two-generation toxicity study on the copepod model species tigriopus japonicus
TRANSCRIPT
Chemosphere 72 (2008) 1359–1365
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Two-generation toxicity study on the copepod model species Tigriopus japonicus
Kyun-Woo Lee a, Sheikh Raisuddin a, Dae-Sik Hwang b, Heum Gi Park c, Hans-Uwe Dahms d, In-Young Ahn e,Jae-Seong Lee a,*
a Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791, South Koreab Department of Molecular and Environmental Bioscience, Graduate School, Hanyang University, Seoul 133-791, South Koreac Faculty of Marine Bioscience and Technology, College of Life Sciences, Kangnung National University, Gangneung 210-702, South Koread Institute of Marine Biology, National Taiwan Ocean University, Keelung 202, Taiwane Korea Polar Research Institute, Korea Ocean Research and Development Institute, Incheon 406-840, South Korea
a r t i c l e i n f o a b s t r a c t
Article history:Received 28 January 2008Received in revised form 21 March 2008Accepted 10 April 2008Available online 3 June 2008
Keywords:Tigriopus japonicusLife cycle testMetalsEndocrine disruptors
0045-6535/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2008.04.023
* Corresponding author. Tel.: +82 2 2220 0769; faxE-mail address: [email protected] (J.-S. Lee).
Previous studies on the intertidal copepod Tigriopus japonicus have demonstrated that it is a suitablemodel species for the assessment of acute toxicities of marine pollutants. In order to standardize T. japo-nicus for use in environmental risk assessment involving whole life cycle exposure, we tested nine pol-lutants for their effects on growth and reproduction during a two-generation life cycle exposure test.Nauplii (F0) were exposed to a range of concentrations of each chemical in a static renewal culture sys-tem. Broods of the second generation (F1) were subsequently exposed to the same concentrations for onefull life cycle. Of the seven traits (nauplius phase, development time, survival, sex ratio, number of clutch,nauplii per clutch and fecundity), only the length of the nauplius phase and development time showed agreater sensitivity to chemical exposure. Between the two sensitive traits, the period of the naupliusphase was more sensitive than cohort generation time. Biocides significantly increased the maturationperiod of nauplii as well as copepodids in F0 generation. In this study, it was demonstrated that T. japo-nicus could also be used in reproduction and life cycle tests and it provides an opportunity for testing thechronic and subchronic toxic effects of marine pollutants. Further validation and harmonization in amulti-centric study involving other laboratories of the region will strengthen its use as a supplementto existing model species.
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1. Introduction
Several copepod species have been used in marine ecotoxicitytesting and biomonitoring (OECD, 2006; Kusk and Wollenberger,2007; Raisuddin et al., 2007). Kusk and Wollenberger (2007) spe-cifically proposed four marine copepod species, namely, Acartiatonsa, Amphiascus tenuiremi, Nitocra spinipes, and Tisbe battagliai,for testing of endocrine-disrupting chemicals (EDCs). Templetonet al. (2006) used A. tenuiremis in assessing the toxicity of single-walled carbon nanotubes (SWNTs). A standardized, full life cyclebioassay with the estuarine copepod A. tenuiremis (ASTM methodE-2317-04) has been used in some studies (Bejarano et al., 2004;Templeton et al., 2006). Life cycle and multi-generation tests areconsidered important, especially for a holistic risk assessment ofEDCs and persistent organic pollutants (POPs). However, moretesting using different species would be necessary to reach a con-clusion on the effects of those chemicals. Therefore, there is need todevelop new methods and test new model species for this purposeto meet regional regulatory requirements.
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Using the intertidal harpacticoid copepod, Tigriopus japonicus,we studied the acute toxicities of metals, biocides and EDCs, andobserved that it has the potential to be used as a benchmark spe-cies for marine environmental testing in Western Pacific coastal re-gions (Lee et al., 2007a). Recently, EDCs and POPs have becomechemicals of particular interest for the general public and scientistsalike, since their impact on human and environmental health hasbecome apparent (Jenssen, 2003, 2006; Breitholtz et al., 2006; Kal-lenborn et al., 2007; Lohmann et al., 2007). Therefore, concerted ef-forts have been made to develop and standardize both acute andchronic tests for the evaluation of the impacts of these chemicals,especially on marine biota (Breitholtz et al., 2006; Kusk and Wol-lenberger, 2007). As a test species, T. japonicus has several advanta-ges, such as distinct sexual dimorphism, nauplius and copepodidstages, high fecundity, and a short life cycle (Lee et al., 2006,2007b; Seo et al., 2006; Raisuddin et al., 2007). It showed measur-able toxic responses to an array of compounds, notwithstandingthe fact that it thrives at a wide range of temperatures and salini-ties. Among the various copepod species used in toxicity testing,the greatest number of genes involved in detoxification and stressresponse pathways have been cloned and sequenced from T. japo-nicus (Raisuddin et al., 2007). In a two-generation toxicity test
1360 K.-W. Lee et al. / Chemosphere 72 (2008) 1359–1365
involving estrogenic compounds, Marcial et al. (2003) reportedthat two endpoints, namely, the naupliar phase duration anddevelopment time for adults were most significantly affected inT. japonicus.
