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NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
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INTRODUCTION
The Mount-Norberg three brood toxicity test for Ceriodaphnia is
convenient for testing complex mixed effluents on site. At 25°C, the
production of three broods is typically completed in seven days [1],
In involving ecosystem characteristics into toxicity testing, it is
desirable to simulate temperatures and levels of trophy (food availability)
characteristic of the systems receiving the test effluents. Thus we have
observed and selected four characteristics of Ceriodaphnia tests which
deserve further attention:
a. what is the necessary duration, or the minimum number of broods
produced, to obtain significant results when using warm versus
cold control waters?
b. what temperature extremes are useful in such modified, ecosystem
simulating tests?
c. how can information on the synchronous release of Ceriodaphnia
neonates be used to simplify such tests, in the hope of
increasing the information obtained at little additional cost.
d. should the chronic endpoint be determined using the number of
young/female or the intrinsic growth rate (r)?
This paper presents answers to these questions.
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EXPERIMENTAL PROCEDURE
Ceriodaphnia were mass cultured as reported elsewhere [1], The
natural algae and bacteria in control waters from Lakes Erie and Superior
were supplemented with a water suspension of activated yeast; 0.05 ml of
yeast suspension was added with fresh control water every day. Individual
animals were cultured in 15 ml pyrex beakers containing 10 ml of control or
test solution; test cultures were started with neonates of £. affinis/dubia
less than 24 hr. of age. The species of cladoceran employed was identified
by Dorothy 3erner of Temple Univ.
Test animals were maintained in cabinets (Percival 1-60) with
temperature control of - 0.5 °C under a maximum of 7000 lux (700 f.c.) of
1ight.
Life tables were calculated using the methods of Birch [2].
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EXPERIMENTAL RESULTS
Duration of Observation
In the relatively food-rich waters of Lake Erie, three broods were
produced in control water in 28 days at 18°C (Fig. 1). The same number of
broods were produced in experimental containers to which was added 10, 25
and 50% by volume complex mixed effluent from the Raisin River; the
characteristics of that effluent are not necessarily pertinent to this
discussion. What is vital to see is that differences in the controls and
experimental treatments were not significant until 28 days, with the
production of three broods. Time and treatment effects were investigated
with a 2-way analysis of variance (A N 0 V A) with replication; the
significance of the F value is shown in Table 1.
In the relatively food-poor waters of Lake Superior, three broods were
produced in control water in 7 days at 25°C. Again time and treatment
effects were investigated with a 2-way ANOVA and shown in Table 1. Two
broods produced in 6 days in controls were significantly different in
numbers young/female from same measure in experimental treatments involving
effluent water from a refinery.
Clearly the concept of a three brood test is valid under different
conditions of food and temperature. The time for production of three
broods is increased greatly at lowered temperatures, as is the total
production of offspring; as will be seen below. Reproductive measures were
made over the normal lifetime of the test animals, so that a true measure
of net reproductive rate was available from our life tables.
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Useful Temperature Extremes
Ceriodaphnia is extremely ubiquitous, occuring in a wide variety of
environments. For those who plan to use it to examine the impact of
complex effluents upon natural aquatic systems observation of the net
reproductive rate R0 at ambient temperature may be more indicative of toxic
impact than observation of R0 at 25°C. That is, during decreased ecosystem
metabolism at low temperatures, complex effluents may exhibit, reduced
toxicity.
By raising animals at temperatures lower than the suggested 25°C [1],
we have determined that a modified Mount-Norberg toxicity test may be run
at temperatures as low as 12°C (Table 2). At 18°C we found 113 young/
^/lifetime of 77 days. At 12°C we found only 13 young/2 /I ifetime of 24
days, and at 6°C only 2 young/?./I ifetime of 24 days. Cultures at 18°C
were maintained at photoperlods of 16 L/80, whereas animals grown at 12°C
and 6°C were kept in continuous light (24L) to stimulate ovulation.
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Synchronous Release of Neonates
Test animals grown at constant temperature (18°C) and started with
neonates of similar age {< 24 hours) exhibited synchrony in the release of
neonates at .approximately 7 day intervals (Figure 1). Young were released
synchronously regardless of toxicity level {% complex effluent added to
control water). Thus toxicity greatly effects the numbers of young
produced per female per time, but not the development time and subsequent
release of those neonates (Table 3).
