Hydrobiologia 313/314 : 231-247, 1995 .

J. Ejsmont-Karabin & R. M. Pontin (eds), Rotifera VII . 231

©1995 Kluwer Academic Publishers . Printed in Belgium.

Rotifers in ecotoxicology : a review

Terry W. Snell l & Colin R. Janssen 2

School of Biology, Georgia Institute of Technology, Atlanta, GA 30332-0230, USA
2 Laboratory for Biological Research in Aquatic Pollution, State University of Ghent, J . Plateaustraat 22, B-9000
Gent, Belgium

Key words : rotifers, ecotoxicology, pollution, LC50s, reproduction, enzymes

Abstract

In the past five years the use of rotifers in ecotoxicologial studies has substantially increased . This greater interest
has been due to the central role of rotifers in freshwater planktonic communities, the ease and speed of making
quantitative measurements of mortality and reproduction, their sensitivity to common pollutants, the commercial
availability of cysts, and the existence of reliable, standardized protocols . The main endpoints used in ecotox-
icology studies are reviewed, including mortality, reproduction, behavior, cellular biomarkers, mesocosms, and
species diversity in natural populations . For each endpoint, published studies are cited, along with the compounds
investigated, duration of exposure, and the LC50s, EC50s or NOECs reported . Rotifers have been included as
part of a standardized mesocosm and in several large-scale, outdoor mesocosm studies . A critique of rotifer use
in ecotoxicology is offered and it is concluded that the scientific basis for including rotifers as part of a battery of
ecotoxicological tests is well established .

Introduction intense (Bogdan & Gilbert, 1982), sometimes affect-

The number of standard toxicity tests accepted by var-
ious international organizations for determination of
the toxicity of chemical, effluents, and sediments is
very low (Persoone & Janssen, 1993) . For the past
3 decades, regulatory agencies have relied to only a
single taxon of freshwater cladocerans for most tox-
icity assessments . Indeed, acute and chronic toxicity
tests with daphnids are currently one of the few fresh-
water invertebrate tests that are formally approved by
the USEPA, EEC, and OECD for regulatory testing
(Persoone & Janssen, 1993) . Although rotifers were
suggested as test animals in the early 1970s (Schaef-
fer & Pipes, 1973 ; Buikema et al ., 1974), only in the
last decade, and especially in the last 5 years, has the
use of rotifers for ecotoxicological studied increased
significantly .
Rotifers are useful as models in ecotoxicology
because they often play a key role in the dynamics
of freshwater and coastal marine ecosystems (Wallace
& Snell, 1991) . Rotifer grazing rates can be seasonally
ing algal species composition and water quality . With
their high assimilation efficiencies, rotifers convert a
considerable portion of their food into biomass, mak-
ing it available to higher trophic levels (Starkweather,
1987) . The rapid turnover rates of rotifer populations
allows them to contribute significantly to nutrient recy-
cling in aquatic habitats (Markarewicz & Likens, 1979 ;
Ejsmont-Karabin, 1983). Rotifers at times strongly
compete with microcrustacean zooplankters for food
(Gilbert, 1985) and serve as prey for other rotifers like
Asplanchna (Gilbert & Stemberger, 1985), cyclopoid
and calanoid copepods (Williamson, 1983), insect
larvae (Moore & Gilbert, 1987), and fish (O'Brien,
1979) .
The life cycle of rotifers contains both asexual and
sexual phases (Wallace & Snell, 1991) . The product
of sexual reproduction is an encysted dormant embryo
called a cyst (resting egg) (Gilbert, 1974 ; Pourriot &
Snell, 1983) which has great utility in ecotoxicology .
Cysts allow test animals to be stored dried for sever-
al years . Hatching is fairly synchronous and can be
232
initiated by hydrating the cysts at 25 ° C in light for
about 24 h . Sufficient animals becoming available for
testing within a few hours of the first hatching . The
neonate rotifers hatch in physiologically uniform con-
dition and, because of the ease of shipping cysts, the
same strain can be used worldwide . Rotifers and some
anostracan crustaceans are the only animals for which
cysts are commercially available for toxicity testing
(Persoone, 1991) .
Another feature of the rotifer life cycle that facili-
tates their use in ecotoxicology is the short life cycle
and rapid reproduction . Generation times of rotifers are
among the shortest of those of any metazoan . Repro-
duction in most rotifer toxicity tests is asexual and
females typically produce 20-40 offspring . Life table
analysis can be conducted on several rotifer species
(Janssen et al ., 1993) and simpler, faster methods for
estimating population growth rate have been intro-
duced (Snell & Moffat, 1992 ; Janssen et al., 1994) .
The small size of rotifers allows them to be cultured
in tl volumes, so toxicity can be assessed with very
small amounts of test compound .
Standardization of test procedures is important in
ecotoxicology so that data collected in different labs
and at different times can be compared . One of the most
rigorous programs for standardization is that of the
American Society of Testing and Materials (ASTM),
which approve protocols after several rounds of peer
review. An ASTM standard acute toxicity test for fresh-
water using Brachionus calyciflorus and for marine
waters using B. plicatilis has been published (ASTM,
1991) . This protocol was the subject of a large interna-
tional intercalibration exercise (Persoone et al ., 1992) .
A variety of other test methods has been published
using as endpoints reproductive rate (Snell & Moffat,
1992 ; Janssen et al., 1994a), ingestion rate (Ferran-
do et al., 1993 ; Juchelka & Snell, 1994), swimming
activity (Janssen et al ., 1993 ; Janssen et al., 1994b),
and enzyme activity (Burbank & Snell, 1994 ; Mof-
fat & Snell, 1994) . None of these, however, has been
subjected to an ASTM-style review.
This paper is organized according to the toxicolog-
ical endpoints that have been employed most often in
rotifer studies . The first endpoint described is mortal-
ity, followed by reproduction, behavior, cellular and
molecular biomarkers, mesocosms, and natural popu-
lation studies .
Mortality
The toxicity of a large number of environmental con-
taminants has been assessed using toxicity tests with
rotifers (Table 1) . As early as the 1970s, when aquat-
ic toxicology was still in its infancy, Buikema et al.
(1974) and Schaefer & Pipes (1973) used short-term
mortality tests with Philodina acuticornis and P rose-
ola, respectively, to evaluate the toxicity of heavy met-
als . The majority of the acute toxicity studies, however,
has been performed with members of the genus Bra-
chionus . Capuzzo (1979b), for example, studied the
effects of chlorinated cooling waters on the short-term
mortality response of B. plicatilis . Halbach et al. (1983)
used acute toxicity tests with B. rubens to screen the
toxicity of pentachlorophenol prior to their life table
and population studies . A standardized 24-h LC50 test
protocol with B. rubens and B. plicatilis, using cysts
to obtain test animals was proposed by Snell & Per-
soone (1989a, 1989b) . The development of these cyst
based acute toxicity tests was a major breakthrough for
routine toxicity assessments because it eliminates the
need for continuous culture (Snell & Persoone, 1989a ;
Janssen & Persoone, 1991) . Based on the same princi-
ple, Snell et al . (1991 a) published an acute test with B.
calyciflorus, assessing the acute toxicity of 28 chemi-
cals . They also examined the influence of factors such
as cyst age, temperature, and salinity on the sensitivity
of the test animals .
The procedure of cyst-based rotifer tests can be
summarized as follows : 18 h prior to the test for B. caly-
ciiorus and 28 h for B. plicatilis, cysts are transferred
to synthetic fresh or saltwater medium and incubated
at 25°C . After hatching, 0-2 h old neonates are col-
lected and exposed to a toxicant dilution series . Tests
are conducted in 24-well plates (Snell et al., 1991 a) or
36-well plates (Persoone et al ., 1993), in which 3 x 10
or 6 x 5 rotifers, respectively, are exposed to each test
concentration . After 24 h, the number of alive and dead
rotifers is counted and the LC50 calculated .
The application of these cyst-based acute rotifer
tests has also been stimulated through the develop-
ment of `toxicity test kits' . These toxkits contain all
materials necessary to perform an acute toxicity test,
i .e . the rotifer cysts, hatching and test containers, and
media to prepare the control and toxicant dilutions . The
interlaboratory reproducibility of these rotifer testkits
was evaluated in a large intercalibration exercise in
Europe and North America which yielded interlabo-
ratory coefficients of variation of 25-68%, depending
on the test species and geographical area of the inter-


