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Tingting Ma, Wei Zhou, Li’ke Chen, Longhua Wu, Peter Christie, Haibo Zhang, Yongming Luo
Toxicity effects of di-(2-ethylhexyl) phthalate to Eisenia fetida at enzyme, cellular and genetic levels
www.plosone.org
www.plosone.org
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2.6589 0 Td
(Co.)Tj
1.6158 0 Td
(Ltd.,)Tj
2.0352 0 Td
(Shanghai,)Tj
4.1272 0 Td
(China.)Tj
2.8857 0 Td
(The)Tj
1.7801 0 Td
(Dako)Tj
2.3981 0 Td
(LSAB)Tj
2.4945 0 Td
(peroxidase)Tj
4.5184 0 Td
(kit)Tj
1.2813 0 Td
(was)Tj
1.7007 0 Td
(obtained)Tj
3.7304 0 Td
(from)Tj
-34.6391 -1.2982 Td
(Dako)Tj
2.3924 0 Td
(A/S,)Tj
1.9332 0 Td
(Glostrup,)Tj
3.9911 0 Td
(Denmark.)Tj
4.2747 0 Td
(The)Tj
1.7801 0 Td
(Fluo-3/AM)Tj
4.7622 0 Td
(kit)Tj
1.2812 0 Td
(for)Tj
1.389 0 Td
(intracellular)Tj
5.057 0 Td
(Ca)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 479.622 569.707 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2+)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 489.7133 565.285 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(concentration)Tj
5.8166 0 Td
(and)Tj
-34.7864 -1.3039 Td
(the)Tj
1.4683 0 Td
(Rhodamine)Tj
4.8982 0 Td
(123)Tj
1.6441 0 Td
(kit)Tj
1.2813 0 Td
(for)Tj
1.3889 0 Td
(mitochondrial)Tj
5.9641 0 Td
(membrane)Tj
4.5694 0 Td
(potential)Tj
3.7587 0 Td
(difference)Tj
4.2065 0 Td
(were)Tj
2.1203 0 Td
(sourced)Tj
3.3619 0 Td
(from)Tj
-34.6617 -1.2982 Td
(Becton)Tj
2.999 0 Td
(Dickinson/BD)Tj
6.0207 0 Td
(Biosciences)Tj
4.8076 0 Td
(Pharmingen,)Tj
5.4084 0 Td
(San)Tj
1.6781 0 Td
(Diego,)Tj
2.846 0 Td
(CA.)Tj
1.7915 0 Td
(Guaiacol)Tj
3.7756 0 Td
(glyceryl)Tj
3.3052 0 Td
(ether)Tj
-32.6321 -1.2983 Td
(\()Tj
/F16 1 Tf
0 Tc
()Tj
/F9 1 Tf
-0.004 Tc
(98%,)Tj
3.2654 0 Td
(C)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 239.3007 524.5795 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(10)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 246.3874 526.2803 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(H)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 254.0976 524.5795 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(14)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 261.1842 526.2803 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(O)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 268.6677 524.5795 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(4)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 272.2393 526.2803 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(\),)Tj
0.7823 0 Td
(monoclonal)Tj
5.006 0 Td
(mouse)Tj
2.8629 0 Td
(anti-human)Tj
4.972 0 Td
(anti-HSP)Tj
3.9231 0 Td
(70,)Tj
1.3946 0 Td
(Triton)Tj
2.8233 0 Td
(X)Tj
0.8731 0 Td
(and)Tj
1.7348 0 Td
(bovine)Tj
2.9253 0 Td
(serum)Tj
-34.52 -1.3039 Td
(albumin)Tj
3.5716 0 Td
(\(BSA\))Tj
2.6363 0 Td
(were)Tj
2.1258 0 Td
(obtained)Tj
3.7304 0 Td
(from)Tj
2.2053 0 Td
(Sigma)Tj
2.6816 0 Td
(Chemical)Tj
4.0252 0 Td
(Co.,)Tj
1.8425 0 Td
(St.)Tj
1.2189 0 Td
