Acute Toxicity Tests Using Rotifers.
111. Effects of Temperature, Strain, and
Exposure Time on the Sensitivity of
Brachion us plica tilis
TERRY W. SNELL, BRIAN D. MOFFAT
Division o f Science,
University of Tampa,
Tampa, Florida 33606, USA
COLIN JANSSEN and GUIDO PERSOONE
Laboratory for Biological Research in Aquatic Pollution
State University of Ghent,
J . Plateaustraat 22,
B -9000Ghent, Belgium
ABSTRACT
As part of the development of a standardized acute toxicity test, the effect of cyst age,
strain, temperature, and exposure time on the toxicity of 21 chemicals to the estuarine
rotifer Bruchionus plicutilis was investigated. Toxicity was chemical specific, with LCsos
ranging from 0.061 mg . L-' for mecury to 598 mg . L-' for 2,4-dichlorophenoxyacetic
acid. Intralaboratory coefficients of variation averaged 11%, a t least three times lower
than for other aquatic invertebrate acute tests. The age of rotifer cysts stored up to 27
months had no effect on the sensitivity of test animals, but significant differences in
sensitivity were detected among rotifer strains. Test temperatures of 25,30, and 35°C
generally yielded lower LC,,s than at 20°C. LCsosdecreased by 80-90% for cadmium and
pentachlorophenol when toxicant exposure time was increased from 24 to 72 h. Life table
analysis of rotifer survival in the controls revealed that 72 h is the longest acute test
possible without feeding. A comparison of the sensitivity of the rotifer test to that of
sea urchin (Arubmia punctulutu) early embryo, sea urchin sperm cell, MicrotoxB, and
Myszdopsis buhia tests revealed comparability for several compounds. However, no species is consistently the most sensitive to all compounds.
INTRODUCTION
Biomonitoring has become an important tool in assessing the impact
of toxicants in aquatic environments and several recent developments
have promoted its integration into monitoring programs. In 1984, the
Environmental Toxicology and Water Quality: An International Journal
Vol. 6,63-75 (1991)
CCC 1053-4725/91/01063-013$04.00
0 1991 John Wiley & Sons, Inc.
64ISNELL ET AL.
U.S. Environmental Protection Agency (EPA) began allowing the use
of biological testing as part of the National Pollution Discharge Elimination System (NPDES) (US. EPA, 1984). Improvements in the precision of toxicity tests have progressed (Rue et al. 19881,and standardized
tests have been developed for both marine and freshwaters that
are easy to execute with minimal technical instruction (U.S. EPA,
1985).
There are, however, several areas where standardized toxicity
tests warrant improvement. Currently, test animals are obtained from
stocks that must be maintained as live cultures. Technician time
for stock culture maintenance is expensive and adequate numbers of
test animals in uniform physiological condition cannot always
be provided. Lack of physiological and genetic uniformity among test
animals causes problems with test reproducibility (Dorn and Rodgers
1989). Acclimating animals to test conditions adds significantly
to the time required to complete a toxicity assay. Typically 2-7
days of acclimation time, followed by 1-4 days of toxicant exposure, are required to complete an aquatic invertebrate acute toxicity
test.
A standardized acute toxicity test using the estuarine rotifer
Brachionus plicatilis has been described that eliminates many of the
problems mentioned above (Snell and Persoone 1989).Test animals are
obtained by hatching dormant eggs called “cysts,” which have a shelf
life of at least two years. No acclimation time is required for test
animals, greatly reducing the set-up time for the assay. Assay costs are
substantially reduced because there is no stock culture maintenance.
Test animal availability is never a problem since they are obtained
simply by hatching cysts. Variability among test animals is also reduced, which considerably lowers test failure rate and promotes test
reproducibility between laboratories. Because rotifers are small,
assays can be conducted with very small volumes of medium, making them ideal for toxicity identification evaluations. These exceptional features make the rotifer test a powerful tool for assessing
aquatic toxicity and its use in aquatic toxicology is likely to
expand.