In this study, we determined the toxicity of nine common con-taminants (three EDCs, three biocides, and three trace metals) ofenvironmental concern on seven life cycle traits across two gener-ations of T. japonicus.
2. Materials and methods
2.1. Copepod maintenance
T. japonicus has been cultured in our laboratory since 2005. Theoriginal stock culture was obtained from Dr. H.G. Park, Faculty ofMarine Bioscience and Technology, College of Life Sciences, Kang-nung National University, Gangneung (South Korea). The identityof the test species has been confirmed by the similarity of mito-chondrial genome sequences (Jung et al., 2006). T. japonicus wasmaintained and cultured in UV-treated, filtered (1 lm) natural sea-water (32 ppt salinity) at 20 ± 1 �C under a 12 h light to 12 h darkcycle. The alga Tetraselmis suecica was provided as food. The algawas cultured at 20 ± 1 �C, with 24 h light exposure (4000 lx) inConwy medium (Walne, 1974) using filtered seawater (1 lm meshof glass fiber filter) sterilized by autoclaving. Initially, approxi-mately 5000 adult copepods were cultured in a 2 l beaker contain-ing seawater (32 ppt salinity) in an incubator maintained at20 ± 1 �C for 20 h.
2.2. Test chemicals
Nine chemicals of environmental concern were tested for theireffects on various attributes of reproduction and growth. These in-cluded three heavy metals: Cu [copper (II) sulfate pentahydrate(CuSO4 � 5H2O, Sigma, USA, > 99% pure)], AsIII [sodium m-arsenite(NaAsO2, Sigma, USA, 94% pure)], AsV [disodium hydrogen arse-nate heptahydrate (Na2HAsO4 � 7H2O, Sigma, USA, > 98% pure)];three EDCs: tributyltin (TBT, Supelco, USA, 99.9% pure), polychlori-nated biphenyl, PCB (Aroclor 1254, Supelco, USA, 99.9% pure), 4-t-butylphenol (BP, Sigma, USA, 99.9% pure); and three biocides:endosulfan (Riedel de Han, Germany, 99.9% pure), Alachlor (Supe-lco, USA, 99.9% pure) and Molinate (Chem Service, USA, 99.4%pure).
2.3. 96-h acute toxicity of arsenicals
Acute toxicity values of all toxicants, except arsenicals havebeen reported previously by Lee et al. (2007a) (Table 1). We deter-mined the 96-h LC10, and LC50 values of the two arsenicals (AsIII
Table 1LC10, LC50, and 95% confidence intervals (CI) for adult Tigriopus japonicus exposed tovarious chemicals in 96-h acute toxicity test
Chemicals LC10 (95% CI; mg/l) LC50 (95% CI; mg/l)
Heavy metals Arsenic-III 5.6 (3.85–8.20) 9.7 (5.57–16.92)Arsenic-V 9.9 (6.72–14.63) 17.2 (11.57–25.67)Copper 2.2 (1.33–3.49) 3.9 (2.60–5.87)
Biocides Endosulfan 0.04 (0.023–0.062) 0.07 (0.046–0.111)Alachlor 4.1 (2.78–6.12) 7.3 (4.80–10.95)Molinate 19.4 (12.78–29.35) 35.5 (21.95–57.50)
EDCs Tributyltin 0.03 (0.021–0.047) 0.05 (0.029–0.108)PCB mix 0.83 (0.36–1.96) 2.5 (0.59–10.88)4-t-butylphenol 7.2 (2.96–17.58) 22.0 (4.76–101.50)
Values of all chemicals, except AsIII and AsV, are based on Lee et al. (2007a). Arsenicwas dissolved in distilled water.
and AsV) using acute toxicity tests and probit analysis as describedpreviously (Lee et al., 2007a).
