Environmental Toxicology and Chemistry, Vol. 27, No. 11, pp. 2397–2402, 2008䉷 2008 SETAC
Printed in the USA
0730-7268/08 $12.00 ⫹ .00

Hazard/Risk Assessment
STAGE-DEPENDENT DIFFERENCES IN EFFECTS OF CARBARYL ON POPULATIONGROWTH RATE IN JAPANESE MEDAKA (ORYZIAS LATIPES)SHOSAKU KASHIWADA,*†‡ HARUKI TATSUTA,† MAIKO KAMESHIRO,†㛳 YOSHIO SUGAYA,†TARA SABO-ATTWOOD,‡ G. THOMAS CHANDLER,‡ P. LEE FERGUSON,§ and KOICHI GOKA††Environmental Risk Research Center, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan‡Department of Environmental Health Sciences, Arnold School of Public Health, §Department of Chemistry and Biochemistry,
University of South Carolina, Columbia, South Carolina 29208, USA
㛳The Institute of Environmental Toxicology, Uchimoriya-cho 4321, Johso, Ibaraki 303-0043, Japan( Received 12 February 2008; Accepted 22 April 2008)
Abstract—Fish embryo toxicology is important because embryos are considered more susceptible than adult fish to the effects of
toxic chemicals. Recently, fish embryo bioassay was proposed to replace the conventional fish acute toxicity chemical test of theOrganization for Economic Cooperation and Development guidelines because it offers the advantages of fewer reagents, easy
handling, and efficient data production. To accelerate the establishment of a chemical toxicity database for the protection ofenvironmental and human health, we need to determine whether the conventional toxicity test can safely be replaced by such fishembryo toxicity tests. For instance, it is unclear how the presence of the chorion moderates the toxic effects of some chemicals.
If such chemical toxicities do differ between embryos and, for example, the larval stage, then different toxic effects should appear
in later life. We tested the later-life effects of the neurotoxic insecticide carbaryl at sublethal concentrations (0 [control] and 5 and
10 mg/L) in embryos and posthatch larvae of the freshwater fish medaka, Oryzias latipes. Although embryos exposed until hatching
showed multiple developmental malformations and reductions in subsequent survival rates over three months, no significant reduction
was observed in tolerance to starvation for 7 d and in intrinsic population growth rate (r). Exposure of larvae for 96 h resulted indose-responsive vertebral fracture, significant reduction in tolerance to starvation for 7 d, and reduced three-month survival rate;r was reduced significantly and consistently. These results suggested that posthatch larvae were more susceptible than embryos tocarbaryl exposure and that the toxic cascades may differ between larvae and embryos. The influences of carbaryl exposure on
population growth rate differed significantly with developmental stage.
Keywords—Carbaryl

MedakaPopulation growth rate

adult acute toxicity test. Very limited data sets are available onother fish—namely, the medaka (Oryzias latipes) and fatheadminnow (Pimephales promelas) [3]. This alternative fish embryo toxicity test is being run under Registration, Evaluation,
Authorization, and Restriction of Chemicals in the European
Union (http://ec.europa.eu/environment/chemicals/reach/reach㛮
intro.htm) to accelerate the toxicity assessment of chemicals
because there are 30,000 existing hazardous chemicals that need
toxicity testing [3]. It is important that a toxicity database be
established to characterize the effects of chemicals on either
embryos or adult fish; however, toxicologists have failed to use
simplified individual toxicity data such as the median lethal
concentration (LC50) and ecological indicators (i.e., uncertainty
factors) to assess the ecological risks of chemicals [4]. Acuteand chronic toxicity may reduce the intrinsic growth rate of a
population and may therefore increase the probability of population extinction. However, there is no scientific consensus on
the extrapolation of individual lethal toxicity data to intrinsic
population growth rates. If we are to successfully develop more
ecologically relevant risk assessments of hazardous chemicals,
then it is important that we use indices for assessing population
dynamics such as intrinsic population growth and carrying capacity [4].
If embryo toxicity data are to be used instead of conventional fish toxicity data, then we need to understand the commonalities and differences between embryo and larval toxicity
tests because there are likely physiological and toxicological
gaps among embryos, larvae, and adult fish. For example, the