233

Table 1 . Summary of acute toxicity studies with rotifers . The LC50s are in mg.1 - 1 .
Test species Chemical Exposure LC50 Reference
(mg-1 -1 )
B, plicatilis free chlorine 30 min 0.18 Capuzzo, 1979a
chloramine 30 min 0 .02
potassium 24 h 22 .2 Persoone et al ., 1989
dichromate
sodium 24 h 40 .1
laurylsulphate
pentachlorophenol 24 h 1 .36 Snell & Persoone, 1989a
sodiumdodecyl- " 4 .42 "
sulphate
free NH3 17 .7
malathion 45 .5 "
copper 0 .13
cadmium 56 .8 "
pentachlorophenol 1 .9 Snell et al ., 1991b
chlorodinitro- 2 .0 "
benzene
sodiumdodecyl- 5 .6
sulphate
dichlorophenoxy- " 598
acetic acid
acetone 75
chloroform 2 .4
phenol > 400
xylene " 496
hexane 156
NaOC1 1 .2 "
diesel fuel 345
free ammonia 38 "
" tributyl tin 0 .30
copper 0 .063 "
mercury " 0.061 "
cadmium 39
zinc > 4.8
nickel > 20
silver 0.12
selenium 17 "
lead > 4.0 "
toluene, hexane, - Ferrando & Andreu-
xylene, benzene Moliner, 1992
trichlorfon 274.9 Ferrando & Andreu-
Moliner, 1991
fenitrothon 8 .87 "
chlorpyrifos 10.7
lindane " 33 .9 "
3,4-dichloroaniline 57 .5
ethanol 36840 Calleja & Persoone, 1992
11
ethylene glycol " 91319 "

Continued on p. 234
234

Table 1 . Continued .
Test species Chemical Exposure LC50 Reference
(mg .1 -1 )
B. plicatilis methanol 24 h 49680"
pharmaceutical " - Calleja & Persoone, 1992 ;
compounds Calleja, 1994
Philodina roseola chromate 96 h 5 .5 Shaefer & Pipes, 1973
11
arsenate 96 h 8 .2 "
P. acutiocormis cadmium 24 h 6 .2 Buikema et al., 1974
cobalt " 27 .8 "
copper " 1 .9 "
lead " 56 .2
" mercury " 1 .0 "
" nickel " 7 .6 "
chromate " 31 .6 "
silver " 5 .3
zinc " 1 .2 "
ammonium " 1140 "
chloride
phenol " 382 "
Dicranophorus phenol, 24 h to 6 no Erben, 1978
forcipatus acetophenone, days LC50's
mestiyloxide,
styrene,
methylstyrene,
benzene, cumene,
ethylbenzene,
toluene, acetone
Brachionus patulus DDT 48 h Rao & Sarma, 1986
Bs rubens phenol " 600 Halbach et al., 1983
pentachlorophenol 0.16 "
4-chloroaniline " 100 "
4-nitrophenol 6.3
B. calyciJlorus pentachlorophenol 24 h 0.62 Snell & Persoone, 1989b
sodiumdodecyl- " 1 .35 "
sulphate
free ammonia " 3.21 "
malathion 35.3
copper 0.19 "
cadmium 0.81 "
furadan 2.00 Dad & Kant Pandya,
1982
malataf " 2.66 "
11
pentachlorophenol " 1 .2 Snell et al ., 1991a
chlorodinitro- 1 .3 "
benzene
sodiumdodecyl- " 1 .4 "
sulphate
dichlorophenoxy- " 117 "
acetic acid