2.4. Concentration ranges
A wide range of concentrations of toxic chemicals were used.With the exceptions of endosulfan and TBT, all other chemicalswere exposed at four concentrations of 0.1, 1, 10, 100 lg/l. Endo-sulfan and TBT were tested at concentrations of 0.01, 0.1, 1, 10 lg/l. In our previous acute toxicity study, endosulfan and TBT werefound to be more toxic than many of the above-mentioned toxi-cants (Lee et al., 2007a). Therefore, we tested a lower concentra-tion range for these two chemicals. Selection of concentrations isalso based partly on the studies reported by Marcial et al. (2003)and Kwok and Leung (2005) on T. japonicus. EDCs and pesticideswere dissolved in dimethyl sulfoxide (DMSO, Sigma, USA, 99.9%pure), and metal salts in ultra-pure distilled water. Stock solu-tions were prepared using a previously described procedure(Lee et al., 2007a). The maximum concentration of DMSO wasmaintained at 60.001%. In the control groups, solvents were usedin equal concentrations to match their levels in the treatedgroups. All treatments were conducted simultaneously at20 ± 1 �C.
2.5. Exposure
Ten newly-hatched nauplii (<24 h after hatching) per concen-tration were transferred to 12-well tissue culture plates (SPL LifeSciences, Seoul, South Korea) with a 4 ml working volume inthree replicates (total 30 nauplii). These nauplii were culturedwith the above-mentioned conditions until adult females devel-oped egg sacs. Test solutions were renewed (�50% of the workingvolume) daily and T. suecica was added at a density of approxi-mately 5 � 104 cells/ml. Developmental stages were observed dai-ly under a stereomicroscope with a scattering light (OlympusSZX9, Japan) and recorded to calculate the time of development(i.e. from nauplii to copepodite, and from nauplii to adults withegg sacs). The sex ratio and survival (%) were determined afterthe maturation of all copepods. The maturation period in controlwas on an average 14 days. However, in exposed groups it varied.The development of egg sac was considered as the time of matu-ration. To measure the fecundity (offspring production), the num-ber of clutches produced and the number of nauplii per clutch ofan adult female, six females bearing an egg sac per concentrationwere individually transferred to a new 12-well culture dish (4 mlworking volume). These females were cultured under the above-mentioned conditions for 10 days. T. suecica was provided at2 � 104 cells/copepod/day. Test solutions were renewed dailyand the resulting nauplii and unhatched clutches were countedand removed under the stereomicroscope. For the experimentwith the second generation (F1), 10 nauplii (F1) produced by eachfemale (F0) with the first or second brood per concentration weretransferred to 12-well tissue culture plates (4 ml working volumein three replicates, total 30 nauplii each treatment). The experi-ment and exposure conditions were the same as those used forthe F0 generation test.
2.6. Statistical data processing
All data were analyzed by one-way analysis of variance (ANO-VA). If significant (P < 0.05) differences were found by the ANOVAtest, Duncan’s multiple range test was used to rank the groups(Duncan, 1955; Zar, 1999). Data are presented as themeans ± standard error (S.E.). All statistical analyses were con-ducted using SPSS version 12.0 (SPSS Inc., Michigan Avenue, Chi-cago, Illinois, USA).
K.-W. Lee et al. / Chemosphere 72 (2008) 1359–1365 1361
3. Results
3.1. 96-h acute toxicity of arsenicals
The acute 96-h LC10, and LC50 levels of AsIII in T. japonicus were5.6, and 9.7 mg/l, respectively (Table 1). Similar values for AsVwere observed to be 9.9, and 17.2 mg/l, respectively (Table 1).
3.2. Effects in the F0 generation
Exposure to most of the toxicants resulted in an increase in theduration of the nauplius phase at most concentrations tested(Fig. 1). This change was significant (P < 0.05) when compared withthe control. Only in the case of Cu at the lowest concentration of0.1 lg/l, was the change not statistically significant. Endosulfanand TBT, which were tested at concentrations 10 times lower thanthose of other toxicants, also showed significant (P < 0.05) effectson this parameter at all concentrations. The effect on duration ofthe nauplius phase was more pronounced in the case of exposureto EDCs (BP, PCB and TBT). TBT, in particular, caused a pronouncedincrease in the length of the nauplius phase at low concentrations(Fig. 1). At 100 lg/l of PCB all copepods did not grow to the copepo-dite stage and died (Table 2).