INTRODUCTION

Toxicity testing in adult fish is currently performed worldwide in accordance with the Organization for Economic Cooperation and Development (OECD) chemical test guideline 203[1]; however, earlier life stages, such as the embryo and larva,
are known to be more sensitive to chemicals. Because chemical
exposure in the early life stages can cause vital and irreversible
damage in later lives, we need to understand the toxic effects
of chemicals on fish embryos and larvae if we are to successfully
regulate hazardous chemicals on the market and manage theirecological risks. Fish embryo toxicity testing is recognized asimportant because it enables the use of microplates; this means
fewer reagents, easier handling, and data production with higher
efficiency than with conventional testing. Recently, the German
government nationally standardized a 48-h sewage-testing assay
in zebrafish (Danio rerio) embryos and submitted it for standardization to the International Organization for Standardization[2; http://www.iso.org/iso/iso㛮catalogue/catalogue㛮tc/catalogue㛮
detail.htm?csnumber⫽37368]. Furthermore, a modified fish embryo test for the regulation of commercial chemicals has been
submitted by the German government to the OECD as an alternative to the conventional acute fish toxicity test [3]; the new
test is based on the reliable correlation of a single data set
between the zebrafish embryo toxicity test and the conventional* To whom correspondence may be addressed(shosaku@gwm.sc.edu).
Published on the Web 5/22/2008.
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S. Kashiwada et al.

Table 1. Effects of carbaryl exposure on development of medaka embryos
Concn. of carbaryl
Biomarker
Tubular heart (%) at embryo development stage 37a
Blood clots in ducts of Cuvier (%) at stage 37
Pericardial edema (%) at stage 37
Heart rate per 15 s at stage 37
Hatch rate (%)
Time to hatch (days)
Spinal deformity (%)
Death rate 24 h posthatch (%)
Body length of posthatch embryos (mm)abc

0 mg/L (control)

5 mg/L

10 mg/L

0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
34.4 (0.6)
94.7 (2.5)
8.7 (0.2)
0.0 (0.0)
5.4 (2.5)
4.3 (0.03)

14.8 (6.4)
0.0 (0.0)
0.0 (0.0)
32.7 (0.7)
100 (0.0)
8.9 (0.3)
2.7 (2.7)
9.3 (3.4)
3.7 (0.02)c

41.2 (14.2)b
1.4 (1.4)
11.1 (4.4)
26.6 (0.4)c
91.9 (3.3)
9.3 (0.2)
5.8 (2.7)
6.8 (3.7)
3.4 (0.02)c

Embryo development stage 37 is appropriately 6 d postfertilization, as judged in the control [18]. Standard errors are in parentheses.
Analysis of variance versus control: p ⬍ 0.05.
Analysis of variance versus control: p ⬍ 0.001.

liver—a typical target organ—is not developed well enoughto protect against xenobiotics in embryos and larvae. Because
fish embryos have a chorion to protect themselves, they areless sensitive to chemical exposure than are dechorionatedembryos and larvae [5,6]. The chorion plays a protective role
against chemical exposure, and the barrier function of the egg
chorion increases with the lipophilicity of the chemical [3]. In
heavily contaminated aquatic environments, aquatic organisms
would be considered damaged in terms of viability and longevity compared with those in a reference area. Several studies
have revealed the impacts of pesticides on behavioral and life
history traits. For instance, carbaryl and other neurotoxic pesticides decrease swimming ability in rainbow trout [7] andincrease the vulnerability of the trout to predation [8]. Carbaryl
is a known aquatic environmental contaminant that has beenused extensively in agriculture for pest control [9] as a neurotoxic acetylcholinesterase and cholinesterase inhibitor[8,10]. We therefore consider it a suitable model chemical forfish ecotoxicological studies. Although the modes of action of
carbaryl are well known, risks to fish populations are still
unclear. We examined whether sublethal exposure of Japanese
freshwater fish medaka (O. latipes) embryos or larvae to carbaryl caused different reductions in later viability and population growth rate.
MATERIALS AND METHODS