Because of such reproductive synchrony, it should be possible to
utilize toxicity tests of very long duration to maximize information on
effluents with low concentrations of a slow acting contaminants. While
neonates from individual females kept in individual containers might be
counted every ? days (at 18°C), it would still be necessary to feed at
least every two days. However, feeding is not as time consuming as
counting offspring. Likewise, the renewal of contaminated effluent in
cultures would have to be rescheduled. Certainly counting neonates at
seven day intervals would provide valuable information on the size of
broods later than the third brood, and at little additional cost.
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Use of Intrinsic Growth Rates (r)
The measures of natality and survivorship employed in toxicity testing
are currently under serious debate, C. Stefan of EPA-Duluth asked us to
consider whether the instantaneous growth rate (jr) constitutes a better
measure of toxic inhibition of natality than the net reproductive rate
(R0). Estimates of r_ from truncated life tables are plotted (Figure 2) for
the same data set shown in Figure 1. While lxmx is cyclic, as young were
produced synchronously, we observe that jr increased for the first 15 days.
Thereafter the slope of £ was constant. In normal 1ife-table calculations,
jr is determined at the termination of the lifespan of the adult population.
In using truncated life tables we are prone to produce artificially low
estimates of r_ if the asymptote has not been reached. Yet because of the
constant slope of r_ it is possible to estimate _r at the normal termination
of the lifetable from a value determined about day 11-15, a shorter time
than the 28 days required in using natality (mx). If valid at ed in future
studies, this method might be used to abbreviate the necessary period of
observation necessary for toxicity testing.
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CONCLUSION
The three brood seven day toxicity test using Ceriodaphnia grown at
25°C became a twenty-eight day test at 18°C. Varying temperature will
become an important part of this test as it is applied to complex
ecosystems. Generally Ceriodaphnia can be grown at temperatures of 12 to
25°C; too few neonates were produced at 6°C for useful test results. The
synchronous release of neonates by Ceriodaphnia females may be used in
determining when to sample offspring and reduce the cost of testing complex
effluents. The intrinsic growth rate of Ceriodaphnia may be used early in
a test to predict the growth rate at the termination of a normal adult
femal e's 1 ifespan.
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FIGURES
Figure 1. Variation in the product of survivorship (lx) and natality (mx)
for Ceriodaphnia raised over 40 days in control and experimental (10% and
50% effluent) cultures.
Figure 2. Variation in the intrinsic growth rate (r) of Ceriodaphnia
calculated from truncated life tables from day 1 through 40 for control and
experimental cultures.
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ACKNOWLEDGMENTS
Work on this project in the laboratory of O.C. McNaught was supported
by a cooperative grant (810775) from the US-EFA, Environmental Research
Laboratory at Duluth supervised by M. Gessner. Larry Jacobson kindly
performed the statistics.
REFERENCES
1. Mount, 0.1. and T.J. Norberg. A seven-day life-cycle Cladoceran
toxicity test. M.S.
2. Birch, L.C. • 1948. The intrinsic rate of natural increase of an
insect population. J. Anim. Ecol. 17:15-26.
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TABLE 1. Time of observation needed for significant treatment effects.
Environments
f ' ¦¦ " " "" " 1 '¦ — ' * "" ' 1 "
Lake Erie (October 1983) Lake Superior (September 1982)
Observation
Period of
Test (days)
7
14
21
28
4
5
6
7
Time
NS
NS
NS
p<.10
NS
NS
p<.005
p<.G05
Treatment
leve.
NS
NS
NS
p<.10
NS
NS
p<.005
p<.005
Interaction
NS
NS
NS
NS
NS
NS
p<.005
p<.005
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TABLE 2. Decreasing net reproductive rate (R0) with temperature (°C), (for
Ceriodaphnia grown in L. Erie water supplemented with yeast).
Temperature RQ (Photoperiod)
18°C 113 young/? 16L/8D
12 13 24L/0D
6 2 24L/00
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TABLE 3. Decrease in net reproductive rate (RQ) of Ceriodaphni a with
increasing amounts of complex effluent from Raisin River, at
18°C. (October 1983).
Effluent
from Site# Control 10% effluent 25% effluent 50% effluent
8 54 32 8 15 ind ind"1
10 54 22 46 49
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0
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8
7
6
5
4
3
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10% EFFLUENT
50% EFFLUENT
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TIME (days)
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