AsV
Concentration (μg/L)
0.0 0.1 1.0 10.0 100.0
Day
0
4
8
12
16
AsIII
0.0 0.1 1.0 10.0 100.0
Day
0
4
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16
Cu
0.0 0.1 1.0 10.0 100.0
Day
0
4
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16
N-CN-A
Molinate
Concentra
0.0 0.1 10
4
8
12
16
Alachlor
0.0 0.1 10
4
8
12
16
Endosulfan
0.00 0.01 0.0
4
8
12
16
aab bcb c
ab b b b
ab b b b
ab b
ba
ab b
ba
a
b bc
ba
a a a a a
a a a a a
a a a a a
Fig. 1. Effect on the nauplius phase (nauplius to copepodid, N–C) and development timchemicals at 20 �C. Significant differences over control values are indicated by a differentindicate a significant difference in observations between the two groups (P < 0.05). Valu
All biocides (alachlor, endosulfan and molinate) caused a signif-icant (P < 0.05) increase in the generation time for adults of the F0
generation (Fig. 1). The effects of EDCs were random. BP at 0.1 and100 lg/l, PCB at 0.1 and 10 lg/l, and TBT at 0.01 and 1 lg/L caused asignificant increase (Fig. 1). However, none of the metals tested re-sulted in any significant effect on generation time for adults of theF0 generation.
Endosulfan at 10 lg/l caused a significant decrease in fecundityand the number of nauplii/clutch (Table 2). Survival in all othertoxicants showed no significant (P > 0.05) effect at any concentra-tion on any other traits of the F0 T. japonicus, except for a significant(P < 0.05) reduction in survival at higher concentrations of TBT andPCB.
3.3. Effects in the F1 generation
The effects of the tested toxicants on the two traits (length ofnauplius phase and cohort generation time for adults) did notshow any pattern in the F1 generation (Fig. 2). Only AsIII at higherconcentrations resulted in a significant reduction in the naupliusphase (P < 0.05) compared to that of the unexposed group. A trendof random increase in the time of the nauplius phase was observedin some EDC- and biocide-treated copepods. However, a significant
tion (μg/L)
.0 10.0 100.0
BP
Concentration (μg/L)
0.0 0.1 1.0 10.0 100.00
4
8
12
16
.0 10.0 100.0
TBT
0.00 0.01 0.10 1.00 10.000
4
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16
10 1.00 10.00
PCB
0.0 0.1 1.0 10.00
4
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16
bc
b b b
bc
b b b
bc c
b b b
a
b b b
b b
a
ab
abbc c
d
c
aab
bab
a
b b bc
b
aab ab
b
e (nauplius to adult, N–A) of F0 generation Tigriopus japonicus exposed to differentcharacter on the bar. a,b,c Values with the same color bars with different characters
es are shown as means ± SE.
Table 2Fecundity, sex ratio and survival rate of F0 generation Tigriopus japonicus exposed to different chemicals at 20 �C
Chemical Survival (%) Sex ratio (F/M) Number of clutch Number of nauplii/clutch Fecundity/10 days
Control 90.0 ± 10.00 4.7 ± 2.13 2.8 ± 0.17 27.8 ± 4.42 82.5 ± 14.26DMSO 93.3 ± 6.67 4.1 ± 2.47 2.8 ± 0.17 33.1 ± 1.33 93.7 ± 6.29Cu (lg/l) 0.1 90.0 ± 0.00 1.5 ± 0.23 3.0 ± 0.00 26.5 ± 1.97 79.3 ± 5.92
1 96.7 ± 3.33 2.5 ± 0.76 3.0 ± 0.00 27.3 ± 0.83 82.0 ± 2.5010 86.7 ± 6.67 3.4 ± 1.80 2.5 ± 0.34 29.0 ± 1.73 72.0 ± 10.38100 86.7 ± 3.33 1.7 ± 0.20 3.0 ± 0.00 28.0 ± 1.79 84.0 ± 5.36