(Louis,)Tj
2.6475 0 Td
(MO.)Tj
2.0806 0 Td
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1.4003 0 Td
(other)Tj
2.3471 0 Td
(chemicals)Tj
-32.5131 -1.2982 Td
(used)Tj
2.058 0 Td
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2.1259 0 Td
(analytical)Tj
4.0024 0 Td
(grade)Tj
2.4435 0 Td
(reagents)Tj
3.5433 0 Td
(purchased)Tj
4.3369 0 Td
(from)Tj
2.2054 0 Td
(the)Tj
1.4683 0 Td
(National)Tj
3.6907 0 Td
(Pharmaceutical)Tj
6.4119 0 Td
(Group)Tj
-32.2863 -1.2983 Td
(Chemical)Tj
4.0252 0 Td
(Reagent)Tj
3.4355 0 Td
(Co.,)Tj
1.8368 0 Td
(Ltd.,)Tj
2.0353 0 Td
(Shanghai,)Tj
4.1272 0 Td
(China.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 458.1354 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Quantitative)Tj
5.4992 0 Td
(analysis)Tj
3.7843 0 Td
(of)Tj
1.0441 0 Td
(DEHP)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 441.1275 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(The)Tj
1.7801 0 Td
(analysis)Tj
3.3278 0 Td
(of)Tj
1.0148 0 Td
(DEHP)Tj
2.8517 0 Td
(concentrations)Tj
6.1795 0 Td
(in)Tj
1.0374 0 Td
(soil)Tj
1.5988 0 Td
(after)Tj
2.0352 0 Td
(incubation)Tj
4.5524 0 Td
(for)Tj
1.3833 0 Td
(28)Tj
1.1736 0 Td
(days)Tj
1.9956 0 Td
(was)Tj
1.7007 0 Td
(carried)Tj
3.0388 0 Td
(out)Tj
-33.6697 -1.2982 Td
(according)Tj
4.1839 0 Td
(to)Tj
1.0261 0 Td
(Ma)Tj
1.5477 0 Td
(et)Tj
0.9411 0 Td
(al)Tj
0.9014 0 Td
([)Tj
0.83 0.64 0.02 0 k
(19)Tj
0 g
(].)Tj
2.075 0 Td
(DEHP)Tj
2.8516 0 Td
(in)Tj
1.0375 0 Td
(10)Tj
1.1735 0 Td
(g)Tj
0.686 0 Td
(of)Tj
1.0148 0 Td
(soil)Tj
1.5987 0 Td
(was)Tj
1.7008 0 Td
(extracted)Tj
3.8834 0 Td
(with)Tj
1.9956 0 Td
(a)Tj
0.6576 0 Td
(total)Tj
2.0013 0 Td
(volume)Tj
3.1974 0 Td
(of)Tj
1.0148 0 Td
(70)Tj
1.1736 0 Td
(mL)Tj
-34.6618 -1.3039 Td
(acetone:hexane)Tj
6.3268 0 Td
(\(1:1)Tj
1.7348 0 Td
(v/v\))Tj
1.7972 0 Td
(three)Tj
2.2677 0 Td
(times)Tj
2.3867 0 Td
(in)Tj
1.0375 0 Td
(a)Tj
0.652 0 Td
(water)Tj
2.4321 0 Td
(bath)Tj
1.9899 0 Td
(at)Tj
0.9524 0 Td
(25C)Tj
2.2224 0 Td
(and)Tj
1.7348 0 Td
(reduced)Tj
3.4412 0 Td
(in)Tj
1.0375 0 Td
(the)Tj
1.4683 0 Td
(flask)Tj
2.0466 0 Td
(by)Tj
1.1792 0 Td
(rotary)Tj
-34.7071 -1.2983 Td
(evaporation)Tj
4.9832 0 Td
(to)Tj
1.0262 0 Td
(12)Tj
1.6837 0 Td
(mL)Tj
1.5818 0 Td
(\(350)Tj
1.9842 0 Td
(mbar,)Tj
2.5795 0 Td
(40C)Tj
2.228 0 Td
(water)Tj
2.4265 0 Td
(bath,)Tj
2.2166 0 Td
(80)Tj
1.1679 0 Td
(rpm\))Tj
2.2677 0 Td
(after)Tj
2.0353 0 Td
(centrifugation)Tj
5.8903 0 Td
(at)Tj
0.9525 0 Td
(1)Tj
0.6973 0 Td
(500)Tj
1.6441 0 Td
(rpm.)Tj
-35.3648 -1.2983 Td
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3.5319 0 Td
(chromatography)Tj
6.8768 0 Td
(purification)Tj
4.9379 0 Td
(was)Tj
1.7008 0 Td
(performed)Tj
4.4674 0 Td
(in)Tj
1.0374 0 Td
(a)Tj
0.652 0 Td
(glass)Tj
2.0863 0 Td
(column)Tj
3.2938 0 Td
(\(1)Tj
/F17 1 Tf
1.0375 0 Td
[()]TJ
/F9 1 Tf
0.788 0 Td
(26)Tj
1.1736 0 Td
(cm\))Tj
1.8028 0 Td
(with)Tj
-33.3862 -1.2982 Td
