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In vitro and in vivo acute toxicity of fenpyroximate to flounder Paralichthys

olivaceus and its gill cell line FG

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 Aquatic Toxicology 92 (2009) 76–85

Contents lists available at ScienceDirect

Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox

In vitro and in vivo acute toxicity of fenpyroximate to flounder Paralichthys

olivaceus and its gill cell line FG
Na Na, Huarong Guo ∗ , Shicui Zhang, Zhaojie Li, Licheng Yin
Department of Marine Biology, Ocean University of China, Qingdao 266003, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Fenpyroximate, an acaricide, is widely used in the prevention of acarids (mites) in plants. In this study, the
Received 13 July 2008 in vitro and in vivo acute toxicity of fenpyroximate was examined using the marine flounder Paralichthys
Received in revised form 16 December 2008 olivaceus and its gill cell line (FG). The 48h-IC50 (95% confidence limits) values of fenpyroximate in the
Accepted 20 December 2008
FG cells were 890 (790–990) nM, 950 (881–1019) nM and 1250 (1159–1341) nM, and 96h-IC50 (95% con-
fidence limits) were 480 (388–572) nM, 490 (454–526) nM and 510 (469–551) nM, for methyl thiazolyl
Keywords:
tetrazolium (MTT) assay, neutral red (NR) uptake and cell protein assay, respectively. The 48h- and 96h-
Fenpyroximate
LC50 (95% confidence limits) values of fenpyroximate in living flounders were 28.84 (14.28–58.26) nM
Flounder
Paralichthys olivaceus
and 11.74 (6.06–22.8) nM, respectively. This indicated that fenpyroximate was highly toxic to both floun-
Cell line ders and FG cells. Moreover, comparisons of the ratios of average 48h-IC50 to 48h-LC50 and average
Toxicity 96h-IC50 to 96h-LC50 showed that the length of exposure time did not significantly affect the cor-
relation between the FG cells and living flounders in the acute toxicity estimation of fenpyroximate
provided the selected exposure time is the same. Thus, we suggest that FG cells could be a good bioas-
say system in rapid estimation of the corresponding LC50 values of pollutants to living fish, instead
of whole living fish. Histopathological examinations showed that liver and gill were the major target
organs of fenpyroximate, especially the damage of gill tissues may account much for the high lethality of
exposed flounders. Consistent with the histopathological observations, analysis of the activities of two key
detoxification metabolism-related enzymes, glutathione S-transferase (GST) and cytochrome P4501A1
(CYP1A1)-dependent ethoxyresorufin-O-deethylase (EROD), in the liver and gill tissues of exposed floun-
ders indicated that liver has much higher detoxification capacity than gills, and this contributes to the
higher tolerance of liver to the toxicity of fenpyroximate in the exposed flounders. Fenpyroximate can
initially induce a quick and significant increase of the activities of the antioxidant enzymes superoxide
dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in all the exposed FG cells, and liver and
gill tissues of exposed flounders. Upon continuation of the exposure the enzyme activities were inhibited,
implying the occurrence of oxidative stress in the exposed fish cells and the possible interruption of the
mitochondrial respiratory chain which involves redox reactions by fenpyroximate.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction found that fenpyroximate could inhibit the activity of mitochon-

Fenpyroximate (1,1-dimethylethyl(E)-4-[[[[(1,3-dimethyl-5-ph-
enoxy-1H-pyrazol-4-yl) methylene]amino]oxy]methyl]benzoate)
is a potent acaricide widely used in China for the control of
phytophagous mites in orange, apple, peach and pear orchards.
Good efficacy against all developmental stages of the spider mites
has been reported (Stumpf and Nauen, 2001). Fenpyroximate is
so highly selective that it shows little or no toxicity to insects,
animal-parasitic mites, and soil-living mites (Konno et al., 1990).
Motoba et al. (1992) reported the toxic mechanism of fenpyroxi-
mate in two-spotted spider mite (Tetranychus urticae Koch). It was
∗ Corresponding author. Tel.: +86 532 82032439.
E-mail address: huarongguo@ouc.edu.cn (H. Guo).
drial NADH-ubiquinone oxidoreductase (complex I) and interrupt
the electron transport and ATP production of redox respiratory
chain, and this ultimately led to the effects against spider mites.
The species-specific toxicity of the compound is generated, since
the sensitive organisms fail to detoxify fenpyroximate via hydroxy-
lation to metabolite A followed by transesterification to metabolite
B (Motoba et al., 2000).
Studies on the metabolism and degradation of fenpyroximate in
plants, soil and water under laboratory conditions have been car-
ried out. It has been reported that the half-life of fenpyroximate
in the peel of the sprayed Citrus trees is 38.4 days, more than 10
days in the field soils, more than 180 days in the water and 2.6
days for photodegradation in aqueous solution (JMPR, 1995). How-
ever, there is up to now no data giving the environmental levels for
fenpyroximate.