Continued on p. 235


235

Table 1 . Continued .
Test species Chemical Exposure LC50 Reference
(mg.1 - ' )
B. calyciflorus acetone 24 h 51 11

11
chloroform 2 .0
phenol > 150
11
3,4-dichloroaniline 62
toluene 113 11

hexane 68
11
xylene 33
benzene > 1000
11
trichlorofon 47 11

fenitrothion 6 .7
chlorpyrifos 12
NaOCI 0 .37 11

diesel fuel 63
11
free ammonia 4.6
tributyl tin 0.19
11
copper 0 .026
11
mercury 0 .060
11
cadmium 1 .3
zinc 1 .3 11

nickel 4 .0
11
silver 0 .0075
selenium 16
aluminium > 3 .0 11

lead > 4 .0
six heavy metals Couiliard et al., 1989
endosulfan 5 .15 Femandez-Casalderrey et
al ., 1992
11
diazinon 29 .2
11

11
methylparathion 29 .2
malathion 33 .7
benthiocarb 6 .50 11

11
fenitrothion 6 .68 Ferrando & Andreu-
Moliner, 1991
11
chlorpyrifos 11 .9
11
parathion 29 .2
lindane 22 .5 11

11
3,4 dichloroaniline 61 .5 11

11
toluene, toluene, Ferrando & Andreu-
hexane, xylene, Moliner, 1992
benzene
50 MEIC 1 Calleja et al., 1993
chemicals, mainly Calleja, 1994
pharmaceutical
compounds
11
50 surfactants Janssen et al., unpubl.
data

Continued on p. 236

236
Table 1 . Continued.
Test species Chemical
B. calyciflorus river sediments
river sediments
11
surface waters &
river sediments
industrial effluents
11
effluents,
sediments, solid
wastes,
monitoring wells,
sludges from
water treatment
plants
industrial effluents
1 MEIC : Muticentre Evaluation of In Vitro Cytotoxicity.
calibration exercise (Persoone et al., 1992) . The acute
toxicity test with B. plicatilis and B. calyciflorus using
cysts to obtain test animals was accepted as a standard
test procedure by the American Society for Testing and
Materials (ASTM, 1991) .
In recent years, several workers have used B. caly-
ciflorus and B. plicatilis for various types of toxicity
assessments . The toxicity of sediment pore waters and
elutriates of contaminated sediments originating from
the St. Lawrence River have been evaluated with B.
calyciflorus neonates obtained from cultures (Couil-
lard et al., 1989 ; Sloterdijk et al ., 1989) . Moses &
Wade (1992 a&b) have assessed the potential use of
the cyst-based B . calyciflorus test and a light emitting
bacterial test (Microtox) for the acute toxicity screen-
ing of reservoir surface waters and sediments . Similar
comparative studies to assess the use of rotifer toxicity
tests for effluent monitoring were performed by Van der
Wielen et al . (1993) and Muna et al . (1994) . Compre-
hensive evaluation of approximately 400 environmen-
tal samples using cyst-based toxicity tests (including B.
calyciflorus) for the toxicity assessment of solid waste
elutriates, monitoring wells, effluents, sediment pore
waters, and sewage treatment sludges was recently
completed by by Persoone et al . (1993) . These authors
concluded that cyst-based toxicity tests with rotifers
and crustaceans, in general have a sensitivity which is
comparable to that of the conventional acute toxicity
test with Daphnia sp .
Exposure LC50 Reference
1)
(mg.1_
24 h Couillard et al., 1987 ;
Couillard et al., 1989
11
- Sloterdijk et al., 1989
" h Moses & Wade, 1992
a&b
Van der Wielen et al.,
1992
Persoone et al ., 1993
- Muna et al ., 1994
The acute toxicity of numerous pure chemicals has
been assessed using rotifers . The 24-h LC50s of 28
contaminants including heavy metals and organic com-
pounds was reported for B. calyciflorus (Snell et al .,
1991a) . LC50s spanned 5 orders of magnitude from
0.008 mg 1 -1 for silver to more than 1000 mg 1-1
for benzene . Using the same test protocol, Ferran-
do & Andreu-Moliner (1992) investigated the acute
toxicity of 5 petroleum derivatives to both B. calyci-
florus and B . plicatilis and Fernandez-Casalderrey et
al . (1992) examined the susceptibility of B . calyci-
florus for various types of pesticides . In another study,
the same authors showed that the organochlorine pes-
ticide endosulfan had a 24-h LC50 of 5 .2 mg 1 -1 and
tended to bioaccumulate in the rotifers during the first
24-48 h of exposure, followed by a gradual decrease
(Fernandez-Casalderrey et al ., 1991) . The use of acute
toxicity tests with B . calyciflorus and B . plicatilis as
part of a battery of alternative tests to warm-blooded
vertebrates has been investigated by Calleja & Per-
soone (1992) and Calleja et al. (1993) . These authors
assessed the acute toxicity of 50 chemicals (mainly
pharmaceuticals) with a battery of 6 acute toxicity
tests and constructed models, using multivariate statis-
tical techniques, for predicting human acute systemic
toxicity based on the aquatic animal tests . (Calleja
(1994) concluded that a battery of standardized aquat-
ic non-vertebrate tests (including the acute rotifer test),
combined with a set of physico-chemical properties of
the test compounds, has the same or better ability to