Collection of fertilized eggs and hatched larvae of medaka
The small laboratory fish species medaka (O. latipes) has
a small body (3–4 cm in adults), a hardy nature (wide temperature and salinity tolerances), and a short generation time
(two to three months); therefore, it is frequently used to investigate waste water toxicology [11], endocrine disruptors[12], liver carcinogenesis [13], germ cell mutagenesis [14],gene mutagenesis [15], and developmental and functional genomics [16]. The medaka orange-red strain that we used was
obtained from broodstock at the National Institute for Environmental Studies (Ibaraki, Japan). Details of cultivation conditions are given in our previous publications [17,18]. Briefly,
breeding groups of medaka were fed Artemia salina nauplii
twice daily and maintained under a 16:8-h light:dark cycle at
26 ⫾ 0.5⬚C in a culture room. After female medaka had
spawned eggs, the external egg clusters were removed. Fertilized eggs were selected and rinsed and then placed in embryo-rearing medium (ERM) [19]. Eggs in development stage
10 (early blastula stage) [20] were isolated and subsequently
used for exposure. Medaka larvae were obtained by the in-cubation of embryos at 26 ⫾ 0.1⬚C in an incubator (Multi
Thermo Incubator MTI-202, Tokyo Rikakikai, Tokyo, Japan)
under a 16:8-h light:dark photoperiod until hatching and subsequently used for exposure. Fish and eggs were treated humanely and with regard for the alleviation of suffering.

Exposure of embryos and larvae
Carbaryl (methyl carbamic acid-1-naphthyl ester, technicalgrade purity ⬎99%) was purchased from Wako Pure Chemical
Industries (Osaka, Japan). The U.S. Environmental Protection
Agency database (AQUIRE, http://cfpub.epa.gov/ecotox) reports the lethal concentration of carbaryl to be approximately
20 mg/L in temperate fish. The sublethal concentrations that
we used were defined as 5 and 10 mg/L. Carbaryl was dissolved in dimethylsulfoxide (Wako) to 0, 5, and 10 mg/ml as
stock solutions, and then each stock solution was diluted with
ERM at a ratio of 1:1,000 to make 0, 5, and 10 mg/L, respectively, of carbaryl–ERM solution. The concentration ofdimethylsulfoxide in each carbaryl solution was 0.1% (v/v).
Five milliliters of each carbaryl solution (0, 5, and 10 mg/L
carbaryl) were prepared in five glass scintillation vials (20-ml
volume), to each of which 15 egg embryos were added andexposed until hatching or 14 d. For the larval exposure experiment, five 100-ml solutions of carbaryl at each concentration (0, 5, and 10 mg/L) were prepared in five glass dishes(volume 200 ml, diameter 100 mm, depth 50 mm), to each ofwhich 15 larvae were added and exposed for 96 h. Embryos
and larvae were incubated at 26 ⫾ 0.1⬚C in an incubator (Multi
Thermo Incubator MTI-202) under a 16:8-h light:dark photoperiod. Test solutions were renewed daily. Embryos and larvae were exposed simultaneously.

Estimation of exposure effects and postexposure incubation
Embryos were observed daily through the glass vials undera dissecting microscope (Olympus SZ-ET, Tokyo, Japan). To
assess embryo toxicity, a number of biomarkers—percent of
occurrences of tubular heart, blood clots in the ducts of Cuvier,and pericardial edema in embryos; heart rate, hatch ratio, andtime to hatch in embryos; and percent occurrence of spinal
deformity, death rate, and body length in posthatch larvae—
were measured and summarized (Table 1). Posthatch larvae
were anesthetized with ice-cold water, and their body lengths
were immediately measured under a dissecting microscope
equipped with a micrometer. After measurement, all larvae(five groups of 15 larvae per embryo treatment) were immediately moved to fresh ERM (26⬚C), which was aerated for 5

