOXICITY TESTING

The following procedure may be modified slightly to suit the nature of the laboratoryfacilities and the effluent samples, but any changes must meet minimum specificationsindicated in this text. All effluent samples must be collected, handled, shipped, andstored according to the procedures outlined in Appendix 2. Test chambers can beany reasonable size, but must be constructed of glass, and must have a similarsurface area to volume ratio (± 10%) to the 25 x 150 mm test tubes. This preventsany discrepancies in results due to excessive evaporation of the test solution, andvariability in oxygen transfer across the water surface. The test tubes also facilitatecounting the organisms in turbid samples. The number of neonates per test chambermust not exceed 1 neonate/ 15ml of test solution. A minimum of ten Daphnia perconcentration is required.

Dissolved oxygen concentration, pH, hardness, and conductivity must be measured inone tube of each test concentration immediately before and after the test isconducted. It is advisable to use a duplicate set of test concentrations for waterchemistry analysis.

Prepare effluent tubes in quadruplicate (or more). The dilution water must be thesame water used to initiate the main and brood cultures. Place 50, 25, 13, 7, 3, and0 ml into 25 x 150 mm screwtop, glass test tubes. Top up all the test tubes to a finalvolume of 50 ml using dilution water which has been aerated overnight. This givesfour replicates of a dilution series with 100, 50, 26, 14, 6 and 0 percent effluent. Thisdilution series can be modified to suit the sample, but it must have a concentrationseries with at least 5 concentrations which include the 0% and 100% effluentconcentrations. If 100% mortality occurs at the lowest test concentration above 0%


effluent, then the test must be repeated with a lower concentration range. If

pretreatment of the effluent sample is desired for the purpose of isolating the toxicconstituents in the effluent (e.g. aeration or pH adjustment), then these tests must beconducted In addition to an untreated sample.

Into each test tube pipette, in <0.5 ml of water, 3 randomly chosen neonate Daphnia,releasing each under the surface of the test solution. This ensures that the neonatesare not trapped by surface tension at the air/water interface, and no bubbles aretrapped under the carapace causing them to float. Add the neonates proceedingfrom the lowest to highest concentrations to prevent changes in effluentconcentrations due to contamination from the pipette. Do each replicate separately,rinsing the pipette in dilution water between replications. Immediately check theneonates and replace any which are floating or are injured. Record the time the testbegins and return the rack of tubes to the water bath.

Observe the Daphnia, without removing them from the tubes, at 1, 2, 4, 24, and 48-hafter the beginning of the test. Record observations such as circling, floating, andimmobility at each time period (see report form Appendix 3). After 48-h remove anyindividuals which are immobile and examine them under a light microscope for a heartbeat. Record the number which are immobile or dead separately for eachconcentration of effluent. If there is more than 10% mortality in the control (0%effluent) then the test must be repeated.

Clean all glassware using a suitable detergent and scrub brushes. Follow this with anacid wash using 5% H2SO4 for > 24-11, and rinse thoroughly with distilled water.

8. ESTIMATING LC50 VALUES

Calculate an LC50 value with 95% confidence limits using a computer with softwarefor statistical analyses (Stephan 1977). Most programs use probit analysis (Hubert1986) with the least squares method or successive repetitions of the maximumlikelihood function to achieve a line of best fit to transformed data points (Hubert andSchoch, 1984). The LC50 value is read from this line by interpolation and representsthe median lethal concentration of the toxicant. The method used to determine theLC50 value must be reported with the results. Where an immobility response isobserved the median effective concentration (EC50) must be calculated in a similarmanner to the LC50, using the immobility and the mortality data combined.

If there are control mortalities < 10% during the experiment, these deaths areassumed to be the "natural" mortality rate, and Abbott's formula (Abbot, 1925) mustbe used to correct the mortality rates at the other exposure levels. The adjusteddeath rates are then used in the probit analysis.

in addition to the computer calculations, a probit line can be drawn as an option.Begin by plotting the data points on graph paper similar to that in Appendix 3. Noticethat the percent effluent is on a logarithmic scale (x-axis) and the percent effect is ona probit scale (y-axis). Since there is no 0% or 100% lines on the y-axis, then circlethe lowest or highest value available (2% and 98%) and use arrows to indicate their


true values (Appendix 4). Do not plot more than two consecutive 0% or 100% effectson the graph.