AsIII (lg/l) 0.1 93.3 ± 6.67 3.6 ± 1.72 2.8 ± 0.17 27.6 ± 3.73 77.8 ± 11.561 96.7 ± 3.33 2.7 ± 0.67 2.5 ± 0.34 29.3 ± 2.11 76.0 ± 13.1110 100.0 ± 0.00 2.2 ± 0.93 2.7 ± 0.21 25.7 ± 2.77 69.7 ± 10.07100 100.0 ± 0.00 3.4 ± 0.57 3.0 ± 0.00 25.6 ± 2.56 76.8 ± 7.69
AsV (lg/l) 0.1 93.3 ± 6.67 2.4 ± 0.80 2.8 ± 0.17 25.9 ± 2.75 75.5 ± 10.281 93.3 ± 3.33 2.6 ± 0.46 3.0 ± 0.00 26.9 ± 1.88 80.7 ± 5.6310 83.3 ± 3.33 1.6 ± 0.70 2.7 ± 0.21 24.6 ± 3.14 67.3 ± 10.69100 80.0 ± 0.00 1.2 ± 0.23 2.7 ± 0.33 24.0 ± 2.93 65.0 ± 12.31
Endosulfan (lg/l) 0.01 93.3 ± 3.33 1.9 ± 0.30 3.0 ± 0.00 23.1 ± 1.94 69.3 ± 5.830.1 86.7 ± 6.67 3.8 ± 1.64 2.5 ± 0.34 24.8 ± 2.21 59.8 ± 8.151 100.0 ± 0.00 1.6 ± 0.38 2.8 ± 0.17 26.6 ± 1.96 74.0 ± 4.0010 100.0 ± 0.00 3.4 ± 0.57 2.3 ± 0.33 15.5 ± 1.97* 34.7 ± 5.17*
Alachlor (lg/l) 0.1 90.0 ± 0.00 4.4 ± 1.95 2.5 ± 0.34 27.0 ± 1.42 68.3 ± 10.601 93.3 ± 6.67 6.6 ± 2.43 3.0 ± 0.00 26.6 ± 1.34 79.8 ± 4.03
10 93.3 ± 3.33 1.9 ± 0.30 2.8 ± 0.17 29.0 ± 0.72 82.0 ± 4.63100 90.0 ± 5.77 1.9 ± 0.54 3.0 ± 0.00 24.3 ± 1.82 72.8 ± 5.45
Molinate (lg/l) 0.1 96.7 ± 3.33 1.6 ± 0.31 2.8 ± 0.17 25.6 ± 1.92 71.5 ± 4.221 100.0 ± 0.00 2.4 ± 1.06 3.0 ± 0.00 22.0 ± 3.06 65.8 ± 9.18
10 100.0 ± 0.00 2.6 ± 0.74 3.0 ± 0.00 22.2 ± 2.01 66.5 ± 6.02100 100.0 ± 0.00 3.6 ± 2.72 2.7 ± 0.33 25.9 ± 2.55 66.8 ± 9.30
TBT (lg/l) 0.01 73.3 ± 12.02 5.0 ± 1.53 2.8 ± 0.17 22.3 ± 6.26 66.0 ± 19.220.1 63.3 ± 12.02* 3.6 ± 1.27 2.3 ± 0.33 30.1 ± 1.99 71.8 ± 12.541 73.3 ± 12.02 2.7 ± 0.60 2.7 ± 0.21 25.7 ± 4.31 71.8 ± 14.16
10 60.0 ± 17.32* 0.8 ± 0.15 2.5 ± 0.34 24.1 ± 1.27 62.2 ± 10.33
PCB (lg/l) 0.1 86.7 ± 6.67 2.2 ± 0.42 2.4 ± 0.40 23.1 ± 3.68 60.6 ± 15.121 76.7 ± 6.67 4.0 ± 1.04 2.3 ± 0.33 32.3 ± 1.40 74.0 ± 9.14
10 66.7 ± 3.33* 1.5 ± 0.23 2.8 ± 0.17 28.1 ± 2.73 81.0 ± 10.55100 – – – – –
BP (lg/l) 0.1 86.7 ± 8.82 3.9 ± 2.08 2.3 ± 0.42 25.1 ± 4.76 63.5 ± 18.051 80.0 ± 5.77 2.6 ± 0.52 3.0 ± 0.00 28.2 ± 2.84 84.7 ± 8.52
10 76.7 ± 3.33 5.3 ± 1.20 3.0 ± 0.00 27.8 ± 2.40 83.3 ± 7.21100 90.0 ± 5.77 2.9 ± 0.35 2.2 ± 0.31 30.7 ± 2.37 67.5 ± 11.14
Significant changes versus respective solvent control are indicated by asterisk (*) indicating P < 0.05. Values are means ± SE.
1362 K.-W. Lee et al. / Chemosphere 72 (2008) 1359–1365
(P < 0.05) effect was observed only at a high concentrations ofmolinate (0.1 and 10 lg/l), TBT (0.1 and 1 lg/l), and BP (0.1, 1and 10 lg/l).
Effects of the tested toxicants on the maturation period in the F1
generation of T. japonicus showed no generalized response (Fig. 2).Cu at concentrations of 1 and 10 lg/l caused a significant reductionin this trait, whereas alachlor, molinate, PCB, TBT and BP showed asignificant increase (P < 0.05) in the maturation period in the F1
generation of T. japonicus at some concentrations. However, the ef-fect was not concentration-dependent (Fig. 2).
Only the lowest concentration of endosulfan caused a signifi-cant decrease in the numbers of nauplii/clutch (Table 3). In thenumber of clutches, BP at the highest concentration (100 lg/l)caused a significant decrease. No other traits in the F1 generationof T. japonicus showed any significant change in response to toxi-cant exposure at all concentrations tested (Table 3).