identify compounds capable of causing human toxic-
ity as conventional rodent acute tests . Van Leeuwen
et al . (1992) used several species of rotifers together
with 17 other aquatic species, to examine quantitative
structure-activity relationships (QSARs) for predicting
the no-effect concentrations at the ecosystem level of
102 narcotic pollutants .
Reproduction
The measurement of rotifer reproductive rates has a
long history in aquatic ecology . Consequently, the
application of reproductive measurements as toxico-
logical endpoints was relatively straightforward . Most
applications have used static or static-renewal cul-
tures . Asexual reproduction has been quantified using
life table techniques on isolated females or population
growth parameters have been estimated from log-phase
populations (Table 2) . In a few papers carrying capac-
ity (K) was used as an endpoint.
Interest in using rotifers in ecotoxicological studies
was stimulated by Halbach and his colleagues (Hal-
bach et al ., 1981 ; Halbach et al ., 1983 ; Halbach, 1984) .
They were the first to point out that rotifer population
dynamics could be used to amplify small, sublethal
effects and developed deterministic population dynam-
ics models to predict the growth of laboratory popula-
tions under toxic stress . Halbach (1984) advocated the
use of a descriptive model for ecotoxicological tests
based on B . rubens and B. calyciflorus populations .
He used r and K as endpoints as well as the frequency
and amplitude of population oscillations . The data sug-
gested that this latter parameter was the most sensitive
endpoint. Halbach's group was developing approaches
to investigating toxicant effects on competition among
brachionids and Asplanchna predation at the time of
Halbach's untimely death. Kooijman & Metz (1984)
also modelled rotifer population dynamics for the use
in ecotoxicology . Using B . rubens, they showed that
the effect of toxicant stress on rotifer populations is
strongly dependent on their feeding status .
Rao & Sarma (1986) examined DDT toxicity to
B . patulus using a life table approach . They measured
all of the traditional population parameters including
r, net reproduction (R0), life expectancy, and genera-
tion time (T), as well as reproductive value (Vi ) and
residual reproductive value (V,*,) . All parameters but T
significantly declined with increasing DDT concentra-
tion, however, reproductive parameters were affected
at lower levels than survivorship . Three-fold lower
food levels reduced EC50s by about 70% . The DDT
237
stress also changed the reproductive tactics of B . patu-
lus by shifting maximum reproductive values to older
age classes .
The need for rapid, inexpensive methods for esti-
mating chronic toxicity led to modification of these
techniques for quantifying rotifer population growth
rates . Snell & Moffat (1992) devised a 2 day popula-
tion growth test using r as endpoint . The test with B.
calyciflorus at 25 ° C encompassed 33% of the lifespan
and captured > 90% of the total contribution to r . Two
thirds of the reproduction was attributable to parental
females and 1/3 to F1 females . The parameter r was
a sensitive measure of toxicity as it was reduced in a
dose-dependent manner with all toxicants investigated .
Based on the analysis of the acute/chronic ratios of 11
chemicals, Snell & Moffat found evidence of chronic
toxicity for only five compounds . The rotifer proto-
col required 70% less effort to quantify reproductive
effects of toxicity than the standard USEPA method
with Ceriodaphnia .
Janssen et al . (1994a) investigated the effects
of temperature and food level on the demographic
response of B . calyciflorus to four toxicants . They
found that LOECs increased about 4-fold as food con-
centration increased from 5 x 10 4 Nannochloris ocula-
ta cells ml - I to 10 6 cells ml- 1 . These authors pointed
out that laboratory toxicity tests are typically conduct-
ed at much higher food levels than those occurring in
natural environments . They argued, however, that the
high laboratory food levels might not be important for
general screening purposes since the change in LOECs
typically was less than 0 .5 an order of magnitude over
a 4-fold food range . In a second paper, Janssen et al .
(1994b) showed that life table experiments for B . caly-
ciflorus at 25°C could be abbreviated to 4 days and
retain good estimates of r . By day four, 95% of r was
determined and the correlation coefficient between r
determined after four days and that of the full life table
(- 8 days) was 0 .98 (P < 0 .01) . The LOECs for four
toxicants were the same whether the four day or full life
table estimates of r were used . The parameter r was not
always the most sensitive endpoint ; Ro sometimes had
a lower LOEC . These authors described a 3 day popu-
lation growth test to estimate r that is similar to that of
Snell & Moffat (1992) . The main differences are test
duration (3 vs . 2 days), food level (1 vs . 3 x 10 6 N. ocu-
lata cells ml -1 ), and source of algae (live cultures vs .
agar disks) . Recent observations have suggested that
the lower food level is better for either test since algae
can interact with toxicants altering their effects . Also
log-phase algae cultures yield higher quality cells than


238

Table 2 . Toxicity assessment using reproduction endpoints . The "end" column indicates the population growht endpoint ;
the "exp" column indicates the duration of toxicant exposure . NOEC is the no observed effect concentration in mg 1- I
and EC50 is the toxicant concentration where 50% inhibition occurs . All data are for females . "Life" is exposure for
the entire lifespan (H 7 days), K is carrying capacity .
Test species Chemical End Exp NOEC EC50 Reference
B. plicatilis pentachlorophenol r 48 h 0.5 - Juchelka & Snell, 1995
" 125 - "
phenol
copper 0 .01 "
cadmium "
free ammonia r life 2 .1 13 .2 Yu & Hirayama, 1986
chlorine r 48 h 1 - Capuzzo, 1979
chloramine " 1 _ ..
B. calyciflorus pentachlorophenol r 48 h 0 .11 0 .27 Snell & Moffat, 1992
" 25 59 "
phenol
dimethyl phenol 2 8 .6 "
trinitrotoluene " 2 .3 4 "
" 20 99 "
xylene
cadmium " 0.04 0 .07 "
copper " 0 .02 0 .026 "
chromium " " 2 2 .9 "
diazinon 8 11 "
chlorpyrifos 0 .23 0 .36 "
2,4 D " 58 128 "
pentachlorophenol mictic 48 h 0 .03 Snell & Carmona, 1994
cadmium " 0 .02 "
" naphthol " " 0 .40 - "
naphthol r 0 .40 "
chlorpyrifos mictic 0 .20 "
pentachlorophenol r 72 h 0.8 - Janssen et al ., 1994
dichloroaniline " 20 -
copper " 0.005 "
lindane " 10 "
dichloroaniline r life 2.5 - Ferrando et al., 1993
lindane " 10 "
methylparathion r life 1 Femandez-Casalderrey et al .,
1993
pentachlorophenol K 28 d 0.1 - Janssen, 1992
dichloroaniline " 2.5 - "
copper " 0.0025 -
" " - "
lindane <10
B . rubens dichloroaniline r life 1 - Kooijman & Metz, 1984
. dichromate " 10 -
potas
pentachlorophenol r life 0 .1 - Halbach, 1984
B . patulus DDT r life 0.015 0.016 Rao & Sarma, 1986
11 1. K 15 d 0.020 Rao & Sarma, 1990