Our objectives in this paper are to elaborate on several aspects of
the rotifer response to toxicants. These include (1)expanding the data
base of chemicals for which acute toxicity has been characterized, (2)
determining the effect of cyst age on neonate sensitivity to toxicants, (3)
comparing the sensitivity of different B . plicatilis strains to reference
toxicants, (4) investigating the effects of temperature on toxicant sensitivity, (5) examining how the duration of exposure to toxicants affects
ACUTE TOXICITY TESTS USING ROTIFERSI65
sensitivity, and (6) characterizing survival in controls to determine the
maximum duration for the test.
MATERIALS AND METHODS
Static acute toxicity assays were conducted with the euryhaline rotifer
B . plicatilis Muller (BP), Russian strain. Range finding and definitive
tests consisted of a series of five toxicant dilutions and a control.
Treatment solutions were made by diluting specific volumes of a
toxicant stock solution with ASPM synthetic seawater (Guillard,
1983). ASPM at 15 ppt salinity consists of 11.31 g NaC1, 0.36 g KC1,
0.54 g CaCl,, 1.97 g MgC1, . 6H,O, 2.39 g MgSO, . 7H20, 0.17 g
NaHCO, in one liter of deionized water, adjusted to pH 8.0. Deionized
base water was obtained from a Barnstead NANOpure I1 system with
one pretreatment, one high-capacity, and two ultrapure cartridges.
The toxicant stock solutions for copper, cadmium, lead, mercury,
nickel, selenium, and silver were prepared from atomic absorption
standards obtained from Sigma Chemical Company, except mercury,
which was obtained from Aldrich Chemical Company. Sodium pentachlorophenate (PCP), SDS, and sodium hypochlorite (NaOC1) stocks
(CDNB),
also were obtained from Aldrich. l-Chloro-2,4-dinitrobenzene
2,4-dichlorophenoxyaceticacid (2,4-D),and phenol were obtained from
Sigma. Free ammonia stock solutions were prepared from NH&l from
Mallinckrodt. Acetone and chloroform were obtained from Fisher Scientific and tributyl tin from M&T Chemicals. Hexane and xylene were
obtained from Union Chimique Belge. Diesel fuel was obtained from a
local commercial source.
Stock solutions were made to the appropriate concentrations by
first adjusting the pH, then diluting to the appropriate volume in volumetric flasks. One ml of the appropriate toxicant dilution was pipetted
into each well of a 24-well polystyrene plate (Corning). Each column of
wells on the plate consisted of four wells and was loaded with one
toxicant concentration.
Test animals were obtained by hatching cysts that were collected
from laboratory populations. At 25"C, cysts typically began hatching
after 19-20 h of incubation in synthetic seawater and continuous light
of 1000-4000 lux. After 20-24 h, enough neonates were available to
set up an acute test in a multiwell plate. Using a micropipette, about
fifty 0-2 h old neonates were transferred to each well in the bottom
row of the multiwell plate. These wells were used as rinsing wells.
Starting with the control, 10 neonates were transferred from the rinsing
well into each of the three remaining wells of the same column. This
66/SNELL ET AL.
TABLE I
BP LC,,-,s, coefficients of variation (CV), 95% confidence limits
(CL), and sample sizes ( N ) for 21 compounds.a
Compound
PCP
CDNB
SDS
2,4-D
Acetone
Chloroform
Phenol
Xylene
Hexane
NaOCl
Diesel fuel
Free ammonia
Tributyl tin
Copper
Mercury
Cadmium
Zinc
Nickel
Silver
Selenium
Lead
Lc50
1.9
2.0
5.6
598
75
2.4
>400
496
154
1.2
345
38
0.30
0.063
0.061
39
>4.8
>20
0.12
17
>4.0
cv (%)
95% CL
N
9.2
8.12
9.8
1.72
2.39
6.9
5.7
5.3
7.0
28.0
20.5
2.7
4.2
1.7-2.1
1.7-2.2
4.9-6.3
72-78
2.3-2.5
387-605
126-182
1.1-1.3
35-41
0.24-0.35
0.043-0.084
0.059-0.062
37-41
-
6
4
5
1
3
4
1
4
4
4
1
6
9
4
6
6
1
1
3
3
1
-
17.5
27.0
-
-
0.070-0.18
6-28
-
a All LCs0sare in mg . L-l for 24-h acute toxicity tests except
chlorine, acetone, and chloroform, which were l-h tests, and diesel
fuel, which is p L . L-I.
