- Email: [email protected]
Bioconcentration, elimination and metabolism of 2,4-dinitrotoluene in carps(Cyprinus Carpio L.)
Vol. 35, No. 8. pp. 1799-1815, lV97 6 1997 Elsevier Science Ltd All rights reserved. Printed in Great Brilain 0045-6535/V7 $17.00+0.00
Pergamon
Chemosphere,
PII: SO0456535(97)00258-O
BIOCONCENTRATION,
ELIMINATION
2,CDINITROTOLUENE
Pei-Zhen Lang*
IN CARPS(Cypriw
Yi Wangt
Xiao-Ming Zhao*
*Department of Environmental
AND METABOLISM
Dao-Bi Chen*
OF
Carpio L.)
Ning Wang*
Yun-Zheng Ding*
Science, Northeast Normal University
Changchun. P.R. China, 130024 TState Key laboratory of Environmental Aquatic Chemistry, RCEES, CAS, Beijing, P.R. China, 100085 (Received in Germany 15 August 1996; accepted 14 May 1997)
ABSTRACT Bioconcentration curves of 2,4-dinitrotoluene(2,4-DNT) in carps(whole
fish, liver, intestine and muscle)
were investigated using .semistatic system For whole fish, its curve could be described as a gentle peak which began with a rise in concentration to summit or steady state, then declined and reached lower level followed by another steady state. For liver and intestine, their curves both contained two successive peaks, with the second peak followed by slight fluctuation. Bioconcentration factors of 2,4-DNT in whole lish during the first and second steady state were 9.15 and 4.15,( 97.86 and 44.39, based on lipid content), respectively. By logarithmk plotting, two straight-lines with different slopes(3.6 and 0.1 de’) were measured for elimination. According to peaky curves of 2,4-DNT in whole fish, liver and intestine, smaller BCFs than calculated BCFs based on thle regression equations for inert chemicals, and large rate constants of elimination, biotransformation was inferred to have happened in tissues such as liver, intestine, and other tissues. Two mtabolites were separated 6om liver and identified as 4-amino-2-nitrotoluene(4A2NT) and 2,4-diamino-toluene(2,4-DAT) on HPLC, their retention times were 23.1 and 8.8 mitt, respectively. In bioconcentration test of 2,4-DNT in liver, two metabolites and 1799
1800 parent were determined at the sang tinz at intervals, higher concentrations of 4A2NT and 2,4-DAT were found when level of 2,4-DNT declined. Such results demonstrated our inference that metabolism caused the declines in bioconcentration curves. A one-compartment model was set up to simulate the bioconcentration. in which biotransformation adhered to Delayed Enzyme-Catalytic Logarithmic Kinetics. Good fit of model curves with measured values could be observed. 01997 Elsevier Science Ltd
INTRODUCTION In recent years, bioconcentration of chemicals in tishes accompanied by biotransformation was a focal point of aquatic toxicology, such as the work on lindane and atrazine(l), on organophosphorus insecticide chlorpyrifos(2,3), and on chlorinated anilines(4). In their work, besides determinations of dynamics in uptake and elimination, metabolism was studied, identifications of metabolites were carried out. Furthermore, some appraisals had been put forward to indicate the happen of metabolism during the uptake, for example, deviated BCF than calculated BCF based on regression equations for inert chemicals(5), large rate constants of elimination(6). and so on, but exceptions often happened. With regard to models of bioconcentration, the majority were in view of passive diffusion, which considered bioconcentration as a partitioning of a chemical between the exposure water and the lipid phase of the fish, and they could be characterized mathematically by one- or two-compartmnt
models, but few mechanisms of biotransformation had been introduced into the
establishments of these models(7). Nitroaromatics,
such
as
2,4-dinitrotoluene(2,4-DNT).2,6-dinitrotoluene(2,6-DNT),,~
nitrobenzene(NB), are important industrial chemicals and intermediate products, their big output and widespread applications have resulted in severe pollution in Songhua River and many other rivers. But few reports on bioconcentration, elimination and metabolism of these chemicals in fishes were found. Deneer et aL(8,9) reported BCFs of some nitroaromatics, including that of 2,4-DNT,which was 204(based on lipid content), and 1gBCF was 2.31. In his tests, semistatic system and rapid determination method were used, each test continued only 8 days, the interval was 1 or 2 days, so there were less than 10 samples for each chemical during its uptake. In his paper, Deneer et al. took a sceptical attitude in larger BCF of 2,4-DNT than calculated BCE based on regression equations for inert chemicals, but he didn’t give a reasonable explanation.