algae reconstituted from disks . Consequently, lower effect of toxicants on sexual reproduction has recently
food levels can be used when algae are obtained from been examined in B . calyciflorus by Snell & Carmona
log-phase cultures . (1994) . They showed that relative effect of four tox-
All of the above work has investigated the effect icants was greater on the mictic rate than the amic-
of toxicants on amictic (asexual) reproduction . The tic reproductive rate . Mictic rate was measured as

the number of mictic daughters/total daughters pro-
duced . At 200 pg 1-1 pentachlorophenol, mictic rate
was reduced by 68% compared to the control, whereas
the amictic r was unaffected . Mictic rates were reduced
92%, 52%, and 48% at 25 jig 1 -1 cadmium, 300 µg
1- ' chlorpyrifos, and 800 ttg 1-1 naphthol, respective-
ly. In contrast, amictic r was reduced by 12%, 11%,
and 20%, respectively, by these same toxicants . Mixis
was inhibited at the initial step - production of mictic
females . Fertilization and male production were not as
sensitive to toxicant stress . The conclusion from this
work was that sexual reproduction is more sensitive to
toxic stress than asexual reproduction . This suggests
that harmless toxicant levels as determined by asexu-
al NOECs may not be protective of rotifer life cycles
since cyst production may be inhibited .
Carrying capacity has been used in two papers as an
endpoint for toxicity assessment. Rao & Sarma (1990)
examined the effect of DDT on food limited B. patu-
lus in static cultures that were renewed daily . A 25 ml
culture was inoculated with 40 rotifers and fed either
1 .5 or 3 x 106 Chlorella sp . cells ml -1 at 27°C . Popu-
lations exposed to 30 µg DDT/L at the low food level
went extinct after 7 days and at the high food level
after 15 days . Exposure to 20 µg 1 -1 caused extinction
in the low food treatment after 9 days, but the high
food level persisted with a K value not significantly
different from controls .
Work by Janssen (1992) also utilized K as an end-
point. B . calyciflorus were cultured in a 680 ml flow-
through vessel with 1/2 exchange per day . A screen
over the outlet retained the rotifers so that the popula-
tion became food limited . Cultures were held at 24°C in
the dark and fed 5 x 10 6 Nannochloris cells ml -1 . Cul-
tures were inoculated with 1 rotifer ml - ' and the densi-
ty measured daily . Populations were in lag phase days
0-2 . log phase days 3-8, and stationary phase after
day 9 until the experiment was terminated on day 30 .
The K values for controls averaged 70 rotifers ml - '
with coefficients of variation of 11-15% . Exposure of
populations to 2 .5 jig ml -1 copper had no significant
effect on K, but 10 pg ml -1 reduced K by 50% . The
LOECs for pentachlorophenol, dichloroaniline, and
lindane were 0 .4, 10, and 10 mg 1 -1 , respectively. The
NOECs from life tables using r as an endpoint were
very similar to those using K . Janssen concluded that
since the K experiment did not yield greater sensitiv-
ity than life tables, the greater duration and level of
effort required to complete the K experiment was not
warranted .
239
Behavior
There are advantages to using behavior as an endpoint
for assessing aquatic toxicity. Behavioral responses are
usually very rapid, occurring in minutes rather than
days as for traditional endpoints . Advances in video
microscopy and computer interfacing have made auto-
mated data collection for behavioral endpoints feasible
and quite cost-effective when compared to traditional
toxicity tests . The disadvantages of behavioral end-
points are that it is often unclear whether the effect is
truly adverse . To demonstrate toxicity an adverse effect
must be shown (Rand & Petrocelli, 1985) . However,
some behavioral responses might be temporary, with
test animals making a complete recovery after toxi-
cant exposure is terminated . Behavioral endpoints are
most useful when they can be directly related to widely
accepted adverse effects like reduced survival or repro-
duction . Such a connection is not easy to establish, but
failure to establish the link weakens the usefulness of
behavioral data .
In rotifers, two types of behavioral characteristics
have been used to detect toxicity : swimming activity
and feeding (Table 3) . Swimming activity has been
measured visually by counting the number of squares
entered by a test rotifer in a 30 second observation peri-
od (Snell et al., 1987) . This labor intensive approach
was used by Janssen et al. (1993, 1994) to assess the
effects of copper, pentachlorophenol, dichloroaniline
and lindane on the swimming behavior of B . calyci-
florus . Video motion tracking systems exist that can
perform this task automatically (Coulon et al ., 1983) .
These systems require video camera, computer, digitiz-
ing board, and software capable of making the desired
calculations . Since this equipment costs several thou-
sand dollars, the number of labs able to afford the tech-
nology is limited . As video technology becomes more
affordable, the quantitative analysis of rotifer behavior
is likely to expand as well as its application in toxicity
assessment .
Rotifer feeding behavior is an especially attractive
endpoint for toxicity studies . It can be rapidly and
easily quantified and it can be directly related to repro-
duction . Feeding behavior is typically measured by the
subtraction method which quantifies microalgae con-
centration at the beginning and end of a feeding inter-
val . This approach has been employed by Fernandez-
Casalderrey et al . (1992) and Ferrando et al . (1993) .
The latter authors used log-phase Nannochloris ocula-
ta at 5 x 105 cells ml -1 , quantified by hemocytometer
counts . The rotifer B . calyciflorus was obtained by