procedure was repeated for each treatment, ending with the highest
concentration. Once all neonates were distributed, the plate was placed
in a 25°C incubator in the dark. The number of live and dead animals
was recorded after 24 h. Animals were considered dead if no movement
was observed within 5 s. The number of replicate assays performed for
each toxicant is listed in Table I. The LCsos were calculated by probit
analysis using Statview I1 on a Macintosh I1 computer.
To examine the effects of cyst age on BP sensitivity, cysts were
stored at 4°C in 55 ppt synthetic seawater in darkness for 0,6, 12, 19,
and 27 months, followed by hatching and exposure to PCP. The following PCP dilution series was prepared on the day of cyst hatching: 0.0,
1.0,1.6,2.2,2.8, and 3.4 mg . L-l. Females from one batch ofcysts were
loaded into each well of a 24-well plate containing the PCP dilution
series. LC,, values for each batch of cysts were calculated for each of
three replicate determinations and correlated with cyst age.
ACUTE TOXICITY TESTS USING ROTIFERR67
The sensitivities of three different BP strains to copper, PCP, and
SDS were compared. The strains used were Russian (Rus), Hawaiian
(Haw), and Austrian (Aus). Rus cysts were obtained from Dr. P. Sorgel00s of the State University of Ghent in 1980and originated from salinas
in the Azov Sea region. Haw cysts were obtained from Dr. A. Hagiwara
of the Oceanic Institute in Hawaii. Aus cysts were collected by Dr. T.
Snell from Lake Obere Halbjochlacke, Austria. The time required for
50% cyst hatching were Rus-23 h; Haw-27 h; and Aus-40 h. A
minimum of 2 definitive assays were performed on each strain for all
3 toxicants. Mean LC50 values were compared using analysis of variance
(ANOVA) and Scheffe’s F test to determine whether significant differences exist between BP strains.
The effects of temperature on BP sensitivity to NaPCP and copper
were examined. Range finding tests for both toxicants were conducted
at 10, 15, 20, 25, 30, and 35°C using treatment concentrations previously determined for the 25°C definitive tests. Three definitive tests
were conducted at each temperature for both toxicants. The resulting
LC,, values were compared using an ANOVA and Scheffe’s F test to
identify significant differences in sensitivity.
The effect of the duration of the test on BP sensitivity to cadmium
and NaPCP was investigated. Three replicate 48- and 72-h definitive
tests were conducted for each toxicant. Six 24-h definitive tests were
available from previous experiments on both toxicants. An ANOVA
and Scheffe’sFtest were calculated to compare mean LC5,s. To examine
survival under control conditions, a life table was constructed for unfed
BP at 20, 25, and 30°C. The time of first hatch was recorded and 240
neonates were transferred immediately to a multiwell plate containing
synthetic seawater. The plate was covered with parafilm to prevent
evaporation and placed in an incubator at the appropriate temperature
in darkness. The number of dead animals in each well was recorded
every 6 h and the bodies removed. The mean number of deaths and
standard error for each day were calculated. Mean life span in days as
well as age-specific survival was calculated for all three temperatures.
Results
BP sensitivity to the test compounds varied widely. LC,, values, coefficients of variation, 95%confidence limits, and sample sizes for 21 compounds are reported in Table I. The range of sensitivities for the test
compounds spans about 4 orders of magnitude. Among the metals, four
were very toxic: mercury, copper, silver, and tributyl tin. Cadmium and
selenium were only moderately toxic, whereas lead, nickel, and zinc
were not toxic at their solubility limits in standard seawater. Of the
68/SNELL ET AL.