Fit a line to the data points using data generated during statistical analysis or by eye,allowing the points nearest 50% effect to have the most influence on how the line isfitted, and give less priority to points further from 50%. If one or more concentrationsof effluent, in addition to the 0% and 100% effluent, caused partial effects then theChit test can be used to evaluate the goodness of fit of the probit model to the data.Most good computer programs will calculate a Chit value for your data, but if it is notavailable it can be calculated using the methods described in Hubert (1986) or ASTM(1987). The line is a good fit if the calculated Chit value is less than the tabulatedChit value (Appendix 5) for the determined degrees of freedom.

The toxicity test report must contain complete information on the effluent sample(industry, location of sample site, dates collected, sampling method, etc.), the testparameters (species used, water temperature, dilution water source, etc.), test results(including raw data, chemical parameters, LC50 and EC50, confidence limits andslope), and any pertinent remarks. Copies of a computer program called TOXDATAare available free of charge from the Ministry. Submission of the plot of the probit lineis requested, but not required. See the Toxicity Test Report Sheet in Appendix 3 forall the data required for submission. Report any deviations from this protocol in theremarks section of the report.

Once every month the laboratories conducting Daphnia magna toxicity tests arerequired to submit water chemistry data, which includes heavy metals, NH3, andcommon groups of pesticides for the dilution water. The analysis should be sufficientto calculate an ion balance.

11. REFERENCES

Abbott, W.S. 1925. A method of computing the effectiveness of an insecticide. J.Econ. Entomol. 18:265-267.

APHA. 1985. Standard methods for the examination of water and wastewater. 16thEd. Amer. Publ. Health Assoc., Wash., D.C.

ASTM. 1980. Standard practice for conducting acute toxicity tests with fish,macroinvertebrates, and amphibians. ASTM E 729-80, Amer. Soc. Test. Materials,Philadelphia, Penn.

ASTM. 1980. Aquatic Invertebrate Bioassays. Amer. Soc. Test. Materials, Philadelphia,Penn.

Buikema, A.L., Jr. 1973. Some effects of light on the growth, molting, reproductionand survival of the Cladoceran, Daphnia pulex. Hydrobiologia 41: 391-418.

Buikema, A.L., Jr., J.G. Geiger, and D.R. Lee. 1980. Daphnia toxicity tests. In A.L.Buikema, Jr. and J. Cairns, Jr. [Eds.] Aquatic Invertebrate Bioassays. Amer. Soc.Test. Materials, Philadelphia, Penn. p 48-69.

II Daphnia magna Toxicity Teat

Canton, J.H. and D.M.M. Adema. 1978. Reproducablity of short-term andreproduction toxicity experiments with Daphnia magna, and comparison of thesensitivity of Daphnia magna compared with Daphnia pulex and Daphnia cucullata inshort-term experiments. Hydrobiologia 59: 135-140.

Government of Canada. 1977. Fisheries Act. Fish. Environ. Canada. 35 pp.

Government of Ontario. 1974. The Environmental Protection Act. M.O.E. 96 pp.

Government of Ontario. 1976. The Ontario Water Resources Act. M.O.E. 49 pp.

Hebert, P.D.N. 1978. The population biology of Daphnia (Crustacea, Daphnidae).Biol. Rev. 53: 387-426.

Hubert, J.J. 1987. Bioassay (2nd Ed.). Kendall/Hunt Publ. Co., Toronto. 164 pp.

Hubert, J.J., and J.P. Schoch. 1984. Probit: An interactive program in Basic for probitanlysis. University of Guelph Statistical Series No. 1984-160. Guelph. 18 pp.

Hutchinson, T.C., and P.M. Stokes. 1975. Heavy metal toxicity and algal bioassays.ASTM STP 573: 320-343.