1801 Qn the basis of our earlier work on bioconcentration(l0, 11,12,19), and in the light of characteristics in the development of work on bioconcentration since 1990, the purpose of the present study was to investigate the bioconcentration, elimination and metabolism of 2,4-DNT in carps. Two tests using diirent
concentrations
of chemical in water were carried out, with interval shortened, exposure prolonged to 40 days, 15 samples acquired during each uptake. Two metabolites were found in liver and identified on HPLC. Based on the expetintal
results, n~~hanisrns of bioconcentration and metabolism were discussed, then a model was
established to simulate the uptakes.
MATERIALS
AND METHODS
Materials One year old carps(Cy@na
Curyio L., 5-18g) were supplied by Changchun Aquatic Institute. The
average lipid contents were 9.35%(100 lishes exposed to aqueous concentration at 613.51.@) and 7.08%(100 fishes exposed to aqueous concentration at 75.28@l), respectively. Dechlorinated tap water was used, the pH value averaged 7.6’0.2(n=20),
temperature was 15+1°C(n=40), the content of dissolved oxygen was
maintained at 9.51*0.40mg/l(n=lO). 2&DNT
was purchased horn Aldrich
Corp., its purity was 97%. The chemical was purified before
test. Three standards of metabolites including 2,4-diamino-toluene(2,4-DAT), 4-amino-2-nitro-toluene(4A2NT), and 2,4-dinitro-benzoic acid(S,CDNA), were synthesised in our laboratory. Their purities were tested and verified by tests of melting points, on HPLC, IR, and GC-MS. Gas chrornatography(Shimadzu, Model GC-7AG, with a 6-‘Ni electron capture detector and a CR-3A data processor) was used in chemical analysis. HPLC(Shimadzu LC-6A, with a SPDdAV IR(Matteion, ALPHA-600, USA), and GC-MS(Vc;Quattro, England) were used.
Methods Bioconcentmtion Phase
ultraviolet detector),
1802 200 fishes were exposed to 2,4-DNT which had two different aqueous concentrations, one was 613.5pgA the other was 75.3pg/l. Half of the contaminated water was replaced once a day, the lishes were fed daily with a commercial fish food. After 40 days the exposure was stopped. During the accumulation p’hase, three tishes and four water samples were periodically taken and individually analysed to determine the chemical. In test one, 2,4-DNT in liver and intestine was also determined. In test two, 2,4-DNT in muscle was also determined. Elimination Phase The elimination of 2,4-DNT by fish was studied for 8 days after 10 days’or 40 days’uptake. During the elimination phase, half of the test water was replaced once a day, three fishes were sampled and individualIy analysed at intervals. Research on Metabolism The concentrations of 2&DNT
were maintained(O.l, 1, 4mg/l) in the test. Half of the contaminated
water was replaced daily, the fishes were not fed during the test. Two fishes were periodically sampled, their livers excised at once and individually analysed for metabolites on HPLC. Chemical Analysis Water samples were analysed by extracting 23ml of water with 2ml of petroleum ether. The ether ;phase was analysed gas-chromatographically. The individual fish was killed by a blow on head and homogenised in a mortar, mixed with three times its weight of dry NazSO, and then extracted in a soxhlet apparatus with 30ml of extraction agent(acetone:petroleum=59:41, v/v) for 2 hours. The extract was cleaned up with an Al~O3-column, then concentrated before analysing on gas-chromatography. The glass column(3m~2mm, I.D.) was filled with chromosorb ~~-2250. The exci.stxl livers of fishes were homogenized with 20-25 volun~s of the same extraction agent in a glass tissue homogenizer. The homogenate was centrifuged at 900g for 5 mins, cleaned up with an &&column, then dried with a N?-stream before analysing on HPLC(DOS C- 18 reversed-phase column, wavelength 254nn-1,pH 7.0, temperature 40 C, flow rate 0.6ml/min). Standards of metabolites were determined on HPLC,IR,GC-MS.