(Na)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 211.6913 374.4566 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 215.2629 376.1574 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(SO)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 227.5086 374.4566 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(4)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 231.0236 376.1574 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(,)Tj
0.4478 0 Td
(neutral)Tj
3.0671 0 Td
(Al)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 275.4708 374.4566 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 279.0425 376.1574 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(O)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 286.5259 374.4566 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(3)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 292.2519 376.1574 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(and)Tj
1.7347 0 Td
(neutral)Tj
3.0671 0 Td
(silica)Tj
2.2167 0 Td
(gel)Tj
1.3549 0 Td
(\(from)Tj
2.5512 0 Td
(bottom)Tj
3.1521 0 Td
(to)Tj
1.0261 0 Td
(top\))Tj
1.8822 0 Td
(with)Tj
2.0013 0 Td
(acetone:hexane)Tj
6.3269 0 Td
(\(1:4)Tj
-34.537 -1.3039 Td
(v/v\))Tj
1.7971 0 Td
(before)Tj
2.7383 0 Td
(collection)Tj
4.1159 0 Td
(and)Tj
1.729 0 Td
(reduction)Tj
4.1159 0 Td
(to)Tj
/F14 1 Tf
0 Tc
1.0204 0 Td
(<)Tj
/F9 1 Tf
-0.004 Tc
0.9921 0 Td
(1)Tj
0.6974 0 Td
(mL)Tj
1.576 0 Td
(by)Tj
1.1849 0 Td
(rotary)Tj
2.6532 0 Td
(evaporation)Tj
4.9833 0 Td
(as)Tj
1.0148 0 Td
(described)Tj
4.0535 0 Td
(above.)Tj
-31.4756 -1.2982 Td
(Analysis)Tj
3.5716 0 Td
(was)Tj
1.6895 0 Td
(performed)Tj
4.4616 0 Td
(with)Tj
1.9899 0 Td
(an)Tj
1.1905 0 Td
(Agilent)Tj
3.1464 0 Td
(7890GC-5975)Tj
5.743 0 Td
(MSD)Tj
2.3187 0 Td
(gas)Tj
1.4684 0 Td
(chromatograph-ma)Tj
7.8122 0 Td
(ss)Tj
0.9298 0 Td
(spec-)Tj
-35.5178 -1.2983 Td
(trometer.)Tj
3.9571 0 Td
(During)Tj
3.1351 0 Td
(analysis,)Tj
3.5545 0 Td
(whole)Tj
2.6079 0 Td
(procedure)Tj
4.3143 0 Td
(blanks,)Tj
3.0387 0 Td
(soil)Tj
1.5987 0 Td
(matrix)Tj
2.8744 0 Td
(blanks,)Tj
3.0387 0 Td
(spiked)Tj
2.8119 0 Td
(soil)Tj
1.6044 0 Td
(matrix)Tj
2.8743 0 Td
(and)Tj
-35.41 -1.3039 Td
(parallel)Tj
3.1351 0 Td
(samples)Tj
3.3845 0 Td
(were)Tj
2.1203 0 Td
(all)Tj
1.1508 0 Td
(employed)Tj
4.1385 0 Td
(together)Tj
3.532 0 Td
(with)Tj
2.0012 0 Td
(analysis)Tj
3.3222 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.474 0 Td
(certified)Tj
3.5093 0 Td
(reference)Tj
3.9174 0 Td
(material)Tj
3.515 0 Td
(to)Tj
-36.2151 -1.2983 Td
(ensure)Tj
2.8743 0 Td
(quality)Tj
2.9537 0 Td
(control.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 281.9905 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Earthworms)Tj
5.5039 0 Td
(and)Tj
1.8662 0 Td
(soil)Tj
1.6913 0 Td
(preparation)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 264.9826 cm
BT
/F15 1 Tf
1 TL
-0.004 Tc
0 0 Td
[()16()-4()-3()2()18()4()]TJ
3.0387 0 Td
[()9()8()7()4( )4()]TJ
/F9 1 Tf
2.4264 0 Td
(individuals)Tj
4.6488 0 Td
(purchased)Tj
4.3313 0 Td
(from)Tj
2.2054 0 Td
(a)Tj
0.6576 0 Td
(farm)Tj