ISO. 1982. Water quality - determination of the inhibition of the mobility of Daphniamagna Straus (Cladocera, Crustacea). International Organization for StandardizationISO 6341-1982(E).

Johnson, W.W., and M.T. Finley. 1980. Handbook of acute toxicity of chemicals tofish and aquatic invertebrates. U.S. Fish Wildl. Serv. Res. Publ.No. 137. 98 pp.

Ontario Ministry of the Environment. 1978. Water Management: Goals, policies,objectives, and implementation procedures of the Ministry of the Environment. 67 pp.

Stephan, C.E. 1977. Methods for calculating an LC50. ASTM STP 634: 65-84.

Taub, F.B., and A.M. Dollar. 1968. The nutritional inadequacy of Chlorella andChlamydomonas as food for Daphnia pulex.

Unestam, T. 1973. Fungal diseases of crustaceans. Rev. Med. Vet. Mycol. 8: 1-20.

U.S. EPA. 1987. User's Guide: Procedures for conducting Daphnia magna toxicitybioassays. U.S. EPA Rep. No. EPA/600/8-87/011. 57pp.

Wong, S.L. 1985. Algal assay evaluation of trace contaminants in surface waterusing the nonionic surfactant, Triton X100. Aq. Toxicol. 6: 115-131.

APPENDIX 1Algal Culture Preparation

Bristol's Medium (Hutchison and Stokes, 1975) - use at half strength.

Add 10 ml of each of the following stock solutions to 940 ml of distilled water(macron utrients):

Solution Concentration

NaN03 25.0 g/L

CaC12 .21-120 2.5 g/L

MgSO4. 71120 7.5 g/L

K2HPO4 7.5 g/L

KH2P04 17.5 g/L

NaCl 2.5 g/L

Add 1.0 ml of each of the following stock solutions (minor nutrients) to the litresolution above:

EDTA - Nat

KOH

FeC13. 61120

H3B03

50.0 g/L

31.0 g/L

4.84 g/L + 1 ml H2SO4

11.42 g/L

Micronutrient solution:MnC12. 41120

ZnSO4. 71120

M003

CUSO4. 51120

Co(N03)2. 61120

CoC13. 6H2O

1.44 g/L

8.82 g/L

0.71 g/L

1.57 g/L

0.49 g/L

0.35 g/L

Adjust pH to 7.0 with 10% HCL.

Autoclave at 121 °C, under 1.06 - 1.2kg/cm2 pressure for 30 minutes, and coolthe mixtures.

. Inoculate culture bottles with equal quantities of Selenastrum capricornutum andChlorella fusca, or inoculate them separately.

Incubate algal cultures with continuous, vigourous aeration under constant light, t(8000 lux) (Wong, 1985). Cool white fluorescent light is recommended.

Feed Daphnia with algal cultures between 7 and 14 days old.

Algal cultures can be obtained from the University of Toronto culture collection.

Appendix 2

Protocol for Sampling and Shipping of Effluent

and Legal Toxicity Samples

All samples taken for the purpose of toxicity testing should be accompanied by samplestaken for chemical characterization at the same time. This will assist the interpretation ofpotential causative agents in a complex mixture.

A sufficient record to identify the source of the sample and how it was handled is alsoessential (see Appendix 3).

Sampling containers should be lined with food grade polyethylene or polypropylenebags or made of stainless steel, glass or Teflon*. Grab or composite samples,depending on the variability of the effluent, should be a minimum of two litres forDaphnia (80 L if static trout tests are to be done as well (Protocol to determine the AcuteLethality of Liquid Effluents to Fish, M.O.E., 1983). Any materials coming into contactwith the sample must be clean and non-toxic. The sample containers must be filled andsealed without headspace. There should be no preservatives used in the sample.

The sample must not be frozen, but should be chilled if possible. If more than 48 hourselapse prior to testing, the sample must be refrigerated to between 5 and 15 °C.

The sample is valid for a maximum of seven days including the exposure period of thetest (ie. the testing must begin within five days of sampling).

Samples must be recombined and thoroughly mixed prior to testing.

* Teflon - Reg TM E.I. du Pont de Nemours & Co.