1803
RESULTS Recoveries of Chemical The recovery of the fish analysis of 2,4-DNT was 92.9%(n=lO), the recovery of the determination of concentration of 2,4-DNT in the water was 92.3%(n=lO). The data presented were corrected for these recoveries. Bioconcentration Table 1 and Table 2 gave the concentrations of 2,4-DNT in whole fish during the bioconcentration tests at two different aqueous concentrations. The accumulation data in the period were shown in Fig.1 and Fig.2. The data in Table 1 were more accurate than those in Table 2, because triplicate lishes were sampled in the forrrtsr, while only one fish was sampled in the latter. If the abnormal data on 6 days in Fig.2 was got rid of, which might be resulted horn error in determination, there were many similarities between curves in Fig.1 and Fig.2, such as similar shape, similar tine required to reach steady state, similar time for decline to happen. The bioconcentration factors(BCFs) during the first and the second steady state were 9.15 and 4.15, respectively. Table 3 and Fig.3 gave the concentrations of 2,4-DNT in muscle during the exposure to aqueous concentration at 753yg/l. The peak of muscle was milder than that of whole fish, probably because of the simplicity of muscle, but it could also reflect the metabolism in tissues such as liver, intestine, and other tissues. The accumulation data of 2,4-DNT in liver and intestine during the exposure to aqueous concentration at 613.5l~~/l were presented in Table 4 and shown in Fig.4. Both curves contained two successive peaks, with the
second
peal:
followed by slight fluctuation.
Elimination In Table 5 and Table 6, Fig.5 and Fig.6, the elimination data of 2,4-DNT by fishes after exposure to different aqueous concentrations were given and shown. An expression consisting a composite exponential fnnction(C~O.98e”~~-1.31e”“) was to describe the process because the elimination curve was a biphasic course
1804
Identification of Metabolites Two major nztabolites, 4-amino-2-nitrotoluene(4A2NT)
and 2,4-diio-toluene(2,4-DAT),
were
found in liver extracts of 2 days and 15 days after the fishes had been exposed to 2,4-DNT(concentration at lmg/l), when the concentration of 2.4DNT in liver declined on 2 and 15 days. Peak(o) in Fig.9 was supposed to be 2-amino-4-nitrotoluene(2A4NT)
by comparing its retention tims on HPLC with retention tinr: values of
2A4NT under similar analysis conditions in literature. It was not identified in our test because we have not standard of 2A4NT. Charts of control of liver extract, standards of metabolites, mztabolites in liver extracts (of 2 and 15 days on HPLC were shown in Fig.7, Fig.8 and Fig.9.
DISCUSSION Bioconcentration Factors Table 7 gave the BCFs of 2,4-DNT in whole fish presented in this paper, reported by Deneer et aL(9), and calculated BCFs based on regression equations for inert chemicals. It was shown that BCFs during the first steady state presented in this paper corresponded to the calculated BCFs, while the BCFs during the second steady state was much lower than the calculated BCFs, because of the metabolism taken place after the tirst steady state. The BCFs reported by Deneer et al. should be recognised as our BCFs during the lirst steady state, for his test was continued for less than 10 days, while in our test concentration of 2,4-DNT declined from day 11. In Deneer’s test, the aqueous concentration of 2,4DNT was about 61.7mg/l(that was l/5 of its LCW), the IgBCF value he got was 2.31, which was higher than our values(l.90 or 1.99) determined, but no reasonable interpretation had been given by Deneer et al.
1805 Table 1. Data of biocoucentration test in which aqueousconcentration was 6 13.5 pg/L Standard errors are given in parentheses.