2.1316 0 Td
(in)Tj
1.0375 0 Td
(Nanjing,)Tj
3.702 0 Td
(East)Tj
1.8822 0 Td
(China)Tj
2.6589 0 Td
(were)Tj
2.1203 0 Td
(selected)Tj
3.3505 0 Td
(as)Tj
1.0148 0 Td
(the)Tj
-35.206 -1.2982 Td
(test)Tj
1.5987 0 Td
(organism.)Tj
4.2179 0 Td
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2.2394 0 Td
(were)Tj
2.1203 0 Td
(held)Tj
1.9502 0 Td
(for)Tj
1.3833 0 Td
(one)Tj
1.7008 0 Td
(month)Tj
2.9196 0 Td
(in)Tj
1.0375 0 Td
(the)Tj
1.4683 0 Td
(laboratory)Tj
4.3484 0 Td
(and)Tj
1.7348 0 Td
(were)Tj
2.1203 0 Td
(fed)Tj
1.457 0 Td
(with)Tj
2.0012 0 Td
(cattle)Tj
2.3414 0 Td
(feces)Tj
2.1373 0 Td
(at)Tj
-36.7764 -1.2983 Td
(20)Tj
/F17 1 Tf
1.1678 0 Td
[()]TJ
/F9 1 Tf
0.7937 0 Td
(1C)Tj
1.7518 0 Td
(at)Tj
0.9468 0 Td
(a)Tj
0.6576 0 Td
(moisture)Tj
3.7927 0 Td
(content)Tj
3.2712 0 Td
(of)Tj
1.0148 0 Td
(about)Tj
2.4945 0 Td
(50%.)Tj
2.1543 0 Td
(Healthy)Tj
3.3675 0 Td
(strong)Tj
2.7723 0 Td
(adult)Tj
2.2564 0 Td
(earthworms)Tj
5.0172 0 Td
(with)Tj
1.9956 0 Td
(a)Tj
0.6577 0 Td
(visible)Tj
-34.1119 -1.3039 Td
(clitellum)Tj
3.719 0 Td
(\(350)Tj
/F17 1 Tf
1.9842 0 Td
[()]TJ
/F9 1 Tf
0.788 0 Td
(50)Tj
1.1736 0 Td
(mg\))Tj
1.8425 0 Td
(were)Tj
2.126 0 Td
(selected)Tj
3.3448 0 Td
(24)Tj
1.1736 0 Td
(h)Tj
0.7483 0 Td
(prior)Tj
2.2507 0 Td
(to)Tj
1.0261 0 Td
(use)Tj
1.5307 0 Td
(and)Tj
1.7348 0 Td
(their)Tj
2.1033 0 Td
(gut)Tj
1.5137 0 Td
(was)Tj
1.7008 0 Td
(purged.)Tj
-27.5639 -1.2983 Td
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0.907 0 Td
(typical)Tj
2.863 0 Td
(yellow)Tj
2.778 0 Td
(brown)Tj
2.8403 0 Td
(soil)Tj
1.5987 0 Td
(classified)Tj
3.8211 0 Td
(as)Tj
1.0148 0 Td
(Alfisols)Tj
3.1804 0 Td
(according)Tj
4.1896 0 Td
(to)Tj
1.0205 0 Td
(the)Tj
1.474 0 Td
(USDA)Tj
2.8516 0 Td
(soil)Tj
1.5987 0 Td
(classification)Tj
-31.3339 -1.2982 Td
(with)Tj
1.9955 0 Td
(a)Tj
0.652 0 Td
(pH)Tj
1.508 0 Td
(\(in)Tj
1.3776 0 Td
(water\))Tj
2.7666 0 Td
(of)Tj
1.0148 0 Td
(7.4)Tj
1.4003 0 Td
(was)Tj
1.6952 0 Td
(collected)Tj
3.7417 0 Td
(from)Tj
2.2053 0 Td
(the)Tj
1.4683 0 Td
(top)Tj
1.5421 0 Td
(15)Tj
1.1735 0 Td
(cm)Tj
1.4627 0 Td
(of)Tj
1.0148 0 Td
(a)Tj
0.6519 0 Td
(seldom)Tj
3.1068 0 Td
(disturbed)Tj
4.0308 0 Td
(mountain)Tj
-32.8079 -1.3039 Td
(area)Tj
1.8821 0 Td
(\(32)Tj
1.9049 0 Td
(8')Tj
0.9071 0 Td
(51")Tj
1.491 0 Td
(N,)Tj
1.1849 0 Td
(118)Tj
2.0352 0 Td
(57')Tj
1.389 0 Td
(58")Tj
1.491 0 Td
(E\))Tj
1.1282 0 Td
(at)Tj
0.9524 0 Td
(Qixia)Tj
2.3981 0 Td
(District,)Tj
3.4696 0 Td
(Nanjing,)Tj
3.702 0 Td
(Jiangsu)Tj
3.1522 0 Td
(Province,)Tj
4.0024 0 Td
(East)Tj
1.8822 0 Td
(China.)Tj
2.8857 0 Td
(The)Tj
-35.858 -1.2983 Td
(soil)Tj
1.5987 0 Td
(has)Tj
1.542 0 Td
(a)Tj
0.6577 0 Td
(clay)Tj
1.7744 0 Td
(content)Tj
3.2712 0 Td
(of)Tj
1.0148 0 Td
(1.67)Tj
1.8708 0 Td
(g)Tj
0.686 0 Td
(kg)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 333.7511 178.4692 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(-1)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 339.9307 173.9905 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(,)Tj