1 8
1 6
1 4
LC50
l 2
(mg.1-I)1
8
6
4
2
0
0
5
10
15
20
25
30
CYST AGE (months)
Fig. 1 The effect of cyst age on the sensitivity of test animals to sodium pentachlorophenate (PCP).Data plotted are the means of three replicate tests; vertical lines indicate
standard error.
two pesticides examined, PCP was very toxic and 2,4-D was nontoxic.
The sensitivity of BP to the relatively toxic CDNB, NaOC1, and SDS
was similar. Free ammonia was only moderately toxic as was chloroform, but BP was not sensitive to acetone, phenol, hexane, xylene, or
diesel fuel. Coefficients of variation for the LC,,s of the 21 compounds
indicating a high degree of reproducibility
investigated averaged 11%,
for the BP acute test.
The effect of cyst age on the sensitivity of neonates to PCP was
examined (Fig. 1).Neonates from cysts up to 27 months old did not
differ in their PCP LC,,s. Regression analysis showed there was no
correlation between cyst age and PCP sensitivity ( r = 0.09). The coefficient of variation for LC,,s of cysts of different ages was 3.7%. This is
less than the coefficient of variation among replicate LC,,s from a single
batch of cysts, which for PCP was 9.2%.
Sensitivity to PCP, copper, and SDS differed among the Aus, Rus,
and Haw strains (Fig. 2). Significant differences in the LC,,s of the
three strains were detected using ANOVA and Sheffe’s F test. Mean
PCP LC5,s were 0.63, 0.97, and 1.9 mg - L-* for Aus, Haw, and Rus
strains, respectively. The Rus strain was two times less sensitive to
PCP than the Haw strain and three times less sensitive than the Aus
strain (8’= 38.7, df = 11,p < 0.01). Mean copper LC,,s were 0.035,
0.063, and 0.17 mg . L-’ for the Haw, Rus, and Aus, respectively,
indicating that the Haw and Rus strains were significantly more sensitive t o copper than the Aus strain ( F = 23.1, df = 8, p < 0.01). Mean
ACUTE TOXICITY TESTS USING ROTIFERS/69
lo T
1
LC50
(rng.1-l)
01
0 01
copper
PCP
SDS
Toxicant
Fig. 2 A comparison of the sensitivity of three BP strains to copper, PCP, and SDS.
Data plotted are the means of at least two replicate tests; vertical lines indicate standard
error.
SDS LCs,s were 3.8,4.7, and 5.6 mg . L-l for Haw, Aus, and Rus strains,
respectively. Only the Haw and Rus LC5,p were significantly different
by ANOVA ( F = 9.57, df = 9 , p < 0.01).
BP sensitivity t o copper and PCP was temperature dependent (Fig.
3a and b). No significant difference in copper sensitivity was observed
at temperatures of 15 and 20"C, but a temperature reduction to 10°C
increased sensitivity 20%. Increasing temperatures from 20 to 25,30,
and 35°C increased sensitivity 62,66, and 88%, respectively ( F = 920,
df = 17, p < 0.01). Temperature increases from 10 to 25°C had no
significant effect on PCP sensitivity. However, increasing temperatures
from 20 to 30 and 35°C increased sensitivity 51 and 72%, respectively
( F = 115, df = 17,p < 0.01). The degree of change in toxicant sensitivity with temperature differed for PCP and copper. Copper sensitivity
increased 16%more than PCP sensitivity when temperature was raised
from 20 to 30 or 35°C.
Increasing the duration of the test substantially decreased LC5,s
for cadmium and PCP (Fig. 4a and b). Cadmium LC,, decreased from
39.1 mg L-' for 24-h exposures to 9.2 and 5.2 mg - L-' at 48- and 72h exposures, respectively ( F = 511, df = 8 , p < 0.01). PCP LC50
decreased from 1.9 mg . L-l at 24-h exposures to 0.90 and 0.27 mg - L-l
at 48- and 72-h exposures, respectively ( F = 150, df = 8, p < 0.01).