0166-445X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2008.12.006
 N. Na et al. / Aquatic Toxicology 92 (2009) 76–85
The residual, sublethal and chronic toxicity of fenpyroximate to
the spider mites have been examined (M. Kim et al., 2004; Kim et al.,
2006). Results on the fenpyroximate resistance and susceptibility as
well as the stability and inheritance of the fenpyroximate resistance
in the spider mites have also been reported (Goka, 1998; Y.J. Kim
et al., 2004; Sato et al., 2004). Recently, Irigaray and Zalom (2006)
reported the side effects of fenpyroximate to the predator mites, and
Schäfers et al. (2007) tested the zooplankton avoidance behavior
following exposure to fenpyroximate.
It has been reported that fenpyroximate has low toxicity towards
mammals, except for a recent study in the neuroblastoma cells
inferring a potential linkage of fenpyroximate exposure with
Parkinson’s disease (Sherer et al., 2003, 2007). However, in fish,
both rotenone and pyridaben, another two mitochondrial electron
transport (complex I) inhibitors, are highly toxic (Edwards, 1973;
Ding et al., 2004). However, no toxicity data for fenpyroximate have
earlier been reported in fish.
The flounder Paralichthys olivaceus is a kind of marine benthic
flatfish widely cultured in China, and a continuous flounder gill cell
line (FG) has been established (Tong et al., 1997) and successfully
used to study the toxic effects of environmental pollutants on this
fish species (Li and Zhang, 2001; Xu et al., 2007; Xiao et al., 2007; Su
et al., 2007). Although in vitro cultured fish cells have been found to
be a rapid and cost-effective bioassay system to screen the potential
ecotoxicity of pollutants (Rachlin and Perlmutter, 1968; Kocan et al.,
1979; Bols et al., 1985; Babich et al., 1986; Babich and Borenfreund,
1991; Davoren and Fogarty, 2006), and in most of these studies with
fish cell lines a rather good linear correlation (r > 0.8) was obtained
when comparing the in vitro IC50 values with the corresponding
in vivo LC50 values for living fish (Babich and Borenfreund, 1987;
Castano et al., 1996, 2003; Saito et al., 1991), it has also been doc-
umented that, in many cases, in vitro cultured fish cells are more
tolerant to chemicals, usually with one or two orders of magni-
tudes, than living fish. The deviation between IC50 and LC50 values
became more frequent and more pronounced with increasing tox-
icity of the chemicals (Gülden and Seibert, 2005). Studies on the
reasons for the higher tolerance of cultured fish cells than living
fish are quite rare. Gülden and Seibert (2005) suggested that dif-
ference in the bioavailability of chemicals between the in vitro
cultured fish cells and living fish, at least in part, accounted for
this deviation. However, it must be remembered that cell lines and
living animals are different bio-systems, and the toxic effects are
species and chemical-specific. Recently, Yin et al. (2007) reported
the 96h-LC50 of the herbicide butachlor in the flounder (6.55 nM)
and 24h-IC50 in the cultured FG cells (43.32–44.91 ␮M), indicating
a difference of three orders of magnitude. However, the difference
between these two bioassay systems at the same exposure time is
unknown.
Thus the main purposes of this study are to investigate the in
vitro and in vivo acute toxic effects and the possible toxic mecha-
nism of fenpyroximate to seawater fish using flounder and its gill
cells (FG), and to evaluate the correlation between the above two
bioassay systems in the acute toxicity estimation of fenpyroximate.
The correlation analysis could be helpful for the further use of FG
cells instead of living fish.
2. Materials and methods
2.1. Cell line
The continuous cell line FG was derived from the gill tissues
of flounder (P. olivaceus) in 1993 and maintained according to the