Carbaryl exposure and medaka population growthmin. After all larvae had been confirmed to be alive, eachgroup was moved to 200 ml of fresh ERM in the respective
glass dishes and fed with a rotifer, Brachionus urceolaris, oncea day for 3 d and then with A. salina nauplii. At 7 d posthatch,the larvae were moved to 500-ml acrylic resin aquariums (90⫻ 150 ⫻ 90 mm, height ⫻ width ⫻ depth) in a medaka culture
system (Small Fish Culture System Type Meito-Hikosaka),
Meitosuien, Nagoya, Japan) and fed with A. salina nauplii
three times a day for three months until the medaka reachedsexual maturity. The numbers of surviving medaka were recorded at 30, 60, and 90 d posthatch. Tap water, which was
dechlorinated with an activated carbon filter and temperature
controlled to 26 ⫾ 1⬚C, was supplied (sodium ions 19.3 mg/
L, chloride ions 32.7 mg/L, hardness 76 mg/L, total organiccarbon 0.9 mg/L, pH 7.8) to the medaka culture system.
For the larval exposure experiments, larvae were observed
daily under the dissecting microscope to detect any morphological changes, and mortalities were recorded daily during
the 96 h of exposure. After the exposure period, the larvae
were rinsed with fresh ERM and moved to the medaka culture
system. The number of surviving medaka was recorded at 30,
60, and 90 d posthatch.

Impact of carbaryl on growth of medaka populations
To evaluate the impact of the different carbaryl exposures
on medaka populations, we first estimated r, a summary index
that represents the ability of each population to proliferate.
The index r was estimated by fitting the life table data foreach exposure treatment to the Euler–Lotka equation [21]. The
equation is冘lme ⫽1
t

t

⫺rt

t

where t is age in days, lt is survivorship until age t, and mt is
per-capita fecundity. Since females began to spawn eggs at
age around 75 d posthatch, we used the number of eggs from91 to 105 d posthatch for estimating r. The number of newborn
females is usually taken as mt, but in our experiment we did
not know the exact number of female newborns. Therefore,
we extrapolated the sex ratio data from the exposure experiment (i.e., estimated number of newborn females ⫽ numberof hatched individuals ⫻ sex ratio) and the fecundity of eggs
produced by each replicate in order to estimate mt. The estimated sex ratio was 1:1.283 male to female.
Five mating pairs were randomly selected from each treatment at 90 d posthatch and incubated for two weeks. We thenmeasured the number of eggs spawned and fertilized and obtained the fertilization ratio, and we calculated the arithmetic
mean of fecundity over the pairs in order to estimate mt.
Data were analyzed using Dunnett’s test to evaluate theimpact of carbaryl at each concentration compared with thecontrol at each developmental stage. Subsequently, two-way
analysis of variance (ANOVA) was used to examine the impacts of carbaryl on all biomarkers and r. All calculations
were performed by using a code developed for the statisticalprogram R (http://www.R-project.org).

Effects of carbaryl on embryo osteogenesis and larvalvertebrae
To obtain information on the effects of carbaryl exposure
on osteogenesis (as demonstrated by changes in body length)and on the vertebrae of medaka, additional carbaryl exposuresof the same series of medaka embryos and larvae were per-Environ. Toxicol. Chem. 27, 2008

2399

formed. Larvae hatched from embryos exposed to carbaryl
and directly exposed larvae were immersed in 0.02% calcein(3,3⬘-bis[N,N⬘-bis(carboxymethyl)aminomethyl]fluorescein,
Dojindo Molecular Technologies, Kumamoto, Japan) solution
for 10 min and then rinsed in fresh ERM for 30 min. The
stained larvae were anesthetized with ice-cold water, and wethen observed the effects of exposure on osteogenesis andvertebral structure under a fluorescence microscope (Leica MZ
FL III, Leica Microsystems, Tokyo, Japan) equipped with agreen fluorescent protein filter (wavelength range: excitation
480 nm, emission 510 nm). Fluorescence images were captured
for 100 ms with a digital camera (Leica DC 350FX, Leica
Microsystems) attached to the microscope. To identify the
body shape and skeleton from the fluorescence, regular lightimages were also captured for 5 ms, and both images wereoverlaid. The captured fluorescence was pseudocolored withyellow-green by using the Leica FW 4000 software (Ver 1.0.3,
Leica Microsystems).