0.4478 0 Td
(an)Tj
1.2019 0 Td
(organic)Tj
3.2371 0 Td
(matter)Tj
2.863 0 Td
(content)Tj
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-0.004 Tc
0 0 Td
(O)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 550.7149 458.3055 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 554.2299 460.0062 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(.)Tj
-35.4215 -1.2982 Td
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3.6397 0 Td
(in)Tj
1.0375 0 Td
(absorbance)Tj
4.7395 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4683 0 Td
(reaction)Tj
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(solution)Tj
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(470)Tj
1.6498 0 Td
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(every)Tj
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(60)Tj
1.1736 0 Td
(s.)Tj
0.805 0 Td
(POD)Tj
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3.1181 0 Td
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0.8448 0 Td
(expressed)Tj
4.0932 0 Td
(by)Tj
1.1848 0 Td
(change)Tj
3.0444 0 Td
(in)Tj
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(absorbance)Tj
4.7395 0 Td
(as)Tj
1.0148 0 Td
(U)Tj
0.9525 0 Td
(g)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 404.9007 438.4629 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(-1)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 413.2913 433.9842 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(FW)Tj
1.7121 0 Td
(min)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 446.7968 438.4629 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(-1)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 452.9763 433.9842 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(.)Tj
-24.1 -1.2982 Td
(Two)Tj
2.024 0 Td
(mL)Tj
1.576 0 Td
(of)Tj
1.0148 0 Td
(collected)Tj
3.7417 0 Td
(enzyme)Tj
3.3052 0 Td
(solution)Tj
3.4809 0 Td
(were)Tj
2.1203 0 Td
(added)Tj
2.6646 0 Td
(to)Tj
1.0261 0 Td
(2)Tj
0.6917 0 Td
(mL)Tj
1.5817 0 Td
(of)Tj
1.0147 0 Td
(0.5%)Tj
2.1486 0 Td
(TBA)Tj
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(in)Tj
1.0374 0 Td
(20%)Tj
1.9276 0 Td
(TCA)Tj
2.177 0 Td
(and)Tj
-34.8262 -1.2983 Td
(heated)Tj
2.8517 0 Td
(in)Tj
1.0374 0 Td
(a)Tj
0.652 0 Td
(boiling)Tj
3.0274 0 Td
(water)Tj
2.4321 0 Td
(bath)Tj
1.9899 0 Td
(for)Tj
1.3833 0 Td
(30)Tj
1.1735 0 Td
(min.)Tj
2.0807 0 Td
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1.7801 0 Td
(mixture)Tj
3.3959 0 Td
(was)Tj
1.6951 0 Td
(centrifuged)Tj
4.7847 0 Td
(at)Tj
0.9525 0 Td
(3)Tj
0.6973 0 Td
(996)Tj
/F17 1 Tf
1.6498 0 Td
[()]TJ
/F15 1 Tf
[(\n)]TJ
/F9 1 Tf
1.2018 0 Td
(for)Tj
1.3833 0 Td
(5)Tj
0.6974 0 Td
(min)Tj
-34.8659 -1.3039 Td
(and)Tj
1.7292 0 Td
(immediately)Tj
5.1987 0 Td
(cooled)Tj
2.8573 0 Td
(on)Tj
1.2755 0 Td
(ice.)Tj
1.5591 0 Td
(The)Tj
1.7801 0 Td
(absorbance)Tj
4.7395 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4684 0 Td
(supernatant)Tj
4.9831 0 Td
(at)Tj
0.9525 0 Td
(450,)Tj