APPENDIX 3

TOXICITY TEST SUMMARY REPORT SHEET

Industry / Location :....................................................................................

Region :.......................................................................

Sample # :.......................................................................

Collected By :..........................................................................

Date / Time Collected :................................. Tested :...............................................

Sampling Site :............................................ Method :.........................................

Storage / Preservation Details: ..................................................................................

TEST PARAMETERS

Test Species :.............................................. Life Stage :..................................

Dilution Water Source :................................. Chlorine Residue :................. ug/L

Test Volume :............................................................

EFFLUENT PARAMETERS

WATER CHEMISTRY

Effluent Concentration

D.O.mg/I

Hardnessmg/I

Conductivityumhos/cm

pH

0 hrs 48 hrs 0 hrs 48 hrs 0 hrs 48 hrs 0 hrs 48 hrs

TEST RESULTS

Length of Test (h) :..........................................................

Median Lethal Concentration (LC50) .............................

Upper Confidence Limit: ....................................

Lower Confidence Limit: ....................................

Estimated Slope of the Probit Line: ..................................

Median Effective Concentration :......................................

Upper Confidence Limit :.................................

Lower Confidence Limit: ...............................

Estimated Slope of the Probit Line: ..................................

REMARKS:......................................................................................................................................................................................................................................................................................................................................................................._.........................................................................................................................................._.......................................

Daphnia magna Acute Lethality Toxicity Test Work Sheet

Test #: ............................................. Animals/Container:..........................................

Number of Animals Immobile # Dead Notes

Elapsed Time (h)

A

B

C

D

A

B

C

D

A

B

C

D

A

B

C

D

A

B

C

D

A

B

CD

PERCENTAGE

Z 5 1 0 1 5 20 3 0 40 50 8 0 70 8 0 8 5 9 0 9 5 98 76

7

- - j

Ji

I ;-

55J I M ! 1 11TI R 11 M I 1 11 M U ,

41 A 1 1 11 1 1 1111 11 11 [li l t r. '... : t t = 4

1 t! :IJJ li t I } 1:: ::11 l l;i i;. !:i: ".I li!

! I I I 3 !

-2

- - _i 1 , I. ,fr 1. {rl: I1., lax 1 }

1 r 2-t-

H I

1 i9

_

f !F i t ' wm- 99 _ _ - - t ' " :I :I .' 1.f

'

M :; T .l: aV7 - - -- - - - {t

11 1{ t . i. I: l , ±r

7- ! i 'j' j! 1 :i !1 Ij i r

1 .3

d

- --fit I n iF 1 :J 111. -

4 - - s 1 . = iJt i' i = - = 4

ii iiI J .,

I it

1 1E: m

s - - _ 7 I t 4 1 i8

I t i i t -7

8

- - i' ii is - - -

- t:7

Z - :1 ,U 1 14 11 1 11- 11 ;i..

.

1:f '

t :

- -

-1!

1 It H I M M MIt t tl

i21

'

i t .J

j' in!

till`

i',!1

it';f i!t LIP1

7,0PROBITS

APPENDIX 4Sample Calculation

TOXICITY TEST SUMMARY REPORT SHEET

Industry / Location :.. ..x .. /...

Region ........................1/.......

Sample # :.... row ...................................

Collected By :.......... ........................................

Date /Time Collected :.. Tested

Sampling Site e . ........... Method :........... 1,,7,na.hl ..................

Storage / Preservation Details:...... ...t,q ...l ...... .......

TEST PARAMETERS

Test Species Life Stage :.....iJ91P.4.ct.lc............

Dilution Water Source Chlorine Residue :.... XJ:.... u /L/ /- 9

Test Volume :...........4 Pw/ ...................................

EFFLUENT PARAMETERS

WATER CHEMISTRY

Effluent Concentration

D.O.mg/I

Hardnessmg/I

Conductivityumhos/cm

pH

0 hrs 48 hrs 0 hrs 48 hrs 0 hrs 48 hrs 0 hrs 48 hrsa

0

o o . iy6 11T ,26 z S 2 711

6 $. 9 Fr. 6 /60 /Sy Soo 90 `/ 73

/SG /S`/ // 76

,7 Fl. 3 / G /6 ? I Foe 1;780 7. 7.7

So Y. 6 Fs.1 /? 3 Zs z 7,5-0.0 ( a . q

,ZO S z0 Saio s-/'o 0 ?. s y

TEST RESULTS

Length of Test (h) :................$1...................................