method described by Tong et al. (1997). Briefly, the cells were cul-
tured in minimal essential medium (MEM; Gibco BRL, New York)),
supplemented with 10% bovine calf serum (BCS; Hyclone, Utah),
77
100 IU/ml penicillin and 100 mg/ml streptomycin, in plastic culture
flask (Corning) at 20 ◦ C in an atmosphere of air.
2.2. Fish
Marine flounders (P. olivaceus) with body length of 11 ± 2 cm
were purchased from a local fish farm in Jiaonan, Qingdao,
China. These fishes were acclimatized in aerated natural seawater
(18–22 ◦ C) under an ambient photoperiod in the laboratory for 4
days before use. The fishes were fed with live food twice a day and
starved for 24 h before and during the experiments.
2.3. Chemicals
Fenpyroximate with a purity of 97.5% was provided by Bei-
jing Green Agrosino Cooperation (China). Stock solution of 100 mM
fenpyroximate was prepared in acetone and the maximum final
concentration of acetone in treatment medium was below 0.1%.
Neutral red (NR), methyl thiazolyl tetrazolium (MTT) were from
Sigma company (St. Louis, MO, USA), Polyethylene Glycol 6000 (PEG
6000) was from Solarbio company (Beijing, China). All the other
chemicals are analytical grade.
2.4. Determination of the 48h- and 96h-IC50 values in FG cells
FG cells were harvested and diluted to a concentration of 1 × 105
cells/ml in MEM with 10% BCS. The cell suspension was agitated
and 200 ␮l aliquots were added to each well of 96-well tissue cul-
ture plates. Plates were incubated overnight at 20 ◦ C, and the next
day the medium was removed. Based on the preliminary results,
the cells were re-fed with medium containing 0 (control), 0.1,
0.5, 1, 5, 10, 20 and 30 ␮M fenpyroximate for 48h-IC50 analysis,
or 0 (control), 0.1, 0.5, 1, 5 ␮M fenpyroximate for 96h-IC50 analy-
sis, respectively. Then three endpoints for toxicity, i.e. neutral red
(NR) uptake assay, MTT assay and protein concentration assay were
determined after 48- or 96-h exposure.
NR uptake, as described by Borenfreund and Puerner (1985),
measures the inhibition of cell growth, which is based on the
absorbance of the vital dye NR by living, but not by dead, cells.
After 48- or 96-h exposure, the medium in each well was replaced
by 200 ␮l MEM containing 50 mg/ml NR and incubated in situ for 3 h
at 20 ◦ C. Then the cells were rinsed with PBS to remove the NR dye.
After destaining with 200 ␮l solution consisting of glacial acetic
acid: ethanol: water (1:50:49), absorbance of the solution in each
well was measured at 540 nm with a microplate reader and the IC50
value (concentration of test agent which causes a 50% inhibition of
the control) was determined.
MTT assay, as described by Borenfreund et al. (1988), is based on
the inhibition of mitochondrial succinate dehydrogenase, involv-
ing the reduction of soluble yellow MTT tetrazolium salt to a blue
insoluble MTT formazan product. After 48- or 96-h exposure, the
medium was replaced by 200 ␮l 5 mg/ml MTT in PBS. After incu-
bation for 4 h at 20 ◦ C, the MTT solution was removed carefully,
and the cells were rinsed twice with PBS rapidly, then 150 ␮l/well
dimethylsulfoxide (DMSO) was added to dissolve the purple for-
mazan crystals produced. Then the absorbance at 490 nm was
measured and the IC50 value was calculated.
The cell protein assay, as described by Shopsis and Eng (1985),
investigates the cell growth by the alteration of the total cellular
protein. After 48- or 96-h exposure, the medium was removed, and
the cells were washed with PBS and then lysed in 50 ␮l 0.1 mM
NaOH per well for 1 h at 20 ◦ C. Then 200 ␮l Coomassie Blue solution
(10 mg Coomassie Blue G-250 was dissolved in 5 ml 95% ethanol and
10 ml 85% phosphoric acid, and then diluted to the final volume of
100 ml with distilled water) was added to each well and dyed for
20 min at room temperature. Then the absorbance at 595 nm was
78 N. Na et al. / Aquatic Toxicology 92 (2009) 76–85