240

Table 3 . Toxocity assessment using behavioral endpoints . The end column indicates the endpoints ingestion rate
(ing) or swimming activity (swim) ; the exp column indicates the duration of toxicant exposure . NOEC is the no
observed effect concentration in mg I -1 and EC50 is the toxicant concentration where 50% inhibition occurs . All
data are for females .
Test species Chemical End Exp NOEC EC50 Reference
B. plicatilis free chlorine ing 30 m 1 .0 Capuzzo, 1979
chloramine 11 11
- 1 .0 "
benthiocarb ing 0 .3 - Hirata et al ., 1984
pentachlorophenol ing 60 m 0 .5 - Juchelka & Snell, 1995
phenol 250
cadmium 30
copper 0.1
mercury 0 .01
naphthol 8
chlorpyrifos 0.3 - "
free ammonia swim 15 m - 2 .3 Snell et al ., 1987
B . calyciflorus 3,4 dichloroaniline ing 5 h 30 41 .2 Ferrando et al., 1993
lindane 2 8 .5
pentachlorophenol 0 .5 1 .9
copper 0 .012 0 .034
phenol ing 30 m 250 - Juchelka & Snell, 1994
dimethyl phenol 6
xylene 30
cadmium 0 .25
chromium +6 1
mercury 0 .1
naphthol 0 .63
chlorpyrifos 0 .25
endosulfan ing 5h 1 .25 - Femandez-Casalderrey et al.,
1992
11
diazinon 1 .25 3 .11
copper swim 3h 15 6 Janssen et al., 1993
pentachlorophenol 2 .1 0.5 Janssen, 1992
dichloroaniline 45 .5 - "
lindane 16 .7 5

hatching cysts, and 30 females ml -1 were exposed in
5 ml with no rotation at 25 ° C . Control filtration and
ingestion rates were 5 .6 ± 0 .7 pl female -1 hr-1 and
1602 ± 142 cells female -1 hr 1 , respectively. Increas-
ing toxicant concentrations reduced these rates in a
dose-dependent manner. Rotifer densities of 10, 20,
and 30 ml -1 had no effect, but when female density
was 50 ml -1 , a 55% lower ingestion rate was observed .
Rotifers were allowed to feed for 5 hr, exposed to tox-
icant the entire feeding period. The problem with this
approach is that a 5 hr exposure far exceeds the gut pas-
sage time for B . calyciflorus. Starkweather & Gilbert
(1977) showed that ingested food passes through the
alimentary canal of this species in about 20 min . It is
especially important to set the feeding interval lower
than the gut passage time when radiotracers are used
to quantify feeding . Even when algal cells are indi-
vidually counted, it is possible that some algal cells
pass through the alimentary canal intact, resulting in
an underestimate of ingestion rate . For this reason,
and the high variance associated with hemocytometer
counts, other approaches to estimating toxicant effects
on feeding rate are advisable .
Another approach to quantifying rotifer ingestion
rate and its response to toxicity was described by
Juchelka & Snell (1994). These authors used fluores-
cently labeled, 2 pm microspheres and image analy-
sis to quantify ingestion in B. calyciflorus. Neonates
hatched from cysts were exposed to toxicants for 1 hr in
the absence of algae at 25 ° C . Microspheres were added

