Methods of culturing and performing toxicity

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					Methods of Culturing and Performing Toxicity Tests with
the Australian cladoceran Ceriodaphnia dubia

Cheryl Orr and Sharyn Foster

CSIRO Land and Water
September 1997

Technical Report 20/97
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 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

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

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,

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.

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

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

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).

               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.

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

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 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

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.

Figure 1. Diagram of a haemocytometer (Improved Neubauer - Weber
England). The area pictured appears on both sides.

                  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
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 (days
designated “T” on Figure 2). Transfers and feeding should be done at approximately
the same time each day (eg. 9 am m 2 h). This is achieved using a shortened glass

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.

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     20    30        40   50   60

                   9     19    29        39   49   59

                   8     18    28        38   48   58

                   7     17    27        37   47   57

                   6     16    26        36   46   56

                   5     15    25        35   45   55

                   4     14    24        34   44   54

                   3     13    23        33   43   53

                   2     12    22        32   42   52

                   1     11    21        31   41   51

        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

                       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

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 3 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

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.

Figure 5. An example of a chronic test data sheet detailing the performance of each
individual in the test.
               10           20          30           40           50           60
      1        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           a6
      6        a            a           a            a            a            a9
      7        a4           a12         a10          a14          a12          a
      8        a            a15         a13          a15          a16          a11
                9           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           a4
      5        a9           a7          a8           a7           a6           a11
      6        a            a9          a            a            a            a
      7        a15          a           a10          a11          a7           a13
      8        a            a12         a14          a16          a6           a13
                8           18          28           38           48           58
      1        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           a5
      6        a            a           a            a            a            a6
      7        a12          a11         a14          a10          a11          a1
      8        a13          a12         a16          a13          a14          a10
                7           17          27           37           47           57
      1        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          a3
      6        a            a12         a            a14          a13          a7
      7        a11          a           a13          a            a            a
      8        a13          a13         a            a14          a12          a8
                6           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           a4
      5        a9           a8          a9           a            a9           a8
      6        a            a           a            a            a            a9
      7        a10          a9          a15          a4           a11          a
      8        a7           a16         a15          a5           a11           x
                5           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            a4
      5        a8           a8          a9                        x6           a5
      6        a            a           a                                      a6
      7        a10          a13         a14                                    a
      8        a10          a12         a16                                    a
                4           14          24           34           44           54
      1        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           a4
      5        a7           a6          a8           a8           a9           a8
      6        a            a           a            a            a            a
      7        a14          a8          a13          a10          a7           a7
      8        a13          a9          a11          a12          a8           a7
                3           13          23           33           43           53
      1        a            a           a            a             x           a
      2        a            a           a            a                         a
      3        a            a           a            a                         a
      4        a            a4          a4           a4                        a5
      5        a4           a8          a9           a7                        a9
      6        a11          a           a            a                         a
      7        a            a11         a14          a11                       a14
      8        a9           a14         a9           a11                       a15
                2           12          22           32           42           52
      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           a            a4           a
      5        a            a6          a6           a            a9           a
      6        a9           a           a            a            a            a6
      7        a9           a13         a7           a7           a12          a9
      8        a            a           x10          a8           a10          a
                1           11          21           31           41           51
      1        a            a           a            a            a            a
      2        a            a           a            a            a            a
      3        a            a           a            a            a            a4
      4        a3           a4          a3           a5           a1           a4
      5        a            a           a            a9           x4           a10
      6        a9           a9          a6           a                         a13
      7        a7           a9          a9           a12                       a
      8        a            a           a            a12                       a7

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

Conc      1      2      3       4      5         6    7      8       9     10   No.
(mg/L)                                                                          alive/1
0.0       24     25     30      18     20        25   26     31      19    22   10
1.25      26     18     26      14     18        23   27     29      33    25   10
2.5       14     22     26      25     19        20   32     15      23    32   10
5         12     15     14      8      5         16   12     13      11    15   8
10        2      6      2       0      1         1    5      2       0     4    5
20        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

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.


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.

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