measured. Serial dilutions of 1–100 mg/ml BSA dissolved in 0.1 mM

NaOH were used for the protein standard.
All experiments were performed at least three times, and the
average absorbance at each concentration was calculated and
expressed as the percentage of absorbance of treated cells against
control.

2.5. Determination of the 48h- and 96h-LC50 values in flounders

Based on the preliminary results, a total of 84 flounders, i.e.

7 individuals randomly selected to every group, were exposed to
a final concentration of 0 (control), 8, 16, 23, 32, and 45 nM fen-
pyroximate in seawater for 48 h, or 0 (control), 4, 8, 12, 16, and
32 nM fenpyroximate for 96 h, respectively. The seawater with a
constant concentration of fenpyroximate was changed every 24 h.
Dead fishes were counted and removed immediately every day. All
experiments were repeated at least three times. The LC50 values
were calculated by linear regression analysis.

2.6. Histopathological examination Fig. 1. In vitro cytotoxicity of fenpyroximate to FG cells after 48-h exposure by NR
uptake, MTT assay and cell protein assay. The individual data points are expressed
as the arithmetic mean percentage of control ± S.D. (n = 3).
Based on the 96h-LC50 value of fenpyroximate in the flounders
obtained previously, the surviving fishes after a 96-h exposure to
12 nM fenpyroximate were sampled and used for the histological for 10 min at 4 ◦ C. The supernatants were used for the antioxidant
examinations. Control fishes were processed similarly. Fishes were enzyme activity test.
anesthetized with 50 mg/l MS 222 (tricaine methane sulfonate) All experiments were performed in triplicate. The protein con-
for 2–3 min, and then the gill, liver and kidney tissues were dis- centrations were standardized by the method of Bradford (1976)
sected and cut into two or three pieces and fixed in freshly prepared with bovine serum albumin as standard.
4% paraformaldehyde in 100 mM phosphate-buffered saline (PBS;
pH 7.4) at 4 ◦ C for 8 h. After serial dehydration in ethanol, these 2.8. CYP1A1 enzyme activity assay
were embedded in paraffin and sectioned at 5 ␮m in thickness.
The sections were stained by haematoxylin and eosin, and were The cytochrome P4501A1 (CYP1A1) enzyme activities in the
photographed under a BX51 Olympus microscope. liver and gill tissues of exposed flounders were examined using
a kit purchased from Genmed Scientifics Inc. (http://www.sh-
2.7. Antioxidant enzyme activity assay genmed.com), based on the ethoxyresorufin-O-deethylase (EROD)
activity and development of resorufin with fluorescence spec-
Changes in the activities of superoxide dismutase (SOD), catalase trophotometry. Briefly, the liver and gill tissues were dissected from
(CAT) and glutathione peroxidase (GPX) during the 48-h exposure the surviving flounders after 0 (control), 6, 12, 24, 48, 72 and 96-h
period of FG cells to a sublethal concentration of fenpyroximate exposure to 12 nM fenpyroximate, respectively. After rinsing three
were examined with kits (Nanjing Jiancheng Bioengineering Insti- times in ice-cold 1.15% KCl solution, the tissues were weighed and
tute, China, http://www.njjcbio.com). In the kits we used, SOD homogenized in four volumes of homogenate buffer (0.25 M cane
activity was determined by luciferase chemiluminescence elicited sugar, 10 mM Tris–HCl, 1 mM EDTA, pH 7.4), then centrifuged at
by xanthine oxidase system; CAT activity was determined by the 9000 × g for 15 min at 4 ◦ C. The supernatant was transferred to a
absorbance of the remaining H2 O2 using a colorimetric method; new tube and 50% PEG 6000 was added slowly to a final concen-
and the GPX activity was assayed by the oxidization of glutathione
(GSH) based on the development of a yellow color with 5,5 -
dithiobis (2-nitrobenzoic acid) (DTNB).
In detail, a total of 36 culture flasks (25 cm2 ) were each seeded
with 5 × 105 cells and incubated for 24 h, then exposed to 300 nM
fenpyroximate in medium. The media of 6 flasks were discarded
at 2, 4, 8, 12, 24 and 48 h of exposure. The cells of the 6 flasks
were then detached by trypsinization, gathered into one tube and
washed once with PBS and centrifugated at 2000 × g for 10 min
at 4 ◦ C. Then the cell pellets were re-suspended in PBS (5%; w/v)
for ultrasonication. The homogenates were centrifuged at 3000 × g
for 10 min at 4 ◦ C, the supernatants were pooled and used for the
enzyme activity assays of SOD, CAT and GPX. Blank controls with-
out fenpyroximate in the medium were set up simultaneously for
each exposure endpoints.
The changes of the activities of antioxidant enzymes of SOD, CAT
and GPX in the liver and gill tissues of the flounders exposed to a
sublethal concentration of fenpyroximate were examined similarly.
Liver and gill tissues were dissected from the surviving fishes after
12, 24, 48, 72, 96-h and 6, 12, 24, 48, 72, 96-h exposure to 12 nM
Fig. 2. In vitro cytotoxicity of fenpyroximate to FG cells after 96-h exposure by NR
fenpyroximate, respectively. The sampled tissues were weighed uptake, MTT assay and cell protein assay. The individual data points are expressed
and homogenized in PBS (5%; w/v) and centrifuged at 3000 × g as the arithmetic mean percentage of control ± S.D. (n = 3).
 N. Na et al. / Aquatic Toxicology 92 (2009) 76–85 79

Table 1
In vivo acute lethality of fenpyroximate to flounder after 48- and 96-h exposures.