Median Lethal Concentration (LC50) :.........OTZ'I .........

Upper Confidence Limit :...............sf .,z...........

Lower Confidence Limit: ................ ..........

Estimated Slope of the Probit Line:........... 66...............

Median Effective Concentration :............ V,. 3 ................

Upper Confidence Limit:.......... I f... 0..............

Lower Confidence Limit:......... 19.1.............

Estimated Slope of the Probit Line:...........: 52........

REMARKS:.. CC....eF cp.,44. ,C 7....,,..,. ................... ........... ....... ........................................o.....,........r.......ft....c........................ll :'.. Ii Wx/irC e .... ofatoo, . st.... /.k ....< ....(.1Azr .. QT...., K.....................:

(/

Daphnia magna Acute Lethality Toxicity Test Work Sheet

Test :.1..... Animals/Container:.....:]:..... ,lR.°.: ....

Number of Animals Immobile # Dead Notes

Elapsed Time (h) / Z y Z yyO

A

C D I.W.4W0l

D Z)A

C I

AB

C

/z

D

AB

c

z

D

A / .2

B /3

D

AB Z

Z3

/OD % C : z3 lZ

D .4 .3 3

PERCENTAGE

2X 5 10 1 5 20 30 40 50 80 70 80 8 5 90 9 5 98 76

T 1 1 1 1 1 11 1 11 II II J1 1 11 11 111 1111 111 1 11111 111HI M1 1! 11 11111 11 11111 11 11 .1 1 111 1

e11,

ei7M W f

5 5

a

-- - - t :: ' :i:: I t I `: -. r

33

ET Ti1

et i:, .} . 'it 1(ir i!' .;,. ... I.ll -t - t i -.T _

2Y f

2

[ 'F -

4 1

1 1 t ;I".

r

iti itr It !,fi

h

7 _ = tip ii 'i -t r i s rt - 1 :; t . .Y _t70

8 60

5 50

11,141 11 li11 lt1t 11It U l Wl 11 11111 11 U4 1-1 11 1 t I:: . 40ii i

3_

30t

ON U h li

+! 1 ,

+

2 f 2Dt F i

lr It I0 I M

tH I

-

M

"

;, to96 - - -

-

M. m vi H}iY

I1::

M Hill1

t t- i i9

Y 3 i'I . 1:

66

_:q iti; lltj !'I I' 4tI4' t

55 == _ = 1 [ D IM i 1! '1! 1 14 1

a _ _ _1

if:i.t

1 i

iii iil i ir 1 i - a_

1 1 flU M M 111 1 i4, - 44 4 1

31

3. 14 4 44 ,

t f I1,4 r

1

Jib :.F+ -i-

21f r, 1 r tl, li4 4 -

i 1 tt t ' r:

1.c

T _- I - - 1-1 1 1 . - - - -3.0

too

PROBITS

APPENDIX 5Values of Chf2

Probability of a larger value of x

995 I .990 .975 ! .450 I .900 I .750 I .500 + .250 I .100 .050 I .025 I .010 I .WS

. 0393

.0100

.0717

.207

.412

3.053.574.114.665.23

.03982

.0506

.216

.484

.831

20.7 22.2 24.428.0 29.7 32.435.5 37.5 40.5

.03393

.103

.352

.7111.15

1.842.172.733.333.94

4.575.235.8A6.577.26

26.534.843.2

.0158.211.584

1.061.61

17.318.118.919.820.6

29.137.74G.5

20.821.722.733.824.5

33.742.953.3

.4551.392.373.364.35

5.356.357.348.349.34

10.311.312.313.314.3

20.321.322.323.324.3

25.326.327.328.329.3

39.349.3

19,420.521.622.723.8

24.926.027.128.229.3

30"431.532.633.T34.8

45.658 3Gi.u

17.318.519.821.122.3

23.524.826.027.228.4

29.630.832.033.234.4

35.636.737.939.140.3

51.883.274.4

19.721.022,423.725.0

26.327.828.930.131.4

32.733.935.236.437.7

38.940.141.342.643.8

55.8c7.s79.1

14.416.017.519.020.5

21.923.324.728.127.5

28.830.231.532.934.2

35.536.838.139.440.6

41.943.244.545.747.0

59.371 a83.3

16.818.520.121.723.2

24.726.227.729.130.6

32.033.434.838.237.6

38.940.3al.s43.094.3

45.647.048.349.850.9

63.778.288.4

18.520.322.023.625.2

26.828.329.831.332.8

34.335,737.238.640.0

41.442.844.24.5.64G.9

48.349.G51.052.353.7

66.879.592.0

Appendix 6Culture health and control mortality

If the protocol is carefully followed, the growth, survival, and reproduction of Daphniawill be sufficient to provide a continuous supply of neonates for toxicity tests. The keyto achieving consistent and reproducible test results is to start all tests with neonateswhich have been cultured for several generations under the specified, controlledenvironmental conditions. Deviation from this procedure may alter biologicalcharacteristics of the animals which can affect their response to chemical exposure

Population crashes, the presence of ephippia in cultures, and excessive controlmortalities may still occur for a variety of reasons. The following are some of the morecommon reasons for failed cultures and tests:

1. Contaminants

(a) water

If dilution water has contacted materials other than clean glass or teflon, it may itselfbecome toxic (eg. phthalate esters in new plastic ). All glassware should bethoroughly rinsed several times with distilled water and dilution water after cleaningbut before use. Residual chlorine, nitrates, and amines are contaminants which oftenruin cultures and tests. A standing stock of aerating dilution water, left for a weekbefore use (i.e. "aged") seems to provide better water quality for the biologicalrequirements of Daphnia.

(b) Air

"Toxic fumes" from normal laboratory operations can contaminate cultures, glassware,and dilution water. Ideally, cultures should be physically separate in a "clean" room.However, covering the culture tanks during periods of air contamination (eg. opencontainers of volatile solvents and effluents) should minimize this problem. "Toxicfumes" include laboratory air supplies generated by an oil-lubricated pump. Anactivated carbon -glasswool air trap will remove contaminants from this source andgenerally ensure a cleaner air supply. Use of a diaphram air pump will avoid thisproblem.

2. Daphnia Handling

(a) If animals are exposed to air bubbles in transfer pipets or dropped from above thewater surface into test chambers, poor survival may result. Gentle transfers using apipet with a wide smooth opening are recommended.

(b) Neonate transfers between different batches of water can be very stessful.Undesired mortality can be minimized if the water batches have the sametemperature, pH,and conductivity. Avoid sudden movements of the test chamberswhich could damage neonates by throwing them against the test chamber wall.

3. Culture Health

While it is not possible to exactly specify all the nutritional, physical, and chemicalconditions which optimize culture health, the observed growth, survival, andreproduction of laboratory stocks provide a real indication of successful methods. Ahealthy culture has:

(1) no ephippia present

(2) many young and few adults

(3) individuals with a short time to first brood

(4) many eggs per brood

Isolating and observing a few individuals over a complete life cycle will promotetechnical proficiency in culturing and testing, as well as giving a measure of culturehealth.

Place one neonate (< 24 h old) in a 100 ml beaker filled with a solution of 20% algalfood stock (Appendix 1) and 80% dilution water. Maintain these individuals under thesame conditions as the main cultures. Observe the Daphnia daily for 21 days,recording time to first brood, and average number of neonates produced per brood.After each brood gently transfer the adult, using a large bore glass tube (1 cmdiameter), to a new 100 ml chamber to facilitate counting the neonates. Place theneonates in the main cultures after enumeration. Time to first brood must be less than18 days at 20°C, and the average number of neonates per brood must exceed 30 toindicate a healthy culture (Hebert, 1978).

4 Daphnia magna ACUTE LETHALITY

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