1.8708 0 Td
(532)Tj
1.6498 0 Td
(and)Tj
1.7291 0 Td
(600)Tj
1.6441 0 Td
(nm)Tj
1.5931 0 Td
(was)Tj
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(recorded)Tj
3.7928 0 Td
(and)Tj
1.7291 0 Td
(the)Tj
1.474 0 Td
(MDA)Tj
2.5342 0 Td
(content)Tj
3.2654 0 Td
(was)Tj
1.7008 0 Td
(calculated.)Tj
/F18 1 Tf
-13.3001 -1.2982 Td
(Neutral-red)Tj
5.1534 0 Td
(retention)Tj
4.0932 0 Td
(time)Tj
2.1203 0 Td
(\(NRRT\))Tj
3.6056 0 Td
(assay.)Tj
/F9 1 Tf
3.2088 0 Td
(The)Tj
1.7802 0 Td
(measurement)Tj
5.6691 0 Td
(and)Tj
1.7292 0 Td
(counting)Tj
3.8097 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4683 0 Td
(neu-)Tj
-34.8488 -1.3039 Td
(tral-red)Tj
3.2428 0 Td
(retention)Tj
3.9062 0 Td
(time)Tj
2.0296 0 Td
(followed)Tj
3.6453 0 Td
(the)Tj
1.474 0 Td
(method)Tj
3.3222 0 Td
(of)Tj
1.0148 0 Td
(Ma)Tj
1.5477 0 Td
(et)Tj
0.9411 0 Td
(al.)Tj
1.1225 0 Td
([)Tj
0.83 0.64 0.02 0 k
(10)Tj
0 g
(])Tj
1.8538 0 Td
(using)Tj
2.3867 0 Td
(FCM.)Tj
2.5342 0 Td
(A)Tj
0.9014 0 Td
(coelomic)Tj
3.838 0 Td
(fluid)Tj
-33.7603 -1.2983 Td
(sample)Tj
3.0217 0 Td
(was)Tj
1.6952 0 Td
(mixed)Tj
2.7269 0 Td
(with)Tj
1.9955 0 Td
(a)Tj
0.652 0 Td
(balanced)Tj
3.7644 0 Td
(salt)Tj
1.5647 0 Td
(solution)Tj
3.4809 0 Td
(containing)Tj
4.5298 0 Td
(a)Tj
0.6519 0 Td
(working)Tj
3.5602 0 Td
(NR)Tj
1.5761 0 Td
(solution)Tj
3.4809 0 Td
(of)Tj
1.0148 0 Td
(80)Tj
/F17 1 Tf
1.1735 0 Td
[()]TJ
/F9 1 Tf
(g)Tj
-34.8885 -1.2983 Td
(mL)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 213.5622 334.4314 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(-1)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 221.9527 330.0094 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(\(1:1\))Tj
2.0749 0 Td
(and)Tj
1.7348 0 Td
(determined)Tj
4.8472 0 Td
(on)Tj
1.2756 0 Td
(FCM)Tj
2.3074 0 Td
(every)Tj
2.347 0 Td
(minute)Tj
3.1011 0 Td
(for)Tj
1.389 0 Td
(two)Tj
1.7064 0 Td
(hours.)Tj
2.744 0 Td
(The)Tj
1.7801 0 Td
(NRRT)Tj
2.812 0 Td
(is)Tj
0.8503 0 Td
(the)Tj
1.4684 0 Td
(time)Tj
2.0296 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
-35.6766 -1.2982 Td
(first)Tj
1.8029 0 Td
(interval)Tj
3.2654 0 Td
(where)Tj
2.6475 0 Td
(the)Tj
1.474 0 Td
(ratio)Tj
2.0976 0 Td
(of)Tj
1.0148 0 Td
(coelomocytes)Tj
5.6126 0 Td
(with)Tj
1.9956 0 Td
(fully)Tj
1.9842 0 Td
(stained)Tj
3.0784 0 Td
(cytoplasm)Tj
4.286 0 Td
(exceeds)Tj
3.2655 0 Td
(50%)Tj
1.9275 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
-35.4668 -1.3039 Td
(total)Tj
2.0013 0 Td
(number)Tj
3.4185 0 Td
(of)Tj
1.0148 0 Td
(coelomocytes)Tj
5.6125 0 Td
(counted.)Tj
/F18 1 Tf
-10.8508 -1.2983 Td
(Metallothionein)Tj
6.9504 0 Td
(\(MT\).)Tj
/F9 1 Tf
3.2711 0 Td
(The)Tj
1.7802 0 Td
(determination)Tj
5.9584 0 Td
(of)Tj
1.0148 0 Td
(MT)Tj
1.7234 0 Td
(contents)Tj
3.6283 0 Td
(followed)Tj
3.651 0 Td
(the)Tj
1.4684 0 Td
(instructions)Tj
5.0003 0 Td
(in)Tj
-35.6426 -1.2983 Td
(the)Tj
1.4684 0 Td
(earthworm)Tj
4.6543 0 Td
(MT-Elisa)Tj
3.9572 0 Td
(kit.)Tj
1.5023 0 Td
(The)Tj
1.7859 0 Td
(animals)Tj
3.3278 0 Td
(were)Tj