0.5
1.61(0.42)
0.43(0.03)
3.7(0.98)
1.5
4.65(2.62)
0.51(0.05)
9.1(5.14)
3.0
4.61(1.20)
osqo.03)
9.2(2.39)
5.0
4.12(1.17)
0.54(0.00)
7.6(2.70)
8.0
4.33(0.47)
0.44(0.07)
9.9(1.09)
9.0
4.95(0.62)
0.45(0.07)
10.0 4.13(0.30)
0.51(0.04)
8.1(1.59)
11.0 2.51(0.53)
0.46(0.02)
5.5(1.16)
12.0 1.45(0.21)
0.34(0.02)
4.3(0.62)
14.0 2.90(0.87)
0.43(0.03)
6.8(2.02)
ll.o(l.41)
16.0 1.46(0.08)
0.42(0.03)
3.qO.18)
19.0 l.lg(O.13)
0.28(0.04)
4.2(0.48)
24.0 1.13(0.19)
0.53(0.08)
2.6(0.37)
29.0 1.21(0.22)
0.34(0.11)
3.5(0.66)
40.0 3.53(0.28)
0.24(0.03)
14.7(1.86)
4
01
5
10
f5
10
25
30
5!j
time(d) Fig 1 c, of 2.4DNT in fish vs
a) data of 3 Mm.
timeduring
b) data of 4 water samples.
concenuation was 613.5pg/L
Table 2.
Data of bioconcentrationtest in which
t
t
car
Cd)
(&did
(d) C-h&)
1 2
1.271 1.340
8 11
1.447 2.036
24 29
4 6
1.382 0.299
15 19
0.799 0.682
39
a) data of 1 tish.
C”f
Table 3. Data of 2,4-DNT in muscle of test in which aqueousconcentration was 75.28ugA
aqueousconcentration was 75.28pg’L t
uptake inwhich
C”r
6.Q (P.&T)
t
C”
t (4
cg,
t
cm
QG.‘.@
(4
Q.&g)
W
(wk,
0.773 0.728
1 2
0.858 0.917
8 11
1.700 1.318
24 29
0.804 0996
0.879
4 6
1.376 1.274
15 19
0.885 1.162
39
0.923
a) data of 1 fish.
&
20
IO
30
43
20
10
time(d)
tit=(d)
Fig.2 Gof 2,4-DNT in fish samples vs tirre during
Fig.3 c, of 2,4-DNT in muscle of
uptake test in
fish vs tin-e during uptake test in
which aqueousconcentrationwas
which aqueous concentrationwas
maintajnedat 75.28mg/L
maintainedat 75.28mgk
Table4.
Data of2,4DNT
in liverandintestine of
biioncentration test in which aqueousconcentrationwas 613.5pgA. Standarderrors were given in parentheses. t(d)
~4Mk)
0.5
61.434(0.366)
1.0
87.825(1.075) 61.660(1.100)
E 215 3.0 5.0 6.0 8.0
11.0 12.0 15.0
48.218(0.800) 35.300(8.800) 24X96(3.996) 61&X(0.284)
117.29Q9.210)
ca dnr(P&) 76.18Ot3.620)
107.034(17.034) 53.397(5.503) 48.34q10.719) 47.310(7.coO) 53.334(16.334)
73.615(14.715) 93.610(12.820)
43.848(2X89)
34.560(2.360)
41.146(6.021)
32.091(3.480)
32.067(0.000) 20.036(0.000) 38.689(0.390) 36.692(5.166)
24.134(0.X16) 43.966(16.434) 43.299(1.629) 36.060(0.690) 26.780(2.608) 30.400(7.400) 27.9C0(0.6!IO) 25.85qO.525) 27.667(6.800)
19.0 23.0 27.0 31.0 35.0 38.0
30.400(7.400) 25.878(1.902) 30.%2(3.391)
40.0
31.31l(4.022)
19.511(0.833)
ajdata of 2 livers (intestines)of 2 fishes
I20
2280 3 $40
f2D
g .B 80
I
u
40
0
25
f0
IS
20 *(d)
25
30
Fig.4 Ch and L during the uptake in which1 aqueousconcentxition was maintained at 613.5pg/l
1807 Table 5. Data of elimination by fish after biincentration
Table 6. Data of elimination by fish after biocmcentration test in which the
test in which the
aqueousconcentration was 75.28pgA.
aqueousconcentration was 613SpgA Standarderrors were given in parentheses
0 4 8 11
2.537( 1.050) 1.391(1.208) 1.02qO.499) 1.433(0.386)
24 48 96 192
0.241(0.055) 0.233(0.048) 0.170(0.065j 0.124(0.076)
0 8 24
0.810 0.582 0.127
48 % 192
0.085 0.062 0.014
a) data of 1 fish.
a) data of 2 lishes.
