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Stantec Consulting Ltd.
R.R.2, Nicholas Beaver Road, Guelph
ONTARIO N1H 6H9, Canada

Golder Associates Ltd.
2390 Argentia Road, Mississauga
Ontario L5N 5Z7, Canada

1. Objective and scope of the test method


The primary objective of the 48-hour toxicity test using Daphnia sp. is to evaluate
the acute toxicity of effluents, chemicals, and elutriates on freshwater crustaceans
(in particular, cladocerans, which are common zooplanktonic organisms in
freshwater ecosystems). This test has two predominant uses: (a) regulatory
compliance testing and monitoring of industrial effluents (e.g., pulp and paper and
metal mining sectors); and, (b) one of several toxicity tests used in regulatory test
batteries for ecological risk assessment of commercial chemicals (e.g., OECD
Chemicals Programme, New Substances Notification Regulation (NSNR), Canada,
U.S. EPA Pesticide Registration, United States). Moreover, due to its widespread
use as a regulatory compliance test for aquatic toxicity to invertebrates in many
countries (e.g., Canada, U.S.A., European Union countries), it is also commonly
used as a representative pelagic invertebrate test organism in freshwater toxicity test
batteries supporting environmental monitoring, assessment and ecological risk
assessments where fresh surface waters may be influenced by a variety of organic
and/or inorganic contaminants.
The types of samples tested with this method include: industrial wastewaters,
receiving waters, produced waters, and (pure, both organic and inorganic)
commercial chemicals. The test design, however, is also appropriate for the testing

C. Blaise and J.-F. Férard (eds.), Small-scale Freshwater Toxicity Investigations, Vol. 1, 337-393.
© 2005 Springer. Printed in the Netherlands.

of soil elutriates, where this application is appropriate (e.g., Environment Canada,

1990a). The test is conducted in small beakers or standard test tubes, and often
incorporates replication (e.g., 5 organisms in each of 4 replicates, 10 organisms in
each of 2 replicates, or 3 replicates with a minimum of 10 organisms) and basic
water quality monitoring (i.e., temperature, dissolved oxygen, pH, hardness, and
conductivity). The measurement endpoints generally evaluated are the 48-hour
LC50 (for survival), and the 48-hour EC50 (for immobility). Test organisms are
generally assessed for the two assessment endpoints after each 24-hour interval.
The main attractive features of this test are its simplicity, short exposure
duration, sensitivity, ease with which cultures are maintained, and level of
standardization (these are discussed in more detail below). As a result, it is a
relatively small-scale and highly cost-effective test. When species such as Daphnia
magna are used, another attractive feature is the size of test organisms. Their
relatively large body size, as compared to other cladocerans (such as Ceriodaphnia
dubia), make them highly visible to the naked eye and therefore easier to monitor.


The primary objective of the 21-day toxicity test using Daphnia sp. is to evaluate
the reproductive (multi-generational) toxicity of commercial chemicals on
freshwater crustaceans. This test, in contrast to the acute test described above, is
predominantly used as part of regulatory test batteries for the ecological risk
assessment of commercial chemicals (e.g., OECD Chemicals Programme, Toxic
Substances Control Act, United States, Pest Management Regulatory Agency -
PMRA, Canada). However, the test design has also been used in some innovative
applications relating to effluents and receiving waters (e.g., Moran et al., 1994; see
Section 10.2), as well as long-term assessment of discharge wastewater monitoring
(BEAK, 1985).
The samples tested using this method mainly include commercial chemicals
(both organic and inorganic). The test is conducted in small beakers, plastic tubes
with a mesh bottom, and incorporates replication (i.e., 1 organism per replicate with
a minimum of 10 replicates in a static-renewal test design; or 2 replicates of 10
organisms, or 4 replicates of 5 organisms, each for the flow-through test design) and
water quality monitoring (i.e., temperature, pH, hardness, and conductivity). There
are several biological endpoints that can potentially be used. These are: survival
(21-day LC50), immobility (21-day EC50), young production/fecundity (21-day
ICx/IC50), growth rate (21-day ICx/IC50), biomass (21-day ICx/IC50), and
indicators of a stressed population (e.g., production of males, occurrence of
ephippia; time to first brood).
In addition to those characteristics listed above for the 48-hour acute test design
(Section 1.1), the main attractive feature of the chronic test is the relatively large
number of generations produced by these organisms in a relatively short period
(e.g., 5 batches or 3 generations of young in 3 weeks).