Methods of Culturing and Performing Toxicity Tests withthe Australian cladoceran Ceriodaphnia dubia

Cheryl Orr and Sharyn Foster

CSIRO Land and WaterGriffithSeptember 1997

Technical Report 20/97

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Table of Contents

Page Number

1 Introduction 1

2 Washing containers 2

3 Methods of Culturing 3

3.1 Mass Culture 3

3.2 Culture Media Preparation 3

3.2.1 DMW MQ S 3

3.2.2 Supply 4

3.3 Culture Food - Algae 4

3.3.1 Stock Solutions 4

3.3.2 Algae Media 5

3.3.3 Plates 5

3.3.4 Flasks 6

3.3.5 Concentrating Algae 6

3.4 Culture Food - YCT 9

3.5 Feeding 9

3.6 Transferring 9

3.7 Culture Trays 11

3.8 Initiating a new Culture 11

3.9 Record Keeping 13

4 Methods for Conducting Acute Tests 13

5 Methods for Conducting Chronic Tests 14

6 Reference Toxicant Tests 18

7 Acknowledgments 18

8 References 19

1

1 Introduction

This technical document details the methods used for culturing the cladoceran

Ceriodaphia dubia, as well as its use in both acute and chronic toxicity tests at CSIRO

Land and Water, Griffith. The methods used are adaptations of those of the USEPA

(1991).

The use of aquatic organisms to indicate toxicity is becoming more widespread

because these organisms are often more sensitive to toxicants in water than chemical

means of detection, and as such they give a biological perspective on possible

pollution.

Culturing of water fleas serves to provide a source of organisms for use in both acute

and chronic toxicity tests.

Ceriodaphnia dubia swim with an erratic jerking motion for a period of time and then

remain still, hanging motionless in the water. They are filter feeders feeding on

bacteria, detritus and algae. The life cycle consists of four distinct periods: egg,

juvenile, adolescent and adult. The life span is highly variable and depends largely on

temperature. Average life spans are 30 days at 25oC and 50 days at 20oC (USEPA,

1991).

Reproduction typically begins with a clutch of eggs being released into the brood

chamber. The eggs hatch in the brood chamber, and the juveniles, which are similar in

form to the adult, are released when the female moults. Moulting involves the casting

off of the exoskeleton or carapace.

The time required to produce a first brood varies from 3 to 5 days and appears to be

dependent on body size and environmental conditions.

The growth rate of the organism is greatest during its juvenile stages with the body

possible of doubling in size with each moult. Growth occurs immediately after each

moult while the new carapace is elastic.

2

Following the juvenile stages comes a single adolescent instar. It is during this instar

that the first clutch of eggs reaches full development in the ovary.

Generally instar duration increases with age, but environmental conditions are also a

factor. Four events take place in a matter of minutes at the end of each adult instar.

They are: the release of young from the brood chamber to the outside, moulting,

increase in size and the release of a new clutch of eggs into the brood chamber. The

number of young per brood is highly variable and depends on body size, food

availability and environmental conditions (USEPA, 1991).

The production of males has been reported as resulting from environmental factors

including low water temperatures, high population densities and a decrease in food

availability (USEPA, 1991). Our data, from four years of culturing C. dubia under

constant conditions, suggests that the production of males is unpredictable and can

occur in any brood number (Anderson-Carnahan et al, 1995). Males (see back cover)

can be distinguished from females (see front cover) by their slightly smaller size and

lack of a brood chamber. Females grow to a length of approximately 1 mm

(Anderson-Carnahan et al, 1995), with males achieving a length of approximately 0.8

mm.

The original supply of organisms were provided by the Centre for Ecotoxicolgy

(CET), Gore Hill, Sydney. This is an Australian cladoceran species and was collected

from the Gibbon Pond at the Taronga Park Zoo, Sydney.

2 Washing Containers

The following protocol should be followed for washing all glass and plasticware used

for culturing and conducting acute and chronic tests with Ceriodaphnia dubia. Plastic

cups used for housing test organisms are not washed, but used new and then

discarded.