2.126 0 Td
(placed)Tj
2.7893 0 Td
(in)Tj
1.0374 0 Td
(3)Tj
0.6973 0 Td
(mL)Tj
1.5761 0 Td
(ice-cold)Tj
3.3902 0 Td
(Tris-HCl)Tj
3.8551 0 Td
(25)Tj
1.1735 0 Td
(mmol)Tj
-33.3408 -1.3039 Td
(buffer)Tj
2.6248 0 Td
(\(pH)Tj
1.8425 0 Td
(7.2)Tj
1.4003 0 Td
(at)Tj
0.9525 0 Td
(20C\),)Tj
2.7892 0 Td
(0.1)Tj
1.3947 0 Td
(mmol)Tj
2.6135 0 Td
(phenylmethylsulfon)Tj
7.971 0 Td
(yl)Tj
0.9184 0 Td
(fluoride)Tj
3.3789 0 Td
(as)Tj
1.0148 0 Td
(antiprotease)Tj
5.1023 0 Td
(and)Tj
1.7291 0 Td
(0.5)Tj
1.4003 0 Td
(mmol)Tj
-35.1323 -1.2982 Td
(dithiothreitol)Tj
5.5558 0 Td
(as)Tj
1.0148 0 Td
(the)Tj
1.4684 0 Td
(sulfydryl-protecting)Tj
8.1693 0 Td
(agent)Tj
2.3868 0 Td
(to)Tj
1.0261 0 Td
(prevent)Tj
3.2599 0 Td
(oxidation)Tj
4.0365 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.474 0 Td
(MT)Tj
1.7234 0 Td
(and)Tj
1.7292 0 Td
(then)Tj
-32.859 -1.2983 Td
(homogenized)Tj
5.6465 0 Td
(with)Tj
2.0013 0 Td
(distilled)Tj
3.3845 0 Td
(water.)Tj
2.6532 0 Td
(The)Tj
1.7858 0 Td
(homogenate)Tj
5.1704 0 Td
(was)Tj
1.7008 0 Td
(centrifuged)Tj
4.7848 0 Td
(at)Tj
0.9525 0 Td
(10)Tj
1.1735 0 Td
(000)Tj
/F17 1 Tf
1.6441 0 Td
[()]TJ
/F15 1 Tf
[(\n)]TJ
/F9 1 Tf
1.2019 0 Td
(for)Tj
1.3889 0 Td
(20)Tj
1.1679 0 Td
(min)Tj
1.8595 0 Td
(at)Tj
-36.5156 -1.3039 Td
(4C)Tj
1.7518 0 Td
(in)Tj
1.0318 0 Td
(individual)Tj
4.2916 0 Td
(1.5)Tj
1.3946 0 Td
(mL)Tj
1.5761 0 Td
(microtubes.)Tj
4.9549 0 Td
(The)Tj
1.7801 0 Td
(purified)Tj
3.4129 0 Td
(earthworm)Tj
4.6545 0 Td
(MT)Tj
1.7234 0 Td
(antibody)Tj
3.7644 0 Td
(was)Tj
1.7008 0 Td
(used)Tj
2.058 0 Td
(to)Tj
1.0261 0 Td
(coat)Tj
-35.121 -1.2983 Td
(the)Tj
1.4684 0 Td
(micro)Tj
2.6077 0 Td
(pores)Tj
2.3981 0 Td
(before)Tj
2.744 0 Td
(combination)Tj
5.3461 0 Td
(with)Tj
1.9955 0 Td
(MT)Tj
1.7235 0 Td
(antibody)Tj
3.7644 0 Td
(was)Tj
1.7008 0 Td
(labeled)Tj
3.0273 0 Td
(with)Tj
1.9956 0 Td
(horseradish)Tj
4.9039 0 Td
(peroxi-)Tj
-33.6753 -1.2983 Td
(dase.)Tj
2.1884 0 Td
(The)Tj
1.7857 0 Td
(combined)Tj
4.2406 0 Td
(compounds)Tj
4.9606 0 Td
(were)Tj
2.1203 0 Td
(thorough)Tj
3.9571 0 Td
(washed)Tj
3.1748 0 Td
(and)Tj
1.7291 0 Td
(colored)Tj
3.2259 0 Td
(with)Tj
2.0012 0 Td
(tetramethyl)Tj
4.8132 0 Td
(benzi-)Tj
-34.1969 -1.3039 Td
(dine)Tj
1.9843 0 Td
(as)Tj
1.0147 0 Td
(substrate.)Tj
4.0308 0 Td
(The)Tj
1.7802 0 Td
(optical)Tj
2.914 0 Td
(density)Tj
3.1011 0 Td
(value)Tj
2.3073 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4684 0 Td
(mixture)Tj
3.3902 0 Td
(at)Tj
0.9524 0 Td
(450)Tj
1.6498 0 Td
(nm)Tj
1.5874 0 Td
(was)Tj
1.7007 0 Td
(recorded)Tj
3.7928 0 Td
(and)Tj
1.7291 0 Td
(used)Tj
2.0636 0 Td
(to)Tj
-36.4816 -1.2982 Td
(calculate)Tj
3.6736 0 Td
(the)Tj
1.474 0 Td
(concentration)Tj
5.8167 0 Td
(of)Tj
1.0148 0 Td
(MT.)Tj
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-10.7828 -1.2983 Td
(Heat)Tj
2.2392 0 Td
(shock)Tj
2.5965 0 Td
(proteins)Tj
3.6907 0 Td
(\(HSP)Tj
2.4491 0 Td
(70\).)Tj
/F9 1 Tf
2.3868 0 Td
(HSP)Tj
2.0239 0 Td
(70)Tj
1.1736 0 Td
(in)Tj
1.0318 0 Td
(extruded)Tj
3.787 0 Td