48-h exposure 96-h exposure

a
Fenpyroximate (nM) log N No. of dead fishes Mortality (%) Fenpyroximate (nM) log N No. of dead fishesa Mortality (%)

8 0.903 0 0 4 0.602 0 0
16 1.204 1 14.3 8 0.903 1 14.3
23 1.362 1 14.3 12 1.079 3 42.9
32 1.505 3 42.8 16 1.204 6 85.7
45 1.653 7 100 32 1.505 7 100
a
Data are the average of three duplicate experiments; log N, logarithmic concentration of fenpyroximate.

tration of 8.5%, and then centrifuged at 10 000 × g for 20 min at
4 ◦ C. The microsome pellets were washed with ice-cold KCl buffer
(0.15 M KC1, 10 mM Tris–HCl, 1 mM EDTA, pH 7.4) and then the PEG
6000 precipitation repeated again (Zheng et al., 2002). The pel-
lets were then re-suspended with homogenation buffer (4 ◦ C) and
frozen in liquid nitrogen before EROD activity analysis.
2.9. GST (glutathione S-transferase) enzyme activity assay
2.10. Data analysis
Each experiment was repeated at least three times. Data
obtained were expressed as mean ± SD and evaluated by one-way
ANOVA (SPSS v13 for Windows, tests: least significant difference,
Tukey’s honestly significant difference). The confidence limits (95%)
of IC50 and LC50 values were calculated based on Student’s t-
distribution (Student, 1908; Fisher, 1925).

The GST enzyme activities in the liver and gill tissues of exposed
flounders were determined by the method of Habig et al. (1974),
with a kit purchased from Nanjing Jiancheng Bioengineering Insti-
tute, China. Briefly, liver and gill tissues were dissected from the
survival flounders after 6, 12, 24, 48, 72 and 96-h exposure to
12 nM fenpyroximate, respectively. Then the sampled tissues were
weighed and homogenized in PBS (5%; w/v) and centrifuged at
3000 × g for 10 min at 4 ◦ C. The supernatants were used for GST
activity assay according to the kit protocol.
3. Results
3.1. IC50 and LC50
As shown in Figs. 1 and 2, fenpyroximate was cytotoxic
to FG cells at all tested concentrations in both concentration-
and time-dependent manner. By linear regression analysis, the
48h-IC50 (95% confidence limits) values of fenpyroximate to
the FG cells were 890 (790–990) nM, 950 (881–1019) nM and

Fig. 3. Histopathological changes of the gill tissue of the flounders exposed to 12 nM fenpyroximate for 96 h. (A) Structure of the normal gill filament (control); (B) partial
enlargement of (A) encircled by the quadrate frame; (C) structure of the gill filament exposed to 12 nM fenpyroximate for 96 h; (D) partial enlargement of (C) encircled by
the quadrate frame. Gf, gill filament; Sf, secondary filament of gill; Lc, lamellar epithelial cells; Bm, basement membrane; Er, erythrocyte; Pc, pillar cells; Cc, chloride cell; Bc,
blood congestion; Ld, lifting and detachment of epithelial cells.
80 N. Na et al. / Aquatic Toxicology 92 (2009) 76–85
1250 (1159–1341) nM, and the 96h-IC50 (95% confidence lim-
its) values were 480 (388–572) nM, 490 (454–526) nM and 510
(469–551) nM, for MTT, NR uptake and cell protein assays,
respectively.
As shown in Table 1, the toxic effect of fenpyroximate on the sur-
vival of fishes was also both concentration- and time-dependent.
In the 48-h exposure test, 8 nM fenpyroximate had no lethal toxic-
ity on the flounders. Above 16 nM, exposed fishes began to die. At
45 nM, all exposed fishes died in the 48-h exposure period. After
linear regression analysis, the 48h-LC50 (95% confidence limits) of