4. Discussion
Copepods have many attributes that make them an attractivegroup of organisms for toxicity testing of marine as well as fresh-water chemical pollutants (Gourmelon and Ahtiainen, 2007; Kuskand Wollenberger, 2007; Raisuddin et al., 2007). A standardizedtest involving the estuarine copepod A. tenuiremis is already inuse (ASTM method E-2317-04). However, as emphasized by Lee
et al. (2007a) and also by Gourmelon and Ahtiainen (2007), thereis a need to develop and standardize more toxicity tests for meet-ing the regional environmental specificities and regulatoryrequirements. Four species of marine copepods, namely, A. tonsa,N. spinipes, T. battagliai, and A. tenuiremis have been identified aspotential model species for EDCs (Kusk and Wollenberger, 2007).
In a recent overview, the OECD has highlighted T. japonicus as an-other species for toxicity testing and risk assessment of EDCs (OECD,2006). Previously, Lee et al. (2007a) demonstrated that this marinespecies has a good scope of application in acute toxicity testing ofa wide range of chemicals including EDCs. Marcial et al. (2003)and Kwok and Leung (2005) used T. japonicus in short- and long-term toxicity testing of environmental chemicals including EDCs.In this study, we tested nine chemicals including three EDCs usingT. japonicus in a two-generation toxicity test format. A battery of se-ven traits representing growth and development in the F0 and F1
generations were studied and it was found that two traits, the peri-ods of nauplius stage and maturation, were significantly affected bymost of the tested chemicals. The highest concentration of PCB(100 lg/l) caused 100% mortality in the F0 generation. Previously,Marcial et al. (2003) identified the length of nauplius stage and mat-uration period as the sensitive traits of T. japonicus, responding tomost of the estrogenic compounds. We used different EDCs and alsoincluded metals and biocides in this study to enlarge the scope of thetest and establish the sensitivity of T. japonicus.
As V
Concentration (μg/L)
0.0 0.1 1.0 10.0 100.0
Day
0
4
8
12
16
As III
0.0 0.1 1.0 10.0 100.0
Day
0
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Cu
0.0 0.1 1.0 10.0 100.0
Day
0
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N-CN-A
Molinate
Concentration (μg/L)
0.0 0.1 1.0 10.0 100.00
4
8
12
16BP
Concentration (μg/L)
0.0 0.1 1.0 10.0 100.00
4
8
12
16
Alachlor
0.0 0.1 1.0 10.0 100.00
4
8
12
16TBT
0.00 0.01 0.10 1.00 10.000
4
8
12
16
Endosulfan
0.00 0.01 0.10 1.00 10.000
4
8
12
16PCB
0.0 0.1 1.0 10.00
4
8
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16
bb a a a
abc
a abc
abbcbc ca
bab aba ab
aab b
ab ab
ab aa
b b
a a ab bcc
b
a ab a a
abbabab
cab b ab a
b ba a
b
a
b
a
a a b ac
ab
bcc
ab
ac
ab
d
c
a
c bcab
a a a a a
a
aa a a
Fig. 2. Effect on the nauplius phase (nauplius to copepodid, N–C) and development time (nauplius to adult, N–A) of F1 generation Tigriopus japonicus exposed to differentchemicals at 20 �C. Significant differences over control values are indicated by a different character on the bar. a,b,c Values with the same color bars with different charactersindicate a significant difference in observations between the two groups (P < 0.05). Values are shown as means ± SE.
K.-W. Lee et al. / Chemosphere 72 (2008) 1359–1365 1363
Marcial et al. (2003) observed that changes in parental and F1
generations were not significantly different. However, we observedthat the effects on the F0 generation were more remarkable andconsistent as compared with those observed in the F1. These maybe caused by different classes of tested compounds. However, ingeneral, T. japonicus appears to be a suitable organism for the studyof the effects of EDCs on growth and developmental traits. The ef-fects of a number of environmental EDCs on the expression ofstress response genes have been studied in T. japonicus (Raisuddinet al., 2007). Therefore, mechanisms of action of EDCs are known tosome extent in T. japonicus and their toxicity has been profiled.
In terms of environmental hazards, metals are of great concernin freshwater, marine and estuarine ecosystems and to humanhealth (Sanchez-Hernandez, 2000; Järup, 2003; Jenssen, 2003;O’Driscoll et al., 2005). Estuarine environments and intertidalzones are hotspots for metal pollution (Monserrat et al., 2007).Tigriopus spp. and some copepod species have shown sensitivityto metals (Kusk and Wollenberger, 2007; Raisuddin et al., 2007).Recently, Pedroso et al. (2007a, 2007b) have studied the toxicityof silver and its mechanism in the euryhaline pelagic copepod A.tonsa. They observed that Na+,K+-ATPase plays a key role in silvertoxicity in A. tonsa. The study of the mechanistic aspects may behelpful in proper risk assessments of environmental toxicants.The molecular mechanisms of the toxicities of metals and EDCsin T. japonicus have been studied using gene expression (Lee
et al., 2006, 2007b; Seo et al., 2006; Raisuddin et al., 2007). Suchan approach will enhance the use of model organisms, such asT. japonicus, not only in environmental toxicology, but also in envi-ronmental toxicogenomics.