Effects of carbaryl on larval tolerance to starvation
To obtain information on the effects of carbaryl exposure
on larvae viability under altered environmental conditions, additional carbaryl exposures of the same series of medaka embryos and larvae were performed. Larvae hatched from embryos exposed to carbaryl and directly exposed larvae were
subjected to a starvation tolerance test. Larvae posthatchedfrom exposed embryos were moved to five 200-ml solutionsof ERM, each in a glass dish (diameter 100 mm, depth 50mm) and reared for 7 d without feeding. Larvae exposed tocarbaryl had already been subjected to 96 h (4 d) of starvation
during the exposure period, so they were reared without foodfor only an additional 3 d under fresh and clean conditions tocomplete the 7-d starvation test. After the starvation, the survival rates were measured.
RESULTS

Toxicities on medaka embryos and larvae and survival
With exposure of embryos to carbaryl at 10 mg/L, there
was a significant ( p ⬍ 0.05) increase in the rate of formationof tubular heart, a significant ( p ⬍ 0.001) reduction in heartrate, and a significant ( p ⬍ 0.001) shortening of body lengthcompared with those in the controls; however, there was noeffect on hatch rate (Table 1). Tubular heart formation andheart rate reduction are typical phenomena that occur in medaka embryos in response to chemical exposure [22–24]. Fluorescence microscopy with calcein staining was used to examine
the shortening of body length in posthatch larvae due to disruption of osteogenesis as a result of carbaryl exposure during
the embryo stage (Fig. 1). Fractured vertebrae were observedin medaka larvae exposed to carbaryl solutions for 96 h (Fig.
2A and B), although there were no lethal effects (Table 2).
Vertebral fracture was caused by dislocation due to muscle
cramping (Fig. 2C), and the rate of fracture increased with theexposure dose (Table 2). Shortened body length and fractured
vertebrae disappeared in the adult stage (data not shown).
Carbaryl exposure at the embryo stage had no influence ontolerance to starvation after hatching; however, tolerance wassignificantly ( p ⬍ 0.001) reduced when fish were exposed at
the larval stage (Table 3). Although the exposure period at theembryo stage was much longer than that at the larval stage,
the embryos were less susceptible than the larvae to carbaryl
exposure in terms of starvation.

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S. Kashiwada et al.
Table 2. Rates of survival and vertebral fracture in medaka larvae
exposed to carbaryl for 96 haConcn. ofcarbaryl (mg/L)
0 (control)
5
10
a
b

Total nSurvival (%)

Vertebralfracture (%)

75
75
75

100 (0.0)
100 (0.0)
100 (0.0)

0.0 (0.0)
8.4 (2.5)b28.9 (6.4)bStandard errors are in parentheses.
Analysis of variance versus control: p ⬍ 0.001.

sponse of r was greatly dependent on medaka developmental
stage and carbaryl dose rate.
DISCUSSION

Fig. 1. Morphological effects of carbaryl on medaka osteogenesis.
Posthatch larvae were stained with 0.02% calcein solution immediately after hatching. (A) Larva hatched from control embryo, showinga, pigmentation (indicated by diagonal arrow) on tail; b, neural spines(indicated by two downward arrows); c, vertebral centrum; d, uroneural; e, urostyle; f, parhypural (indicated by a arrowhead); and g,
hypural. (B) Larva hatched from embryo exposed to 5 mg/L carbaryl.
(C) Larva hatched from embryo exposed to 10 mg/L carbaryl. Osteogenesis was disrupted in tails of larvae hatched from embryos
exposed to carbaryl.

Ninety-day survival of medaka was reduced by carbaryl
exposure. The rates were 77.5, 60.8 ( p ⬍ 0.05, in comparison
with control), and 55.9% ( p ⬍ 0.005) for exposure to 0, 5,
and 10 mg/L carbaryl, respectively, at the embryo stage and90.7, 73.3 ( p ⬍ 0.05), and 74.7% ( p ⬍ 0.05) for the same
exposures at the larval stage. Carbaryl exposure had no effecton sex ratio, which was 1:1.283 male to female.