3

Washing procedure:

a) Rinse in tap water

b) Soak in detergent (scrub if needed) for at least a couple of hours, or overnight

c) Soak in acid (10% HCl) for at least 1 hour, or overnight

d) Rinse in Distilled water 3 times

e) Rinse in Methanol if for pesticides

f) Final rinse in MilliQ water.

3 Methods of Culturing

3.1 Mass Culture

Mass cultures are used as a “backup” reservoir of organisms which are used to provide

organisms to initiate cultures. Mass cultures are maintained in 1L beakers with the

water to be used for culturing (see section 3.2 below) and a source of food (see

sections 3.3 and 3.4 below). Culture water and food are replaced on a weekly basis

providing an environment for large numbers of organisms to be produced unchecked.

3.2 Culture Media

A volume of 15 mL of culture media is used for individual cultures. This volume is

placed in a 60 mL plastic “lily” cup (Lily, N.Z). Each batch of water is analysed for

pH, conductivity, Ca2+ and Mg 2+.

3.2.1 DMW MQ S Water

The culture media or water used for culturing is prepared using a dilution of Perrier

(Vergeze, France) mineral water and MilliQ water. This water source is used for its

consistent quality over time and ease of preparation. To replicate the hardness of local

waters, a soft water is used which requires the culture media to be a 10% dilution of

Perrier. A volume of 20L is prepared in a plastic carboy by combining 2L of Perrier

with 18L of MilliQ water. This water is aerated overnight prior to its use. This water

is referred to as DMW MQ S (Diluted Mineral Water MilliQ Soft).

4

3.2.2 Supply Water

As an alternative to DMW MQ S, we have access to local irrigation water which we

collect from the main supply canal. This water is collected by lowering a stainless steel

bucket attached to a rope into the overspill section of the canal. The water is filtered

through a 125 µm mesh filter to exclude food and predators, into a 20L plastic carboy.

We have had no success in culturing C. dubia in either local tap water or rain water.

The presence of contaminants and a lack of nutrients are probably the reason for this.

3.3 Culture Food - Algae

We use two species of green algae, Ankistrodesmus sp. and Raphidocellis subcapitata

(formerly Selenastrum sp), as culture food, as these species are both easy to grow, and

their cell size is suitable for ingestion by C. dubia . Whilst other species may be

suitable for this purpose, these green algae appear to provide sufficient nourishment, in

conjunction with YCT, for our test organism. Both species are cultured separately due

to their differing growth rates and were supplied by CET.

3.3.1 Stock Solutions

The following solutions are prepared in order to culture these algal species:

Solution A

6.08 g MgCl.6H2O

2.20 g CaCl2.2 H2O

12.75 g NaNO3

Combine these compounds and dilute to 500 mL with MilliQ water.

Solution B

7.35 g MgSO4.7 H2O

dilute to 500 mL with MilliQ water.

Solution C

0.552 g K2HPO4

dilute to 500 mL with MilliQ water.

5

Solution D

7.5 g NaHCO3

dilute to 500 mL with MilliQ water.

Solution E

First prepare solutions ε1-ε5 “as follows”

ε1 164 mg ZnCl diluted to 100 mL with MilliQ water

ε2 71.4 mg CoCl2.6H20 diluted to 100 mL with MilliQ water.

ε3 36.6 mg Na2MoO4.2H2O diluted to 1L with MilliQ water.

ε4a 60 mg CuCl2.2H2O diluted to 1L with MilliQ water.

ε4b 1 mL of ε4a is diluted to 10 mL with MilliQ water.

ε5 119.6 mg Na2SeO4 is diluted to 100 mL with MilliQ water.

1 mL of ε1, ε2, ε3, ε4b and ε5 to

92.8 mg H2BO3

208 mg MnCl4.H20

79.8 mg FeCl6.H2O

150 mg Na2EDTA

this is diluted to 500 mL with MilliQ water.

3.3.2. Algal Media

The algal culture solution is prepared by adding 1 mL of each of the stock solutions A,

B, C, D and E to an erlenmeyer flask and diluting to 1L with MilliQ water. A ‘bung’

of cotton wool is placed in the opening of the flask which is then autoclaved (Labec,

Australia) to achieve sterilisation.

All steps involving the transfer of algae are performed in a sterile cabinet. The cabinet

(Biological Safety Cabinet Class II) is irradiated with UV light for at least 20 minutes

prior to cabinet use, and the cabinet is wiped with 70% ethanol before and after use.