In copepods, larval development includes 11 molts and onemetamorphosis from the naupliar phase to the copepodid phase.Molting and metamorphosis are regulated by ecdysteroids(Andersen et al., 2001; Forget-Leray et al., 2005; Dahl et al.,2006). Therefore, a copepod test that takes into account thedevelopment of larval stages and the development of nauplii intocopepodids would be useful. Such a test may reflect disturbancesof molting processes that are controlled by ecdysteroids or juve-nile hormones. In this manner, it might be possible to detect dis-turbances to mechanisms that are necessary for growth anddevelopment.
Since EDCs are a major cause of concern in the marine environ-ment, research on development of tests to evaluate their impact,especially those based on invertebrates, has intensified (Andersenet al., 2001; Hutchinson, 2002; Barata et al., 2004). Identificationof key sensitive traits is a major objective in such studies. Of theseveral traits we tested, only development and growth traitsshowed a consistent measurable response. Therefore, employingT. japonicus in the assessment of the impact of EDCs holds promiseand, with further validation, its use may be recommended in regu-latory policies.
Table 3Fecundity, sex ratio and survival rate of F1 generation Tigriopus japonicus exposed to different chemicals at 20 �C
Chemical Survival (%) Sex ratio (F/M) Number of clutch Number of nauplii/clutch Fecundity/10 days
Control 96.7 ± 3.33 4.0 ± 2.52 2.8 ± 0.17 36.8 ± 2.73 103.2 ± 8.06DMSO 90.0 ± 5.77 3.9 ± 3.03 2.8 ± 0.17 33.4 ± 1.88 94.0 ± 6.55Cu (lg/l) 0.1 90.0 ± 5.77 1.3 ± 0.87 2.8 ± 0.17 33.6 ± 3.87 98.3 ± 14.07
1 96.7 ± 3.33 1.4 ± 0.67 2.8 ± 0.17 32.3 ± 2.29 91.3 ± 8.5210 90.0 ± 5.77 0.6 ± 0.21 2.8 ± 0.17 24.8 ± 5.95 73.8 ± 18.15100 90.0 ± 0.00 0.6 ± 0.17 3.0 ± 0.00 35.2 ± 2.25 105.6 ± 6.73
AsIII (lg/l) 0.1 84.2 ± 5.76 6.2 ± 1.36 2.5 ± 0.34 32.1 ± 2.07 82.2 ± 13.511 90.0 ± 0.00 2.5 ± 0.50 2.8 ± 0.17 33.5 ± 3.10 96.8 ± 12.2610 96.7 ± 3.33 0.7 ± 0.32 2.7 ± 0.21 28.9 ± 2.73 77.8 ± 10.96100 90.0 ± 5.77 1.4 ± 0.49 2.7 ± 0.21 26.8 ± 5.74 74.7 ± 16.53
AsV (lg/l) 0.1 80.0 ± 11.55 3.4 ± 0.81 2.8 ± 0.17 32.3 ± 3.55 94.0 ± 13.181 80.0 ± 11.55 5.6 ± 2.41 2.7 ± 0.21 31.1 ± 3.83 81.5 ± 11.2710 83.3 ± 3.33 2.2 ± 0.39 2.8 ± 0.31 30.0 ± 5.20 80.0 ± 15.31100 100.0 ± 0.00 3.0 ± 0.51 2.7 ± 0.21 27.3 ± 2.90 72.2 ± 9.48
Endosulfan (lg/l) 0.01 90.0 ± 5.77 2.9 ± 2.03 2.7 ± 0.21 21.9 ± 3.69* 60.5 ± 11.420.1 90.0 ± 5.77 1.3 ± 0.20 2.7 ± 0.21 32.9 ± 2.88 89.3 ± 12.061 83.3 ± 3.33 0.7 ± 0.21 3.0 ± 0.00 37.1 ± 1.98 111.2 ± 5.94
10 93.3 ± 3.33 5.9 ± 2.58 2.8 ± 0.17 28.5 ± 3.11 81.0 ± 10.67
Alachlor (lg/l) 0.1 86.7 ± 8.82 2.4 ± 0.81 3.0 ± 0.00 29.7 ± 3.63 89.0 ± 10.881 86.7 ± 3.33 1.8 ± 0.87 3.0 ± 0.00 34.8 ± 3.66 104.4 ± 10.98
10 90.0 ± 5.77 1.4 ± 0.15 2.8 ± 0.17 32.1 ± 3.27 90.7 ± 10.85100 96.7 ± 3.33 0.7 ± 0.06 2.7 ± 0.33 32.4 ± 2.20 84.0 ± 10.03
Molinate (lg/l) 0.1 83.3 ± 8.82 0.4 ± 0.09 3.0 ± 0.00 38.3 ± 1.90 115.0 ± 5.691 86.7 ± 3.33 0.9 ± 0.58 3.0 ± 0.00 25.2 ± 3.72 75.5 ± 11.16
10 96.7 ± 3.33 0.3 ± 0.09 3.0 ± 0.00 33.2 ± 1.71 99.7 ± 5.13100 93.3 ± 3.33 0.8 ± 0.00 3.0 ± 0.00 38.9 ± 1.09 116.8 ± 3.28