(coelomocytes)Tj
5.6126 0 Td
(of)Tj
1.0148 0 Td
(treated)Tj
/F15 1 Tf
2.999 0 Td
[()]TJ
/F9 1 Tf
(.)Tj
/F15 1 Tf
0.9921 0 Td
[()9()8()7()4( )4()]TJ
/F9 1 Tf
2.4265 0 Td
(were)Tj
-35.6199 -1.2983 Td
(sampled)Tj
3.5489 0 Td
(and)Tj
1.7291 0 Td
(determined)Tj
4.8529 0 Td
(by)Tj
1.1792 0 Td
(western)Tj
3.3335 0 Td
(blot)Tj
1.7745 0 Td
(on)Tj
1.2756 0 Td
(day)Tj
1.6384 0 Td
(28)Tj
1.1735 0 Td
(after)Tj
2.0353 0 Td
(exposure)Tj
3.8154 0 Td
(to)Tj
1.0262 0 Td
(spiked)Tj
2.8119 0 Td
(natural)Tj
3.0784 0 Td
(soil.)Tj
1.8255 0 Td
(The)Tj
-35.0983 -1.3039 Td
(immune-peroxidase)Tj
8.2714 0 Td
(staining)Tj
3.4129 0 Td
(protocol)Tj
3.5943 0 Td
(was)Tj
1.7008 0 Td
(used)Tj
2.0579 0 Td
(on)Tj
1.2756 0 Td
(methanol-fixed)Tj
6.3496 0 Td
(cytospin)Tj
3.5943 0 Td
(preparations)Tj
5.2724 0 Td
(after)Tj
-35.5292 -1.2982 Td
(blocking)Tj
3.6793 0 Td
(of)Tj
1.0148 0 Td
(endogenous)Tj
5.0626 0 Td
(peroxidase)Tj
4.5128 0 Td
(with)Tj
2.0012 0 Td
(3%)Tj
1.4513 0 Td
(H)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 384.8881 120.359 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 388.4598 122.0031 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(O)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 395.9433 120.359 cm
BT
/F9 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 401.6692 122.0031 cm
BT
/F9 1 Tf
1 TL
-0.004 Tc
0 0 Td
(as)Tj
1.0147 0 Td
(described)Tj
4.0536 0 Td
(previously)Tj
4.3483 0 Td
([)Tj
0.83 0.64 0.02 0 k
(20)Tj
0 g
(].)Tj
2.0806 0 Td
(Monoclonal)Tj
-31.6627 -1.2982 Td
(mouse)Tj
2.8573 0 Td
(anti-human)Tj
4.9776 0 Td
(anti-HSP)Tj
3.9175 0 Td
(70)Tj
1.1735 0 Td
(were)Tj
2.1203 0 Td
(diluted)Tj
3.0387 0 Td
(1:1000)Tj
2.8232 0 Td
(with)Tj
2.0013 0 Td
(0.25%)Tj
2.6248 0 Td
(Triton)Tj
2.8233 0 Td
(X)Tj
0.8731 0 Td
(in)Tj
1.0375 0 Td
(phosphate)Tj
4.3369 0 Td
(buffer)Tj
-34.605 -1.3039 Td
(supplemented)Tj
5.8507 0 Td
(with)Tj
2.0012 0 Td
(1%)Tj
1.4514 0 Td
(BSA)Tj
1.9615 0 Td
(\(overnight,)Tj
4.6488 0 Td
(4C\))Tj
2.092 0 Td
(followed)Tj
3.6452 0 Td
(by)Tj
1.1849 0 Td
(the)Tj
1.4683 0 Td
(labeled)Tj
3.0274 0 Td
(streptavidin)Tj
4.9889 0 Td
(biotin)Tj
-32.3203 -1.2983 Td
(method)Tj
3.3222 0 Td
(\(Dako)Tj
2.7326 0 Td
(LSAB)Tj
2.5001 0 Td
(peroxidase)Tj
4.5184 0 Td
(kit\).)Tj
ET
Q
q
1 j
1 J
0 w
576 737.1 m
36 737.1 l
36 737.6 l
576 737.6 l
f*
36 741.2598 107.1496 23.6976 re
W* n
q
107.0929 0 0 23.6409 36 741.3165 cm
q
/I0 Do
Q
Q
Q
q
0 0 612 792 re
W* n
1 j
1 J
0 w
7.9999 0 0 7.9999 409.4929 745.7952 cm
BT
/F0 1 Tf
1 TL
-0.005 Tc
0 0 Td
(DEHP)Tj
2.9622 0 Td
(toxicity)Tj
3.2173 0 Td
(to)Tj
1.0418 0 Td
(earthworm)Tj
4.8685 0 Td
(in)Tj
0.978 0 Td
(spiked)Tj
3.0614 0 Td
(natura)Tj
2.778 0 Td
(l)Tj
0.4394 0 Td
(soil)Tj
ET
Q
q
1 j
1 J
0 w
36 48.0002 m
576 48.0002 l
576 47.5002 l
36 47.5002 l
f*
0.83 0.64 0.02 0 k
81.5811 34.9228 m
237.8835 34.9228 l
h
f*
0 g
7.9999 0 0 7.9999 36 36 cm
BT
/F0 1 Tf
1 TL
-0.005 Tc
0 0 Td
(PLOS)Tj
2.8559 0 Td
(ONE)Tj
2.3669 0 Td
(|)Tj
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