fenpyroximate to the flounders was 28.84 (14.28–58.26) nM. In the
96-h exposure test, however, 4 nM fenpyroximate had no lethal
toxicity on flounders, and the corresponding lowest effect concen-
tration was 8 nM, and the 100% lethal concentration was 32 nM. The
96h-LC50 (95% confidence limits) of fenpyroximate to the flounders
was 11.74 (6.06–22.8) nM.
3.2. Histopathological changes of the gill, liver and kidney tissues
of the exposed flounders
Obvious histopathological damages were observed in the
exposed flounder gill tissue (Fig. 3). The secondary lamellae of the
exposed gills swelled and epidermis burst apart. The lifting and
detachment of lamellar epithelial cells and blood congestion widely
occurred, indicating that the respiratory function of the gills was
badly impaired.
In the liver tissue of the exposed flounders, no obvi-
ous histopathological changes were observed in the hepatic
parenchyma cells, but the hepatic biliary system was injured (Fig. 4).
The endothelial cells of bile duct swelled and merged with one
another, resulting in roughness and abnormality of the duct wall.
Fig. 4. Histopathological alteration of the liver tissue of flounders exposed to 12 nM fenpyroximate for 96 h. (A) Structure of the normal liver (control); (B) partial enlargement
of (A) encircled by the quadrate frame; (C) structure of the treated liver exposed to 12 nM fenpyroximate for 96 h; (D) partial enlargement of (C) encircled by the quadrate
frame. Hc, hepatic parenchymal cells; Bd, bile duct; Dw, bile duct wall cell.
In contrast, no obvious histopathological changes were observed
in the exposed flounder kidney (data not shown). Therefore, gill and
liver are two important target organs of fenpyroximate.
3.3. Changes of antioxidant enzyme activities in exposed FG cells
and flounder tissues
In the exposed FG cells, the activities of antioxidant enzymes
SOD, CAT and GPX showed similar responses to a sublethal con-
centration of fenpyroximate during the 48-h exposure period. As
shown in Fig. 5, there was a rapid and marked increase in the activ-
ities of all these enzymes tested within the first 12-h exposure,
and then the enzymes were inhibited and their activities decreased
quickly to and were maintained at a constant level lower than that
of control (P < 0.05). The enzymes differed in the time needed to
reach the peak activity upon exposure, for SOD the time is 2 h, for
CAT 4 h and for GPX 8 h.
In the gill and liver tissues of the exposed flounders, similar
stress responses were observed in the activities of antioxidant
enzymes tested during the 96-h exposure of flounder to 12 nM
fenpyroximate (Figs. 6 and 7). However, there was a clear dif-
ference in the response time and background enzyme activity
between liver and gill tissues. The gill tissue of the exposed flounder
responded much more quickly to fenpyroximate than liver. A signif-
icant increase in the activities of all the three antioxidant enzymes
tested was observed in the gill tissue as early as 6 h of exposure
(Fig. 6), but at least 12 h (for GPX) or 24 h (for SOD and CAT) exposure
was required for the liver tissue to respond (Fig. 7). Nevertheless,
the response time needed for the above three antioxidant enzymes
to reach to their peak activity was about 12 h for SOD, 24 h for CAT
and 48 h for GPX in the gill tissue, and 24 h for GPX, and 48 h for SOD
 N. Na et al. / Aquatic Toxicology 92 (2009) 76–85 81