70ISNELL ET AL.
18
1 6
1 4
LC50 1 2
(mg.1-l)
1
0 8
06
0 4
02
0
0.18
0.1 6
copper
0 14
LC50 0 1 2
(mg.I-') 0.1
0.08
0.06
0.04
0.02
0
10
15
20
25
30
35
Temperature ( OC)
Fig. 3 The effect of temperature on the sensitivity of test animals to copper and PCP.
Data plotted are the means of three replicate tests; vertical lines indicate standard error.
For both cadmium and PCP, 72-h exposures yielded significantly lower
LC,,s than 48-h exposures.
The mean life span of BP hatchlings under nonfeeding conditions
decreased with increasing temperatures (Fig. 5). For temperatures of
20,25, and 30"C, 50% of the original population was still alive after 10,
7, and 5 days, respectively. Since mortality in control treatments of
acute toxicity tests must not exceed lo%, it is important t o determine
the time when 10%of the animals die under control conditions. At 20,
25, and 3WC, 10%mortality occurred after 7,5, and 3 days, respectively.
Acute tests of 72 h duration are therefore possible only at temperatures
of 20 and 25°C. At 3WC, more than 10% mortality occurs in controls
after 72 h.
ACUTE TOXICITY TESTS USING ROTIFERS171
LC50
(mg.1-
.
04 20 2 5
.
.
30
.
.
35
40
.
.
45
,
50
.
55
.
.
.
,
60
65
70
75
LC50
(mg.1-l)
Hours
Fig. 4 The effect of the durationof toxicant exposure on the sensitivity of test animals
to PCP and Cd. Data plotted are the means of three replicate tests for 48- and 72-h
exposures and six replicates for 24-h exposure. Vertical lines indicate standard error.
DISCUSSION
In previous work (Snell and Persoone, 19891, BP assays were conducted
in synthetic seawater made with commercial sea salts. Commercial sea
salts use technical grade chemicals, some of which contain impurities
that can react with toxicants and influence test results. To avoid these
problems, we advocate using ASPM (Guillard, 1983), a defined, research-quality seawater composed of reagent grade salts. The acute
toxicity tests presented in this paper were all conducted in ASPM.
Consequently, the LC,,s we report vary for some chemicals from those
published by Snell and Persoone (1989). For example, PCP and SDS
72ISNELL ET AL.
Percent
Survival
0
2
4
6
8
10 12 14
16
18 20
Age (days)
Fig. 5 Age specific survival in unfed controls at three temperatures. Each data point
is the mean of three replicate populations. Percent surviving ( y axis) refers to the percent
of rotifers surviving to a specific age interval out of an initial population of 240.
LC,,s are 42 and 26% higher, respectively, in ASPM than in seawater
prepared from Instant Ocean salts. In contrast, copper and cadmium
LC,,s are 52 and 31% lower in ASPM, respectively.
The repeatability of the rotifer assay is excellent when compared
to other aquatic invertebrate assays. The average intralaboratory coefficient of variation for LC,,s of the 21 compounds we tested was 11%,as
compared to 35%for freshwater Daphnia magna and D.pulex exposed to
reference toxicants (US. EPA, 1985). For the marine invertebrates
Acartia tonsa and Mysidopsis bahia, the average interlaboratory coefficient of variation was 65%(U.S. EPA, 1985). Acute toxicity tests with
the rotifer BP are therefore much less variable than assays with other
aquatic invertebrates.
An important consideration for the acceptability of any new toxicity test is its sensitivity. The sensitivity of the BP assay compares
favorably with that of other assays commonly used in marine environments. Nacci et al. (1986) examined three rapid screening marine
assays: an early embryo growth test using the sea urchin Arbacia
punctulata, a sperm cell toxicity test also with A. punctulata, and Microtox. They compared these tests to results obtained with Mysidopsis
bahia acute toxicity tests. A summary of their findings appears in Table
IT, which also lists BP LCs0s. These data demonstrate that toxicity is
compound specific and that no species is consistently the most sensitive
or the most insensitive to all compounds.