3.3.3. Plates

Algae are cultured on agar plates to provide cells for addition to liquid media. Media

plates are prepared from a 3% solution of agar diluted in algal culture media. This

solution is autoclaved (Bench top Jaymac autoclave) for 20 minutes and then cooled to

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about 60-80oC. It is poured, in a sterile cabinet, into plates and allowed to solidify

with the lid only partially covering the plate. Upon cooling parafilm is placed around

the plate to seal the base and lid together. Once prepared, plates are stored in a

refrigerator until required.

New culture plates are prepared from existing ones containing adequate cell growth.

Inoculation involves flaming a loop to ensure it is sterile, and upon cooling scraping

cells from an existing plate and redispersing them on a fresh culture plate. Inoculated

plates are resealed with parafilm and placed in a controlled environment at 25oC and a

light intensity of 85 µmol m-2 s-1.

3.3.4. Flasks

Cultures for use in cladoceran feeding are prepared in liquid culture media (section

3.3.2). A scrape from a plate culture is added to this liquid which is placed in an

environmental chamber under the same conditions as previously described. This flask

is mixed daily. After 10-14 days the cultures should be ready to centrifuge (section

3.3.5).

3.3.5 Concentrating Algae

50 mL centrifuge tubes are used to spin the algae with the aim of concentrating the

cells to 3 x 107 cells mL-1 (USEPA, 1991). Both species are spun at 4,600 revolutions

per minute for 10 minutes using a Heraeus Sepatech megafuge 1.0 (swing out rotor

3360, RCF3673g). The supernatant is decanted and stored, and the procedure is

repeated until all has been spun. The concentrated algal cells are thoroughly mixed in

a small amount of supernatant and pooled in a 50 mL measuring cylinder. The volume

is recorded (for example 47 mL) and then a small magnetic mixer is added to stir the

solution. 20 µL of algae is then diluted to 1 mL in a volumetric flask. After mixing

this diluted solution is dispensed under the cover slip of a haemocytometer (see Figure

1) using a pasteur pipette. Individual algal cells are counted until a value of around

100 is achieved. The number of small squares required to achieve this value will vary

depending upon initial inoculation size and culture performance. The number of

squares is recorded. This is repeated until 6 counts have been made, with three on

7

each side of the haemocytometer. The mean and standard deviation are calculated.

For example: 6 x 5 squares are counted to achieve the following values of cells 120,

123, 125, 121, 118, 119. The mean value is 121 and the standard deviation is 2.4.

The value determined for the standard deviation should be no greater then 10 so extra

squares may need to be counted if a higher value is obtained.

From this mean value the following calculation is used to determine the number of cells

present per mL:

121(cell number) x 16(total no. of small haemocytometer squares used for calibration)/5(no. of small squares counted) x 3.968*

(haemocytometer factor) x 50(dilution) x 1000(total cell volume)

= 7.7 x 107 cells/mL

* - The ratio of volume counted to the volume in which the units are expressed

(in this case, 1 mL).

From this value you need to determine what volume you need to dilute the algal

concentrate to, to get a value of 3 x 107 cell/mL. This is found by dividing the known

cells/mL by 3 x 107 and multiplying this by the volume of concentrate.

eg. 7.7 x 107 cells/mL x 47mL

_____________

3 x 107 cells/mL

= 120 mL

This volume is made up by the addition of some of the stored supernatant.

Equal volumes of each concentrated algal species are combined and this solution is

transferred to a labelled glass bottle and stored in the refrigerator until required. We

have found that algal cultures can be stored under refrigeration for up to 1 month.

8

Figure 1. Diagram of a haemocytometer (Improved Neubauer - Weber

England). The area pictured appears on both sides.

9

3.4 Culture Food - YCT

YCT is the name given to a bacterial food which combines Yeast, Cerophyll (a U.S.

brand name of alfalfa) and digested Trout pellets. This substance is prepared as

follows:

Five grams of trout pellets and 1L of Milli Q water are combined in a 1L measuring

cylinder. This mixture is continuously stirred and aerated from the bottom of the

container for 7 days. The digest is made up to 1L and placed in a refrigerator for 1

hour to settle prior to mixing with the other solutions.