and CAT in the liver tissue, respectively. The results obtained also
showed that the background activities of the above three antioxi-
dant enzymes tested in the liver tissue were about 3–6 times higher
than those in the gill tissue of flounder (Figs. 6 and 7).
Taken together, the time needed for the above three antioxidant
enzymes to reach their peak activity was 2–8 h for FG cells, 12–48 h
for flounder gill tissue and 24–48 h for flounder liver tissues, respec-
tively. Thus the in vitro cultured FG cells were much more sensitive
and responded more rapidly than the cells of living flounders to
fenpyroximate.

Fig. 6. The change of the activities of antioxidant enzymes of SOD, CAT and GPX in
gill tissue of flounders exposed to 0 (control) and 12 nM fenpyroximate for 96 h. Data
are expressed as means ± S.D. (n = 3). * Significant difference from control (P < 0.05).

3.4. Changes of the EROD and GST activities in the liver and gill
tissues of exposed flounders

In order to compare the detoxification capacity between liver

Fig. 5. The change of the activities of antioxidant enzymes of SOD, CAT and GPX
in the FG cells exposed to 0 (control) and 300 nM fenpyroximate for 48 h. Data are
expressed as means ± S.D. (n = 3). * Significant difference from control (P < 0.05).
and gill of flounders upon exposure to fenpyroximate, the changes
of the EROD and GST activities were determined (Fig. 8). The results
obtained showed that, (1) the background levels of both EROD
and GST enzyme activities in the liver tissue were much higher
than those in the gill tissue, indicating higher detoxification capac-
ity in liver than in gill; (2) the activities of both EROD and GST
enzyme activities increased significantly in the first 6 h of expo-
sure in both liver and gill tissue of exposed flounders in response
82 N. Na et al. / Aquatic Toxicology 92 (2009) 76–85
Fig. 7. The change of the activities of antioxidant enzymes of SOD, CAT and GPX
in liver tissue of flounders exposed to 0 (control) and 12 nM fenpyroximate for
96 h. Data are expressed as means ± S.D. (n = 3). * Significant difference from control
(P < 0.05).
to fenpyroximate; (3) the time for EROD enzyme to reach to its
peak activity after exposure was similar (6 h) in both liver and
gill. However, after the peak activity the EROD activity in liver
decreased much more slowly to the constant level than that in gills.
This would contribute more detoxification capacity to liver than
to gill upon exposure to fenpyroximate; (4) similar to the EROD,
the time for GST enzyme to reach its peak activity was also the
same (24 h) in liver and gills. However, the GST activity in liver
decreased much more quickly to the control level than that in
gill.
4. Discussion
4.1. Fenpyroximate is highly toxic to marine flounder
Fenpyroximate has moderate oral toxicity on mammals and it
seems that the female is more sensitive to fenpyroximate than the
male (Fujimori, 1995). The oral LD50 values for rats and mice are
245–520 mg/kg bw (body weight). In the oral feeding test of 8 dogs
(male:female = 1:1), only two female dogs died after 5 weeks of
exposure to 50 mg/kg bw fenpyroximate. Similarly, of the tested
pregnant New Zealand white rabbits, two female rabbits died after
4 weeks of exposure to 2.5 mg/kg bw fenpyroximate per day, and the
abortion rate increased in a slight dose-dependent manner. In the
present study, all the living flounders were immature and randomly
selected, so any sex-related toxicity of fenpyroximate in flounders
cannot be evaluated. As shown in Table 2, both the 48h- and 96h-
IC50 and LC50 values obtained for fenpyroximate are lower than
1 ppm. This indicates that fenpyroximate is highly toxic to both FG
cells and living flounders according to the testing criteria for pes-
ticides on the non-target organisms. Therefore, fenpyroximate has
the same toxicity grade on seawater fish as the other two mito-
chondrial electron transport (complex I) inhibitors rotenone and
pyridaben have on freshwater fish. The 96h-LC50 value of pyrid-
aben in zebrafish is 0.012 ppm (Ding et al., 2004), and of rotenone
in rainbow trout 0.026 ppm (Edwards, 1973).
4.2. Exposure time has no effect on the correlation between the
responses of FG cells and living flounders in the estimation of
fenpyroximate toxicity
It is not surprising that the in vitro and in vivo acute toxicity
of fenpyroximate in the FG cells and living flounders is time-
dependent. As shown in Table 2, both the 48h-IC50 and LC50 values
are much higher than the corresponding 96h-IC50 and LC50 , respec-
tively. In our previous study, the 24h-IC50 of herbicide butachlor to
the FG cells and the 96h-LC50 of butachlor to the living flounders
differed by three orders of magnitude (Yin et al., 2007). The differ-
ence in exposure time may account for some of above enormous
deviation.
It is of interest to note that in the present study the ratio of
average 48h-IC50 in the FG cells to 48h-LC50 in the living fish is 35.8
and the ratio of average 96h-IC50 in the FG cells to 96h-LC50 in the
live fish is 41.7. It seems that the length of exposure time did not
significantly affect the relationship between the above two bioassay
systems in the estimation of fenpyroximate toxicity provided the
selected exposure time is the same. In vitro cultured FG cells are
useful for rapid estimation of the toxicity of pollutants and be a good
alternative to replace the in vivo acute lethality test with living fish.
4.3. Toxic mechanism of fenpyroximate to flounder
The acaricidal mechanism of fenpyroximate has been examined
in detail using the two-spotted spider mite. Fenpyroximate can
inhibit the activity of mitochondrial NADH-ubiquinone oxidore-
ductase (complex I) and interrupt the electron transport and ATP
production of redox respiratory chain (Motoba et al., 1992). On the
other hand, fenpyroximate cannot be successfully metabolized and
detoxified by these mites. In contrast, fenpyroximate can be detox-
ified efficiently in the liver tissue of several non-target organisms,
including mammals and carp, instead (Motoba et al., 2000). In the