and rotifers were allowed to feed for 5 min. Feeding
was stopped by anesthetization with tricaine . Rotifers
were placed on a microscope slide and an image was
captured at 50 x magnification . An image analysis
system was used to quantify gut fluorescence which
permitted calculation of the ingestion rate . Ingestion
rates of the microspheres were similar to those report-
ed for live algae . Correlation between ingestion rate
and reproductive rate for 4 toxicants revealed good
correspondence between these parameters, supporting
the ecological significance of this endpoint .
Biomarkers
There has been substantial interest in cellular biomark-
ers as sublethal indicators of toxicity . Several
approaches have been taken with aquatic animals and
two have yielded promising results . The first charac-
terizes the abundance of stress proteins and the second
quantifies in vivo enzyme activity. The stress response
is a highly conserved alteration in gene expression
associated with a variety of stressors (Sanders, 1993) .
The rotifer B . plicatilis expresses a typical stress
response when exposed to heat or some chemical stres-
sors (Cochrane et al ., 1991) . These authors have quan-
tified the abundance of a 58 kD stress protein that is
part of the SP60 family of stress proteins (Table 4) . Ini-
tial experiments used labeling of all newly synthesized
proteins with 35 S-methionine to investigate which pro-
teins were up-regulated after mild heat shock . At 32°-
45°C, the synthesis of SP58 protein was increased
several fold over control levels at 25°C . SP58 abun-
dance was quantified after electrophoretic separation,
Western blotting, and probing with a heterospecific
polyclonal antibody for SP60s . Neonate B. plicatilis
females hatched from cysts were exposed to 25 pg
1` copper (10% of the 24-h LC50) for 18 h and a
5-fold increase in SP58 abundance was observed . A
similar exposure to 30 µg 1 -1 of tributyl tin (10% of
24-h LC50) resulted in a 4-fold induction of SP58 .
Exposure to several other toxicants failed to induce
SP58, including ZnC12, HgCl2, AlC1 3 , sodium arsen-
ate, sodium azide, sodium dodecyl sulfate, and pen-
tachlorophenol .
Cochrane et al. (1994) described another approach
to estimating changes in gene expression during s tress .
i n rotifers . Rather than protein abundance, they mea-
sured the amount of mRNA following stress . They
used the polymerase chain reaction (PCR) to ampli-
fy small segments of the SP60 gene . A 345 base
pair fragment was amplified and sequenced to con-
241
firm its relationship to the SP60 family. A method was
described to extract mRNA, bind it to a nitrocellulose
membrane, and hybridize it with a 32P labelled 345 by
probe obtained from PCR. The procedure is called a
dot blot which can be autoradiographed and the extent
of hybridization quantified . These techniques are now
being applied to investigations of toxicant stress in B.
plicatilis and B . calyciflorus .
The use of in vivo enzyme activity has been recog-
nized as a useful tool for characterizing the response
of aquatic invertebrates to toxicant stress (Janssen &
Persoone, 1993 ; Janssen et al ., 1993) . This technique
has been applied to rotifers using the esterase enzyme
(Moffat & Snell, 1994 ; Burbank & Snell, 1994) . The
general procedure is to hatch neonates from cysts and
expose them to a toxicant for one hour . Then a spe-
cial non-fluorescent substrate is added which releases
a fluorescent product upon cleavage by intracellular
enzymes . Rotifers readily take up the substrate from
the medium and fluorescence localizes at sites in the
rotifer where there is esterase activity . The fluorescence
produced the by esterase can be quantified using a flu-
orometer or by image analysis. The technique works
with B. plicatilis and B. calyciflorus, and other enzymes
like phospholipase A2 also can be quantified (Burbank
& Snell, 1994) .
Mesocosms
Since rotifers are one of the major zooplankton groups
in freshwater communities, it is not surprising that the
effects of various types of contaminants on their pop-
ulation dynamics has been studied intensively, in both
laboratory and field mesocosms . Taub et al . (1989)
proposed a standardized aquatic laboratory microcosm
test in which Philodina along with algae and crus-
taceans, all originating from laboratory monocultures,
are placed in 3 liter containers and exposed to various
concentrations of a test compound . At regular intervals
throughout the 60 day exposure period, the population
densities of each species are measured and the impact
of the compound on the different members of this gno-
tobiotic test system is evaluated . A similar experimen-
tal design, including 2 species of green algae, a fila-
mentous cyanobacterium, 5 species of bacteria, a cili-
ate protozoan, an aquatic oligochaete, and two rotifers
(Phidodina and Lepadella) were used to screen the tox-
icity of 6 environmental contaminants (Sugiura, 1992) .
Various outdoor multispecies test systems have also
been described . Recently, the impact of mixtures of the


242

Table 4 . Summary of toxicity studies with rotifers using cellular biomarkers . The end column indicates the
endpoints abundance of the 58 kD stress protein (SP58) or esterase enzyme activity (ester) ; the exp column
indicates the duration of toxicant exposure, NOEC is the no observed effect concentration in mg 1 - I and
EC50 is the toxicant concentration where 50% inhibition occurs . All data are for females .
Test species Chemical End Exp NOEC EC50 Reference
B. plicatilis copper SP58 18 h 0.013 - Cochrane et al., 1991
tributyl tin 11
0.01
pentachlorophenol ester 60 m 0.3 - Moffat & Snell, 1994
tributyl tin 0 .012
copper 0 .01
mercury 0 .02
zinc 2 .5
free chlorine 1 .1
B. calycii lorus pentachlorophenol ester 45 m 0 .1 - Burbank & Snell, 1994
phenol 5
dimethyl phenol 120
naphthol 30
xylene 120
cadmium 0 .025
copper 0.025
mercury 0.005
diazinon 5
chlorpyrifos 2

agricultural pesticides atrazine and bifenthrin on fresh-
water community responses were studied by Hoagland
et al . (1993) . From the experiments conducted in 5500
liter tanks containing natural plankton assemblages and
a fish species (bluegill), these workers concluded that
the population density of various rotifer species after
an initial increase, was adversely affected by atrazine
and bifenthrin concentrations of 156 and 287 jig I -t ,
respectively . Similarly, Liber et al. (1992) examined
the effects of a commercial formulation of 2,3,4,6-
tetrachlorophenol (Diatox) on zooplankton abundance
in a set of mesocosms . They concluded that several
zooplankton species were adversely affected at 1 .5 mg
1 -t Diatox and that rotifers were more sensitive than
the macrozooplankton. Most published field studies
examine the effects of pesticides because of the envi-
ronmental concerns about the toxicity and persistence
of these products, their strict regulation, and manda-
tory field testing . A concise, non-exhaustive overview
of recent reports using rotifers in mesocosm studies is
given in Table 5 . Havens (1992a, b) used in situ meso-
cosm experiments to assess the impacts of acidification
on the function and structure of algal and zooplank-
ton communities . He observed decreasing zooplank-
ton biomass and productivity with decreasing pH . At
pH levels of 5 .5-4 .5, nearly all crustacean herbivores
became extinct and only cyclopoids and rotifers per-
sisted .
Natural populations
Numerous studies have been published assessing the
effects of various types of pollution on natural zoo-
plankton communities . It is beyond the scope of this
review to summarize all of these works . A few exam-
ples of recent work in this area are given below .
Rotifers have been used as indicators of general water
quality by Ramadan et al. (1963), Sladecek & Tucek
(1975), and Sladecek (1983) . Changes in zooplank-
ton community structure caused by acidification of
Adirondack lakes (New York, USA) was studied by
Siegfried & Sutherland (1992) . These authors conclud-
ed that increasing lake acidity generally reduced the
number of zooplankton species present . The density
of two generalist species, however, one of which was
the rotifer Keratella taurocephala, increased in rela-
tive importance in the acidic lakes . Keller et al. (1992)
investigated the changes in zooplankton composition
during experimental neutralization and early reacid-
ification of an Ontarian lake (USA) . They observed
decreased abundance of the acidophilic rotifers K


243

Table 5 . Summary of mesocosm studies with rotifers .