On day 6 of the digestion, 5 g of Cerophyll (dried powdered cereal leaves) is added to

1 L of MilliQ water and mixed using a magnetic stirrer overnight. This solution is

settled for 1 hour prior to mixing with the other solutions.

On day 7, 5 g of dry yeast is added to 1 L of MilliQ water and mixed immediately on a

magnetic stirrer plate. Solutions are then mixed in a ratio of 1:1:1 after filtration

through a fine Nitex 110 mesh. This mixture is placed into small containers and frozen

until required.

Trout pellets were provided by NSW Fisheries at Narrandera. Yeast (Fleischmann’s)

and dried powdered cereal leaves (Cerophyll substitute) were purchased from a local

health food shop.

3.5 Feeding

Cultures are fed daily to maintain the organisms in optimum condition. Individual

cultures are fed at a rate of 100 µL YCT and 100 µL algae mix per 15 mL of culture

media. Food should be at room temperature and thoroughly mixed prior to dispensing

using a multi pipetter (eppendorf).

3.6 Transferring

Individual cladocerans are transferred to fresh culture media every second day (daysdesignated “T” on Figure 2). Transfers and feeding should be done at approximatelythe same time each day (eg. 9 am m 2 h). This is achieved using a shortened glass

11

pasteur pipette approximately 4mm bore. Organisms are slowly drawn into the pipette

with a small volume of media. When releasing the organism, the tip of the pipette

remains under the media surface. Liquid can be poured from vessel to vessel

containing the organism without fear of damage.

3.7 Culture Trays

Cultures are housed in styrofoam trays with chip board backing. The chip board gives

the tray stability, and the styrofoam provides a cheap, light weight, insulated material

that is easy to work with. Holes are drilled in each tray to house 30 lily cups. Figure 3

presents an example of such trays. Due to their use in combination in chronic tests

(see section 4) the trays have two different numbering systems (Figure 3).

3.8 Initiating a new culture

New cultures are initiated from 3rd or subsequent brood organisms that are < 24 h old

in an existing culture. The performance of the adult providing the young should be

examined when choosing organisms.

As a guide, good brood sizes are:

1st 3-6 young

2nd 6-10 young

3rd > 8 young

Young from the same parent are set up in a culture board vertical row (Figure 1)

requiring 5 young from each parent. This requires the young from 6 adults to be used

to fill a culture. The source of young used in each new culture is recorded. Cultures

are covered with a sheet of glass to prevent dust entering and to reduce evaporation.

Glass sheets are wiped with methanol and dried prior to use. They are placed in an

environmental chamber (Labec, Australia) with the following settings:

Temperature 25oC

Light intensity 8-12 µMol m-2 s-1

Photoperiod 16:8 L:D

The page number of the culture book is used to identify each culture and is recorded

on the glass cover that has been placed on each tray.

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Figure 3. Example of the trays used to house Ceriodaphnia dubia

cultures and tests. Trays are made from styrofoam with a backing of

chip board.

10

9

8

7

6

30

29

28

27

26

20

19

18

17

16

60

59

58

57

56

50

49

48

47

46

40

39

38

37

36

TOP

5

4

3

2

1

25

24

23

22

21

15

14

13

12

11

55

54

53

52

51

45

44

43

42

41

35

34

33

32

31

BOTTOM

3.9 Record Keeping

Information describing the heritage of each culture is accurately recorded in the

culture book. Figure 2 is an example of the information recorded for each culture. On

a daily basis the culture is checked for survival of the adult organisms and any birth of

young. The importance of good record keeping cannot be over emphasised.

This information is recorded using the following symbols (see Figure 2 as an

example):

adult is alive

adult is alive with alive young

adult is alive with dead young

adult is dead

adult is dead and brood is alive

adult is dead and brood is dead

male

female

eggs

Any other relevant observations should also be recorded.