The sensitivity of BP described above is for 24-h acute toxicity tests
ACUTE TOXICITY TESTS USING ROTIFERS/73
TABLE I1
Comparative sensitivities for BP and four marine toxicity testsa
Chemical
PCP
cu
Cd
Ag
Zn
Pb
BP
1.93
0.063
39.1
0.12
>4.8
>4.0
EG
sc
MT
MB
0.3
0.014
13.9
0.179
0.205
32.5
0.9
0.012
38.0
0.051
0.121
5.4
1.0
0.076
11.6
0.595
0.440
1.7
0.141
0.063
0.25
0.498
3.0
aToxicity values are EC,,s for the A. punctulata embryo
growth test (EG), sperm cell viability (SC), and Microtox (MT).
Values for BP and M. bahia (MB) are 24-h and 96-h LC,,s, respectively. All toxicity values are in mg . L-'.
conducted at 25°C with the Russian strain. Our data illustrate that
rotifer sensitivity can be increased by increasing test temperature to
30 or 35"C, or by using a strain more sensitive to the particular compound of interest. Extending the test to 48 or 72 h also results in lower
LCsos.Life table analysis of survival under control conditions indicates
that the maximum duration of the BP acute toxicity test is 72 h at
25°C. Exposures beyond 72 h without feeding lead to control mortality
exceeding 10%and invalidation of test results. However, tests of 48-or
72-h duration work well, and can be used to obtain significantly lower
LC,,s than 24-h tests. Therefore, to some extent the sensitivity of the
rotifer assay is under the control of the experimenter through choice of
exposure conditions. For validation purposes, however, test conditions
should closely approximate the conditions in the natural ecosystem of
interest.
The similar sensitivity of neonates from different batches of cysts
contributes to the reproducibility test. By maintaining test animals in
a dormant state, the likelihood of genetic changes in stock populations
through mutation, selection and drift is diminished. Consistent sensitivity makes test results reproducible over long periods of time.
The large variability in sensitivity among rotifer strains argues
for using standard strains. Only those strains that have been calibrated
with reference toxicants are useful for comparative toxicity evaluations. It is further important that the strain used in tests be clearly
identified in publications.
In general, the sensitivity of aquatic invertebrates increases with
increasing temperatures (Bryant et al., 1985; Cairns et al., 1978; Cotter
et al., 1982;Jones, 1975;MacInnes and Calabrese, 1979;Persoone etal.,
1 9 8 9 ) . This probably results from increased metabolic rates, diffusion
74/SNELL ET AL.
rates, and increased toxicant solubility at higher temperatures. However, the interaction of these effects is complex and sometimes sensitivity does not increase with temperature (Cowgillet aZ., 1985; Stephenson
and Watts, 1984). In rotifers, the results appear more consistent and
increased sensitivity at higher temperature is often reported (Capuzzo,
1979; Schaefer and Pipes, 1973; Persoone et al., 1989). For example,
Persoone et al. (1989) investigated the response of BP to selected toxicants over temperatures of 10-31°C at 25 ppt salinity. For potassium
dichromate and SDS, LC,,s were similar at 10,17, and 24°C. However,
between 24 and 31"C,LC,,s decreased 33 and 18%for potassium dichromate and SDS, respectively. In comparison, we found PCP LC,,s to
decrease 75% between 25 and 35"C, and copper LC,,s t o decrease 68%.
While the effect of higher temperatures on rotifer sensitivity to toxicants seems consistent, the degree to which sensitivity increases is
toxicant specific.
We acknowledge with appreciation the comments of Dr. Christian Blaise, which
improved this paper.
References
Bryant, V., D.M. Newberry, D.S. McLusky, and R. Campbell. 1985. Effect of temperature
and salinity on the toxicity of arsenic to three estuarine invertebrates (Corophium
uolutator, Macoma balthica, Tubifex costatus). Marine Ecol. Prog. Ser. 24129-137.