Impacts of carbaryl on population growth rate
The mean r was significantly different among treatmentsin the case of exposed embryos (one-way ANOVA, F ⫽ 38.18,
p ⬍ 0.001). Dunnett’s tests showed that mean r was significantly lower than in controls when embryos were exposed to
5 mg/L carbaryl (mean absolute difference ⫽ 0.014, p ⬍ 0.05),
whereas it was significantly higher than in controls when embryos were exposed to 10 mg/L carbaryl (mean absolute difference ⫽ 0.076, p ⬍ 0.001). Least-squares regression analysis
revealed that there was a significant positive relationship between dose rate and r (F ⫽ 38.184, p ⬍ 0.001; Fig. 3). Thus,
the impact of carbaryl on r on medaka embryos varied withthe concentration.
In contrast, a significant and constant reduction in mean r
was found among treatments in the case of exposure of medaka
larvae (F ⫽ 34.383, p ⬍ 0.001; Fig. 3). Dunnett’s test indicatedthat the difference in mean growth rate between controls andfish exposed as larvae to 5 mg/L was not significant (meanabsolute difference ⫽ 0.051, p ⫽ 0.35), whereas a significant
difference was detected between the controls and fish exposedas larvae to 10 mg/L carbaryl (mean absolute difference ⫽0.300, p ⬍ 0.001). Two-way ANOVA demonstrated that bothof the main factors—dose rate and developmental stage—hadsignificant effects on r (Table 4). The interaction of these two
factors had a highly significant effect, showing that the re-

We examined whether exposure of embryonic and larval
medaka to carbaryl at sublethal dose rates led to different
reductions in later viability and reproduction. We demonstrated
that carbaryl has growth stage–dependent influences on medaka embryos and larvae. Medaka larvae were more susceptible to the effects of carbaryl than were larvae exposed asembryos in terms of the exposure-starvation test and population growth rate. Embryos exposed to carbaryl at sublethal
concentrations exhibited increased rates of tubular heart, bloodclots, and pericardial edema and a reduction in heart rate,increased rate of spinal deformity, and shortened body length.
These abnormalities have been reported in similar embryo
stages, including in other fish species [24–26]. Weis et al.
reported that spinal deformity caused by carbaryl was morecommon when fish were exposed at the blastula stage than in
the early eight- to 16-cell stages [25,26]. Gonzáles-Doncel etal. also reported that the organogenesis stage of the medaka
embryo was more sensitive than the early stages to permethrin
exposure [22]. Sensitivity to carbaryl appeared to depend on
the developmental stage of the medaka embryos. However, thebiological damage observed after exposure in the embryo stage
was not as severe as that after exposure in the larval stage interms of subsequent starvation testing and especially intrinsic
population growth rate. Interestingly, larvae were more susceptible in terms of these two parameters even though theperiod of larval exposure was only 96 h. In freshwater adult/
immature fish, carbaryl has been reported to cause energy
crises and disruption of cholesterol metabolism [27,28] and to
decrease glycogen and protein concentrations in the muscles
and liver [29].
The results in our present study indicate that the yolk plays
a role in combating exposure damage. The posthatch medaka
larva consumes the yolk more rapidly than does the prehatch
embryo, almost exhausting it almost within 72 h posthatch at
26⬚C (data not shown). If medaka larvae were exposed tocarbaryl and damaged, they would become emaciated by exhausting the yolk to mitigate the damage from chemical stress;
subsequently, they would fall into a state of energy deficit
because at this time they are not exotrophic (i.e., they are stillobtaining nutrition from the yolk alone) and do not have anabundance of nutrients that can help them combat chemical
damage. The larvae therefore had less starvation tolerance thanthe embryos (Table 3), and subsequence effects may eventually
have substantially reduced their r (Fig. 3). Our statistical analyses indicated that the response of r differed significantly withgrowth stage (Table 4). These results suggested that exposureof embryos to carbaryl did not necessarily reduce their population numbers. In contrast, we can expect that the suscep-Environ. Toxicol. Chem. 27, 2008