TBT (lg/l) 0.01 96.7 ± 3.33 6.4 ± 2.09 2.5 ± 0.34 26.9 ± 6.44 75.7 ± 19.540.1 100.0 ± 0.00 3.0 ± 0.51 2.7 ± 0.21 32.4 ± 3.89 89.2 ± 14.571 100.0 ± 0.00 2.4 ± 0.87 2.8 ± 0.17 35.6 ± 1.58 101.7 ± 8.73
10 90.0 ± 5.77 0.8 ± 0.36 2.7 ± 0.33 26.8 ± 2.52 75.5 ± 12.37
PCB (lg/l) 0.1 93.3 ± 6.67 1.4 ± 0.50 2.8 ± 0.20 39.9 ± 2.62 113.6 ± 13.671 100.0 ± 0.00 0.9 ± 0.33 2.8 ± 0.17 34.5 ± 3.46 99.5 ± 13.18
10 100.0 ± 0.00 2.6 ± 0.74 2.7 ± 0.21 32.1 ± 3.67 88.3 ± 14.76100 – – – – –
BP (lg/l) 0.1 93.3 ± 6.67 4.3 ± 2.40 2.7 ± 0.21 34.6 ± 2.57 91.8 ± 9.131 100.0 ± 0.00 3.2 ± 0.83 2.5 ± 0.22 24.2 ± 3.85 61.2 ± 12.39
10 80.0 ± 0.00 0.3 ± 0.15 2.5 ± 0.22 34.4 ± 3.27 88.5 ± 15.04100 100.0 ± 0.00 3.8 ± 2.59 2.0 ± 0.26* 32.8 ± 6.61 72.5 ± 16.4
Significant changes versus respective solvent control are indicated by an asterisk (*) indicating P < 0.05. Values are means ± SE.
1364 K.-W. Lee et al. / Chemosphere 72 (2008) 1359–1365
Toxicity results of chemicals are significantly affected by envi-ronmental variables (Larrain et al., 1998). Although T. japonicus istolerant to wide ranges of temperature and salinity, toxicities ofcertain chemicals have been reported to be affected by these vari-ables. For instance, Kwok and Leung (2005) observed that toxicitiesof Cu and TBT are significantly increased in T. japonicus when cul-ture temperature is increased by 10 �C. They also suggested that, athigher temperatures, animals undergo dormancy. Therefore, envi-ronmental variables and confounding factors have to be consideredwhile developing standard test protocols using T. japonicus.
In summary, we observed that the selected toxicants affectedseveral traits of both the F0 and F1 generations of T. japonicus.The most significant effects influenced the length of the naupliarphase and the maturation time. In most cases, an extension ofthe developmental time was observed. In our previous study (Leeet al., 2007a), we have demonstrated the range and sensitivity ofT. japonicus to various environmental toxicants. In this study, weshowed the effect of nine environmental contaminants on growthand developmental traits in a two-generation test. It is suggestedthat T. japonicus, which has the largest database available for anycopepod on chronic and acute toxicity and gene sequences, isdeserving of consideration after further validation for its use inregulatory toxicology exercises as a supplement to exiting modelspecies. This model species would fill the gaps and meet the needs
for marine pollution monitoring and environmental risk assess-ment, particularly in Western Pacific coastal regions.
Acknowledgments
We thank Dr. Kenneth M.Y. Leung at the University of HongKong for his valuable comments on the previous version of manu-script. This work was supported by the Grants of KOSEF (2006;R0A-2006-000-10155), Sea Grant (2007), and MarineBio 21(2008) provided to Jae-Seong Lee, and also supported by a grant(Monitoring on Environmental Changes at the Korean Arctic andAntarctic Stations; PE08040) from the Korea Polar Research Insti-tute provided to In-Young Ahn.
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