4 Methods for Conducting Acute Tests

Acute tests are performed over a 24 or 48 h period. These tests provide EC50

information on toxicants. Usually 5 concentrations of toxicant and a control source is

used in these tests. 5 replicates of each concentration are used and these cups are

randomly distributed in a tray using a template. The template lists the numbers 1

through 6 randomly along each of 5 rows. Test concentrations are then allocated a

number, and distributed accordingly. Test volume is 15 mL and organisms are not fed

during an EC50 test. For a single test, 150 < 24h old neonates of a 3rd or subsequent

brood are required. These neonates are pooled together from cultures. 5 neonates are

placed in each cup making sure that the pipette is rinsed between each addition to

13

14

avoid possible carry over of toxicant. A glass cover is placed on the top and the tray is

placed in an environmental chamber at the same conditions as previously described

(section 3.8). Figure 4 presents an example of the information recorded at the

beginning of each test. The number of organisms alive and dead at both 24 and 48

hours is also recorded. These figures are tallied to express the number of deaths per

25 organisms. This information is entered into an “EC50” computer program which

determines the EC50 value as well as the upper and lower confidence intervals of this

estimate. This software uses the trimmed Spearman-Karber method for the

determination of the EC50, and was provided to us by the USEPA.

5 Methods for Conducting Chronic Tests

Chronic tests are performed over a 7 to 8 day period to determine possible sublethal

toxic effects to organisms over part of their lifespan. Chronic tests require 60 < 24h

old neonates with 6 neonates provided from each of 10 adults. 5 test concentrations

and a control are used with 10 replicates of each concentration which are randomly

arranged in 2 trays using a template. 15 mL of solution is used in each 60 mL cup and

the organisms are fed 100 µL YCT and 100 µL of algal mix daily. At the same time

each day, organisms are transferred to fresh solutions. The young from each adult fill

all the cups in a horizontal row. This is known as “blocking by parent”, and ensures

that the young from the same adult are exposed to each treatment. Trays are covered

with a glass sheet and placed in an environmental chamber. Figure 5 presents the table

used to record the performance of the test, with the same symbols being used to

describe what is observed. Figure 6 presents the form which is filled out to provide the

other information required about the test. The test continues until 60% of surviving

control organisms have produced at least three broods of young. To be a valid test, at

completion time <20% of control organisms will have died. At test completion the

survival and number of young produced is summarised as presented in Figure 7. This

information is entered into the computer program “TOXSTAT” to determine by

ANOVA (Analysis of Variance) at which concentration no effect was observed

(NOEL, No Observed Effect Level) and the lowest concentration at which an effect

was observed (LOEL, Lowest Observed Effect Level). The TOXSTAT package was

provided by the USEPA.

16

Figure 5. An example of a chronic test data sheet detailing the performance of eachindividual in the test.

10 20 30 40 50 601 a a a a a a

2 a a a a a a

3 a a a a a a

4 a a5 a5 a4 a4 a

5 a4 a9 a2 a10 a9 a66 a a a a a a97 a4 a12 a10 a14 a12 a

8 a a15 a13 a15 a16 a119 19 29 39 49 59

1 a a a a a a

2 a a a a a a

3 a a a a a a

4 a4 a4 a4 a3 a2 a45 a9 a7 a8 a7 a6 a116 a a9 a a a a

7 a15 a a10 a11 a7 a138 a a12 a14 a16 a6 a13

8 18 28 38 48 581 a a a a a a

2 a a a a a a

3 a a a a a a

4 a3 a4 a5 a4 a3 a

5 a8 a10 a9 a7 a8 a56 a a a a a a67 a12 a11 a14 a10 a11 a18 a13 a12 a16 a13 a14 a10