Dorn, P.B. and J.H. Rodgers, J r . 1989. Variability associated with identification of toxics
in national pollutant discharge elimination system (NPDES) effluent toxicity tests.
Environ. Toxicol. Chem. 8:893-902.
Cairns, J., Jr., A.L. Buikema Jr., A.G. Heath, and B.C. Paker. 1978. Effects oftemperature
on aquatic organism sensitivity to selected chemicals. Publ. of Virginia Water Resources Research Center. 1061-88.
Capuzzo, J.M. 1979. The effects of halogen toxicants on survival, feeding and egg production of the rotifer Brachionus plicatilis. Estuary Coast. Mar. Sci. 8:307-316.
Cotter, A.J.R., D.J.H. Phillips, and M. Ahsanullah. 1982. The significance oftemperature,
salinity and zinc as lethal factors for the mussel Mytilus edulis in a polluted estuary.
Marine Biol. 68:135-141.
Cowgill, U.M., I.T. Takahashi, and S.L. Applegath. 1985. A comparison of the effect of
four benchmark chemicals on Daphnia magna and Ceriodaphnia dubialaffinis tested
a t two different temperatures. Environ. Toxicol. Chem. 4:415-422.
Guillard, R.R.L. 1983. Culture of phytoplankton for feeding marine invertebrates, P.
108-132. In C.J. Berg J r . (ed), Culture of Marine Invertebrates. Hutchinson-Ross,
Stroudsberg, PA.
Jones, M.B. 1975. Synergistic effects of salinity, temperature and heavy metals on mortality and osmoregulation in marine and estuarine Isopods (Crustacea). Marine Biol.
30:13-20.
MacInnes, J.R., and A. Calabrese. 1979. Combined effects of salinity, temperature, and
copper on embryos and early larvae of the American oyster, Crassostrea uirginica.
Arch. Environ. Contam. Toxicol. 8553-562.
ACUTE TOXICITY TESTS USING ROTIFERS/75
Nacci, D., E. Jackim, and R. Walsh. 1986. Comparative evaluation of three rapid marine
toxicity tests: Sea urchin early embryo test, sea urchin sperm cell toxicity test and
Microtox. Environ. Toxicol. Chem. 5:521-525.
Persoone, G., A. Van de Vel, M. Van Steertegem, and B. De Nayer. 1989. Predictive value
of laboratory tests with aquatic invertebrates: influence of experimental conditions.
Aquatic Toxicol. 14:149-166.
Rue, W.J., J.A. Fava, and D.R. Grothe. 1988. A review of inter- and intralaboratory
effluent toxicity test method variability. Aquatic Toxicology and Hazard Assessment:
10th Vol., ASTM STP 971, W.J. Adams, G.A. Chapman & W.G. Landis (eds.), Amer.
SOC.
for Testing and Materials, Philadelphia, 190-203.
Schaefer, E.D., and W.O. Pipes. 1973. Temperature and toxicity ofchromate and arsenate
to the rotifer, Philodinu roseolu. Water Res. 71781-1790.
Snell, T.W., and G. Persoone. 1989. Acute toxicity bioassays using rotifers. I. A test
for brackish and marine environments with Brachionus plicatilis. Aquatic Toxicol.
14:65-80.
Stephenson, R.R., and S.A. Watts. 1984. Chronic toxicity tests with Duphniu mugna: The
effects of different food and temperature regimes on survival, reproduction and growth.
Environ. Pollut. Ser. A. Ecol. Biol. 36:95-108.
U S . Environmental Protection Agency. 1984. Development of water quality-based permit limitations for toxic pollutants: National policy. Fed. Reg. 49:9016-9019.
U.S. Environmental Protection Agency. 1985. Methods for measuring the acute toxicity
of effluents to freshwater and marine organisms. W.H. Peltier and C.I. Weber (eds.),
U.S. Environmental Protection Agency.