Carbaryl exposure and medaka population growth2401

Fig. 2. Vertebral fracture in medaka larva exposed to carbaryl. Posthatch medaka larvae were exposed to 10 mg/L carbaryl for 96 h. (A) Control,(B) carbaryl-exposed, and (C) a partial enlarged image from B (white arrowhead indicates vertebral fracture). Exposed larvae were stained with
0.02% calcein solution and observed under a fluorescence dissecting microscope (B and C). Images B and C are overlapping images of regularlight and fluorescence images.

tibility of medaka larvae would have a longitudinal negative
influence on future population growth.
The European Union REACH legislation proposes the bioassay of embryos of zebrafish, medaka, and fathead minnow
as an alternative to fish toxicity testing [3]. Use of zebrafish
embryos for acute toxicity testing has advantages in terms of
cost-, space-, and time-effectiveness because zebrafish can produce greater numbers of embryos than medaka and complete

their hatching within 2 or 3 d (shorter than the 7–10 d required
for medaka). The new zebrafish embryo assay will become analternative to the conventional fish acute toxicity test. Conventional LC50 data have been applied to assessments of theecological risks of hazardous chemicals; hence, LC50 data onembryos will be used as well. It is therefore important that wedemonstrated here that the toxic effects from carbaryl exposure

Table 3. Growth-dependent effects of carbaryl exposure on toleranceof larvae to starvation (7 d)Tolerance

Carbaryl
concn.
(mg/L)

Tolerance to starvation 0 (control)(% survival rate after
5
7 d)
10

Exposure at
Embryo stagea

Larval stageb

95.3 (2.9)
93.9 (1.6)
96.5 (2.1)

98.7 (1.3)
85.3 (5.3)c
81.3 (3.9)da

Embryos were exposed to carbaryl from immediately after fertilization until hatch. Hatched larvae were immediately moved to freshand clean water conditions and kept for 7 d without feeding.
b Immediately after hatching, larvae were exposed to carbaryl for 96
h without food and then moved to fresh and clean water, where they
were kept for a further 3 d without feeding (total 7 d starvation).
Standard errors are in parentheses.
c Analysis of variance versus control: p ⬍ 0.05.
d Analysis of variance versus control: p ⬍ 0.001.

Fig. 3. Population-level effects of carbaryl exposure on medaka exposed as embryos and larvae as measured by the intrinsic rate ofnatural increase.

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Table 4. Two-way analysis of variance table: comparison of the effects
of developmental stage and carbaryl dose level on intrinsic growthrate, r
Factor

df a

MSb

Fc

pd

Dose
Stage
Dose ⫻ stage

2
1
2

0.03590.0286
0.3397

6.787810.818564.22780.00460.0031⬍0.0001a

Degrees of freedom.
Mean squares.
c F statistic.
d Probability of significance.
bof medaka at the embryo stage were lower than from larval
exposure in terms of population growth rate. Carbaryl exposure of embryos and larvae led to different ecological scenarios. The early establishment of a toxicity database to characterize the effects of chemicals on adult fish or embryos istherefore important; it is also important that we pay attention
to population dynamics in the ecological risk assessment of
hazardous chemicals.
Acknowledgement—We thank John D. Stark, Department of Entomology (Washington State University, WA, USA), for his criticalreview. We also thank Yutaka Ogamino, Fusae Oyama, Satomi Karube, Sachie Kawakami, and Kiyoshi Kawabe from Kawakami Agricultural Business, Japan (Tsukubamirai, Ibaraki, Japan), and Mie
Shibuya and Kayoko Shikishima of the National Institute for Environmental Studies (NIES), Japan, for their technical support of thisresearch at NIES. This research was partially supported by the Research-Promoting Grant from NIES and by the U.S. Environmental
Protection Agency Science to Achieve Results (STAR) program(award R-83-2737). It has not been subjected to NIES and U.S. EPA
peer and policy review and therefore does not necessarily reflect theirviews, and no endorsement should be inferred.
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