48
tii(W Fig.5 G of 2,4-DNT in 6sh during elimination vs tirm(after exposure to 613.5pfll).
76
f44
tim$hrj Fig.6 Cf of 2,4-DNT in fish during elimination vs tim$after exposure to 75.28/.@).
192
0
0
4
4
12
m
tikne(min)
16 Uy
36 32
36
ppy
8
A
20
time(mw
of~&diks
Ii 28
hi ZWZi
24
of 2 and 15 days on HPLC.
- Charts -7
12
B
32
\
\C
extiacts
C
onday
40
36
40
onday
Fig.7 Chart of control of liver extract on HPLC. B o
8
4
-2 24. 4:
2A4NT)
4
12
time(ti)
8
16
20
24
28
Fig. 10 Fitted bioconcentration curves of 2.4-DNT in cam.
2
3.
5-
6-
0: unidentifie4l n-etabolite(maybe
o
F ig. 8 Charts of standardsof 2 metabolites on HPLC.
1~
7t
8
c: 2,4-DW
B: 4A2NT
A: 2,4-DAT
0
32
36
40
1809 Table 7. Comparison of BCFs determined and BCFs calculated.
Det. a by author
BCF
Det. a by Deneer et al.(9)
Calc. b
first equil.¢ second equil, c 9.15
4.15
(Calc. b based on lipid content) BCF
97.86
44.39
204
lgBCF
1.99
1.65
2.31
1.90d : 1.99
a) Det. was determined values, b) Calc. was calculated values. c) equil, was equilibrium, d) calculated values obtained from equation of Wolf(5):lgBCF=l.061gKow0.20. e) calculated values obtained from equation of Deneer et al.(8): lgBCF=0.961gKow+0.09
Identification of Metabolites Resuks showed that higher concentrations of two metabolites in livers occurred at the time of 2 days and 15 days during the uptake concentration of 2,4-DNT in water was maintained at lmg/1. The same metabolites were not found in two other tests on biodegration of different aqueous concentrations(0.1 and 4mg/1), probably because of the unfitness of these concentrations for stimulating certain enzymes. The mechanism of metabolism was reduction step by step, but transitional and reactive metabolites such as nitroso-compounds were not found in the tests. The irreversible protein binding of these reactive metabolites might cause greater toxicity to fish.
The enzymes for reduction still need
further work to be identified, however, some researches(ll,12,13) showed that aryl hydrocarbon hydroxylase, nitro-reducing enzymes, and intestinal microflora might be of some importance.
Bioconcentration Curves of Whole Fish, Liver and Intestine It was obvious that peaky bioconcentration curves of liver and intestine influenced the shapes of
1810 curves of whole fish, and the peaks in Fig.2 looked more like those of liver and intestine. Based on our work(12,19) and some reported work(10,14), a relationship among concentrations in whole fish and in
its all tissues was suggested as Equ. 1, namely, the concentration of 2,4-DNT in whole fish might be the weighted averages of concentrations in its all tissues.
Cf,t = ~Ci,t = Ch.t + C~t + C~t + ...
(I)
i
where Cct and C~t were concentrations of cbemical in whole fish and in any tissues at time t, respectively. While C~.t, C~,, and C~t were concentrations in liver, intestine, and muscle at time t(expressed in mg/g whole fish weight), respectively. For example, several parameters such as Cf.,, C~.t, C ~ were calculated according to data in Table 1 and Table 4 in order to check the relationship. After 6 days' uptake, Ck,6 was l17.29mg/g, C~6 was 93.61mg/g, while W~(weight of liver) was 0.29g, W~(weight of intestine) was 0.26g, so Mk.6(amount of 2,4-DNT in liver) was 0.034mg, M~.6(amount of 2,4-DNT in intestine) was 0.024mg. Since the Wf(weight of whole fish) was 15g, the C~.6 and C~6 in Equ.1 were 2.27 and 1.60ro~,/g, respectively, the sum was 3.87mg/g. By con~arison, Cc6 determined in Table 1 was 4.20mg/g, the proportion of 3.87 to 4.20 was 0.90. The calculation was rough because the fish samples were not identical, but it supported the soundness of the relationship in Equ. 1.