histopathological examination of the gills, liver and kidney of the
exposed flounders, it is found that the gill and liver, but not the kid-
ney, are two target organs of fenpyroximate. Gill is directly exposed
to fenpyroximate, and it seems that gill cells are sensitive and have
much lower capability to detoxify fenpyroximate than liver cells.
Lesions including the lifting and detachment of epithelial cells and
 N. Na et al. / Aquatic Toxicology 92 (2009) 76–85
Fig. 8. The change of the enzyme activities of EROD and GST in liver and gill tissues of flounders exposed to 0 (control) and 12 nM fenpyroximate for 96 h. Data are expressed
as means ± S.D. (n = 3). * Significant difference from control (P < 0.05).
blood congestion of the gill filaments were induced. Gill serves as a
major organ for respiration, osmoregulation, nitrogen excretion and
pH regulation. Failure of the gill to function during the acute expo-
sure to fenpyroximate may to some degree account for the high
lethality of the exposed flounders. There is no obvious histologi-
cal change in the hepatic parenchyma cells after 96-h exposure of
flounders to a sublethal concentration of fenpyroximate, indicating
the possible high detoxification capability of liver cells to fenpy-
roximate. However, the destructive effect of fenpyroximate on the
hepatic biliary system was observed. The edema and fusion of the
endothelial cells of the bile duct occurred widely. The mechanism
of the selective toxicity of fenpyroximate to hepatic bile ducts is
unclear. One possible explanation is the lack of detoxicification of
fenpyroximate in the wall cells of the bile duct. Considering the
fact of efficient detoxification of fenpyroximate by flounder liver
tissue, it is not surprising to find that kidney is not affected by the
fenpyroximate.
In order to further confirm the conclusion based on the
histopathological examination, that is, that detoxification capac-
ity accounts for much of the different sensitivity to fenpyroximate
between gill and liver of exposed flounders, the activities of two
important detoxification metabolism-related enzymes, GST and the
Table 2
Comparison of the in vitro IC50 and in vivo LC50 values of fenpyroximate to FG cells and living flounders.
48h-IC50
MTT NR CP Average
nM 890 950 1250 1030
ppm 0.375 0.399 0.526 0.433
83
CYP1A1-containing EROD, were determined in the liver and gill
tissues of exposed flounders. The results we obtained provided
support for the above postulation: (1) the background levels of
both EROD and GST enzyme activities in liver were much higher
than those in gills; (2) the activities of both EROD and GST enzyme
increased significantly in the first 6 h of fenpyroximate exposure
in both liver and gill tissues; (3) after the first 6 h of exposure, the
EROD activity in liver decreased much more slowly than that in gills.
Similar results have also been reported for other aquatic organisms
(Lemaire et al., 1992; Goksøyr and Förlin, 1992; George, 1994; Peters
et al., 1997; Gallagher et al., 2001; Flammarion et al., 2002; Teles et
al., 2003; Abrahamson et al., 2007).
Analysis of the changes of the antioxidant enzyme activities in
the FG cells, liver and gill tissues of flounders exposed to sublethal
concentration of fenpyroximate indicated that fenpyroximate could
induce a rapid and significant increase of SOD, CAT and GPX activi-
ties in both the in vitro and in vivo assays, implying the occurrence
of oxidative stress in the exposed fish cells, suggesting that the
mechanism of fenpyroximate toxicity involves effects on the mito-
chondrial redox respiratory chain (Barrientos and Moraes, 1999;
Motoba et al., 1992). Possibly the oxidative stress overruns the tol-
erance of the fish cells, and eventually the antioxidant enzyme
48h-LC50 96h-IC50 96h-LC50
MTT NR CP Average
28.84 480 490 510 490 11.74
0.005 0.202 0.206 0.214 0.206 0.012
84 N. Na et al. / Aquatic Toxicology 92 (2009) 76–85
activities are inhibited and exposed cells injured. Interestingly, the
time needed for the above three antioxidant enzymes to reach to
their peak activity was 2–8 h for FG cells, 12–48 h for flounder gills
and 24–48 h for flounder livers, respectively. Obviously, FG cells
were exposed directly and responded much more rapidly to the
toxicity of fenpyroximate than the exposed flounder tissues. Also,
the longer time needed for fenpyroximate to be absorbed and trans-
ported into the liver than into the gill, might to some degree account
for the longer response time in the liver than in the gills for these
antioxidant enzymes.
5. Conclusions
Taken together, the main findings in this study are the following:
Fenpyroximate is highly toxic to marine flounder. In vitro cultured
FG cells are good system for rapid estimation of the pollutant tox-
icity. The length of exposure time does not significantly affect the
fenpyroximate toxicity relationship between the FG cells and liv-
ing flounders provided that the selected exposure time is the same.
Gills and liver are two target organs of fenpyroximate, especially
the damage to gill tissue may account for much of the high lethality
of exposed flounders. Differences in detoxification capacity largely
account for the higher sensitivity of gill tissues to fenpyroximate
than of liver tissue. Fenpyroximate can induce a rapid and signifi-
cant increase of the activities of the antioxidant enzymes of SOD,
CAT and GPX both in vivo and in exposed FG cells. After the initial
induction of enzymes, they were inhibited, implying the occur-
rence of oxidative stress in the exposed fish cells, and the possible
interruption of the mitochondrial redox respiratory chain by the
fenpyroximate.
Acknowledgements
We thank Peilong Xu from Qingdao University, China for the
great help in the experiments, and Beijing Green Agrosino Coop-
eration (China) for the kind provision of the fenpyroximate. This
work was funded by the national high technology research and
development program (863 Program No. 2001AA628130) in China.
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