Chemical/type of Type of study Rotifers endpoint Reference
pollution (NOEC-LOEC', range
tested2 )
Atrazine & bifenthrin outdoor ponds abundance Hoagland et al., 1993
mixture (5500 1 tanks) 2 (15-2167 µg 1 -1
atrazine and 15-
3150 ng 1- 1
bifenthrin)
Acidification in situ enclosures biomass and Havens, 1992a
(addition of H2SO4) productivity
'(pH 5 .5-4 .5)
Acidification in situ enclosures mean plankton size Havens, 1992b
(addition of H2SO4) '(pH 5 .5-4 .5)
Polypeptone laboratory abundance, species Sagiura, 1992
multispecies test elimination
2 (100-500 mg 1 -1 )
11
Cu '(0 .4 mg 1 -1 )
Acitic acid (2,4,5-T)
11

DDT
11
Lindane (alfa &
gamma)
Tetrachlorophenol limnocorals abundance Liber et al ., 1992
(diatox) '(0 .75-1 .5 mg 1 -1 )
Fenvalerate in situ enclosures abundance Day et al ., 1987
(125 m 3 ) 1 (0 .05-01 µg 1 -1 )
organic piscicide outdoor tanks abundance Jana et al., 1987
(Brassica latifolia)

taurocephala and Ploesoma lenticulare and increased
abundance of K. cochlearis, Polyarthra sp ., and other
species after neutralization . Four years after neutral-
ization, however, K. taurocephala densities increased
as average lake pH decreased to approximately 5 .5 .
The effects of eutrophication on zooplankters in
general, and rotifers in particular, have been inves-
tigated in a long-term study by Maley et al . (1988) .
From 1969 to 1975 they observed a marked decline in
both crustacean and rotifer biomass and species diver-
sity after fertilization of an experimental oligotroph-
ic lake . Balvay & Laurent (1990), however, noted
an overall net increase in rotifer abundance resulting
from an increase in eutrophication-tolerant species and
colonization by new species, despite the disappear-
ance of eutrophication-sensitive species . Green (1993)
examined rotifer diversity and dominance in freshwa-
ter communities and devised means to relate domi-
nance to environmental stress and pollution .
Critique of rotifer use in ecotoxicology
In general, the performance of ecotoxicity tests is mea-
sured by their ecological relevance, reproduciblity,
reliability, robustness, and sensitivity (Calow, 1993) .
The ecological relevance of rotifers as test animals
was discussed in the introduction . The reproducibility
of acute tests with B. calyciflorus and B . plicatilis has
been extensively documented . For example, Snell et
al . (1991a) tested 28 chemicals on B. calyciflorus and
found intralaboratory coefficients of variation ranging
from 2 .4 to 49% and averaging 10-15% for repeated
tests . Similar results were obtained for B. plicatilis,
with coefficients of variation of 1 .7 to 28% (Snell et
al ., 1991b) . These high intralaboratory reproducibil-
ities have been confirmed by other workers (Ferran-
do & Andreu-Moliner, 1991 ; Janssen et al ., 1993 ;
Calleja et al ., 1993) . Interlaboratory reproducibility
was examined in a large intercalibration exercise using
cyst-based tests (Persoone et al ., 1992) . The coefficient
of variation for copper tested by > 170 labs in Europe
244
and North America ranged from 28-68%, which is
comparable to that found in much smaller intercalibra-
tion exercises with other invertebrates .
Reliability in ecotoxicological terms is the ability to
set up a test at will using test animals of known quality
and to successfully complete it with high probablity
(Calow, 1993) . The use of rotifers hatched from cysts
makes rotifer tests among the most reliable of any
aquatic invertebrate, because problems associated with
culturing of test animals are eliminated . The costs of
cyst-based tests are up to 50% less than conventional
tests (Persoone, 1991) . The commercial availability of
B. calyciflorus and B . plicatilis cysts has promoted the
use of rotifers in ecotoxicology . In fact, the majority
of papers cited in this review used cysts as starting
material .
Factors contributing to the robustness of rotifer
acute tests include small size which requires small
sample volumes and bench space, year-round avail-
ability of test animals (cysts), and highly standardized
test procedures.
A major consideration for the acceptance of a new
test is its comparative sensitivity with conventional
tests . Snell et al. (1991b) compared the sensitivity of
the acute B. plicatilis test with four marine tests for five
metals and pentachlorophenol. They concluded that the
rotifer had comparable sensitivity to most compounds,
but no single species was consistently the most sen-
sitive to all compounds . A similar comparative study
of B. calyciflorus, Daphnia magna, and Pimephales
promelas (fathead minnow) revealed that the LC50s of
the two invertebrates were within one order of mag-
nitude for 8 of the 12 compounds tested (Snell et al .,
1991 a) . Likewise, B. calyciflorus and P promelas dif-
fered by less than an order of magnitude for 9 of 12
compounds . Based on the test of 99 environmental
samples (effluents, solid wastes, sediments), Persoone
et al. (1993) showed that the cyst-based acute test with
B. calyciflorus was in 45% of the cases as sensitive and
in 17% more sensitive than the conventional D . magna
test. A similar comparison between the rotifer test and
the bacterial Microtox test was made for 250 samples .
The Microtox test was more sensitive than the rotifer
test in 68% of the cases and as sensitive in 10% of
the cases . However, comparative sensitivity does not
only mean `more or less sensitive than', but also the
magnitude of the difference and the toxicity detection
capacity should be taken into consideration (Persoone
et al., 1993) .
In conclusion, the mortality and reproduction tests
with B . calyciflorus and B . plicatilis seem to fulfill all of
the requirements to be useful in ecotoxicological test-
ing. Cyst-based rotifer acute tests with these species are
already accepted as standard methods (ASTM, 1991) .
Other protocols based on reproduction, behavior, and
cellular biomarkers have been published and are being
used by several labs . The scientific basis for the accep-
tance of rotifers as part of a battery of ecotoxicological
tests is well established .
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