7 17 27 37 47 571 a a a a a a

2 a a a a a a

3 a a a a2 a4 a

4 a4 a3 a9 a a a

5 a9 a7 a13 a10 a10 a36 a a12 a a14 a13 a77 a11 a a13 a a a

8 a13 a13 a a14 a12 a86 16 26 36 46 56

1 a a a a a a

2 a a a a a a

3 a a a a a a

4 a3 a4 a4 a a2 a45 a9 a8 a9 a a9 a86 a a a a a a97 a10 a9 a15 a4 a11 a

8 a7 a16 a15 a5 a11 x5 15 25 35 45 55

1 a a a x a a

2 a a a a a

3 a a a a a

4 a4 a4 a4 a a45 a8 a8 a9 x6 a56 a a a a67 a10 a13 a14 a

8 a10 a12 a16 a

4 14 24 34 44 541 a a a a a a

2 a a a a a a

3 a a a a a a

4 a4 a a4 a4 a4 a45 a7 a6 a8 a8 a9 a86 a a a a a a

7 a14 a8 a13 a10 a7 a78 a13 a9 a11 a12 a8 a7

3 13 23 33 43 531 a a a a x a

2 a a a a a

3 a a a a a

4 a a4 a4 a4 a55 a4 a8 a9 a7 a96 a11 a a a a

7 a a11 a14 a11 a148 a9 a14 a9 a11 a15

2 12 22 32 42 521 a a a a a a

2 a a a a a a

3 a a a a a a

4 a4 a4 a4 a a4 a

5 a a6 a6 a a9 a

6 a9 a a a a a67 a9 a13 a7 a7 a12 a98 a a x10 a8 a10 a

1 11 21 31 41 511 a a a a a a

2 a a a a a a

3 a a a a a a44 a3 a4 a3 a5 a1 a45 a a a a9 x4 a106 a9 a9 a6 a a137 a7 a9 a9 a12 a

8 a a a a12 a7

18

Figure 7. An example of a summary of results from a Chronic test. Thenumber of young produced by each test organism during the test isrecorded as well as the number of test organisms alive at the end of thetest.

Conc(mg/L)

1 2 3 4 5 6 7 8 9 10 No.alive/10

0.0 24 25 30 18 20 25 26 31 19 22 101.25 26 18 26 14 18 23 27 29 33 25 102.5 14 22 26 25 19 20 32 15 23 32 105 12 15 14 8 5 16 12 13 11 15 810 2 6 2 0 1 1 5 2 0 4 520 2 0 1 3 0 0 4 0 0 0 0

6 Reference Toxicant Tests

On a monthly basis an acute and a chronic test should be performed with a reference

toxicant to determine whether the sensitivity of the organism is changing. These tests

are performed using the chemical sodium dodecyl sulphate (SDS). Acute and chronic

tests are set up as described above and a running tally of results is maintained known as

a control chart. Methods for constructing a control chart are described in Anderson-

Carnahan, (1994). The concentrations of SDS we use in these tests is the same each

month and is as follows: 0.0, 1.25, 2.5, 5.0, 10.0, and 20.0 mg/L.

7 Acknowledgements

This work was initiated by visiting US Scientist Linda Anderson-Carnahan from the

USEPA. Her contribution is gratefully acknowledged, as is the technical assistance of

Praba Pathmananthan, Denise DePaoli, Sue Korth, Rhonda Smith, Gillian Napier and

Geoff McCorkelle. The original supply of Ceriodaphnia dubia from the Centre for

Environmental Toxicology, Sydney is gratefully acknowledged. Help with the

preparation of this document and ongoing support from Kath Bowmer, Wolfgang

Korth and Martin Thomas is greatly appreciated. The assistance of Jane Roberts and

19

Peter Fairweather with proof reading is also acknowledged. For providing us with

photographs of C. dubia, we acknowledge Russ Shiel (MDFRC) and Gillian Napier.

8 References

Anderson Carnahan, L. (1994) Development of Methods for Culturing and

Conducting Aquatic Toxicity Tests with Australian Cladoceran Moina

australiensis. CSIRO Division of Water Resources Seeking Solutions Water

Resources Series No.13.

Anderson-Carnahan, L., Foster, S., Thomas, M., Korth, W., and Bowmer, K.H.

(1995) Selection of a suitable cladoceran species for toxicity testing in turbid

water. Australian Journal of Ecology. 20, 28-33.

USEPA. 1991. Methods for Measuring the Acute Toxicity of Effluents and Receiving

Waters to Freshwater and Marine Organisms (forth edition). C.I. Weber, ed.

Environmental Monitoring Systems Laboratory, U.S. Environmental

Protection Agency, Cincinnati, Ohio. EPA / 600 / 4-90 / 027.

COVER

Front: A female Ceriodaphnia dubia. Actual size, approximately 1 mm.Photograph by Russ Shiel.

Back: A male Ceriodaphnia dubia. Actual size, approximately 0.8 mm.Photograph by Gillian Napier.