Peaky Curves Suggested Metabolism During the Uptake Several inferences had been drawn from our results including peaks in bioconcentration curves of whole fish, liver and intestine, and the identification of metabolites as well. The peaks of liver and intestine represented the metabolism taken place in such tissues. The peaks of whole fish resulted from influences of peaks in its tissues, especially the biotransformation occurred in liver and intestine, as described in Equ. 1. There were two successive peaks both in Fig.2 and in Fig.4, the resemblance was obvious. Lang(12)reported that bioconcentration curves of 2,6-DNT in carps could be regarded as platform roughly, but its data before 4 days fluctuated violently not that error in experiment, but that its uptake curves in liver and intestine also had peaks as those in the present paper, so we drew an inference that metabolism of 2,6-DNT existed too. Gorge and Nagel(1) reported the bioconcentration and
1811 metabolism of lindane and atrazine in early life stages of zebrafish. The uptake curve of atrazine could be described well as a platform, while curve of lindane was similar to that of 2,6-DNT in shape. It was reported that 50% of lindane had been transformed into 12 metabolites within 40 hours, while only 4% of atrazine had been metabolised into 4 metabolites. Yu(15) reported that uptake curve of 2nitrofluorene in grass carp looked like a peak in which level of chemical declined to the half of the smrmfit after 96 hours' uptake. It was supposed that metabolism resulted in the decline. To sum up, we held that happens of metabolism during the uptake could be recognised from three aspects: the peaky bioconcentration curves of chemicals such as NB(19),2-nitrofluorene(15) and 2,4-DNT in whole fish; the peaky curves of chemicals in liver and intestine such as 2,4-DNT, 2,6-DNT(12), dioctyl solium sulfosuccinate(DSS)(16), and 2-ethylhexyl diphenyl phosphate(EHDP)(17); and platforms which had violent fluctuations in curves of chemicals in whole fish, such as 2,6-DNT(12) and lindane(1). Though metabolism had been verified, curves of some chemicals, such as atrazine(1) and chlorinated anilines(4), could still be described well as platforms, probably because of the srmll rates of transforrmtions. In a word, peaky bioconcentration curves of chemicals in whole fish, liver and intestine might suggest metabolism, considering other appraisals including deviated BCF than calculated BCF based on regression equations for inert chemicals, and large rate constants of elimination.
One-Compartment Bioconcentration Model Reflecting Metabolism A tentative idea was put forward in view of mechanism of bioconcentration accompanied with metabolism. During the first steady state, rates of accumulation and elimination(excretion of 2,4-DNT) balanced each other. After this period of accumulation, certain enzymes were activated to metabolise 2,4-DNT into several metabolitcs, so the level of 2,4-DNT declined quickly from the first steady state to the second, which represented another balance between rates of accumulation and elimination(excretion and metabolism of 2,4-DNT). As indicated above, the curve should he divided into two phase, the first from the beginning of uptake to the first steady state, the second from the decline of concentration to the second steady state. A one-compartment bioconcentration model, in which biotransformation adhered to Delayed EnzymeCatalytic Logarithmic Kinetics(20) reflecting metabolism was designed to describe the uptake.
1812
k2
k2, Vnu,x , Xo
phase I: kz=kw
phase II: kz=kr,2+km
where k . k2 were rate constants of accumulation and elimination, respectively. While kp~, kvz_were rate constants of excretion of parent(2,4-DNT) in phase I and phase II, respectively, k~ was rate constant of metabolism V.~x was the maximal transformation rate, and Xo was level of substrate to activate certain enzymes. Process of phase I was described by dCJdt = 0
(2)
dCddt = klCw - kzCf
(3)
Process of phase II was described by dC,,/dt = 0 dCJdt = klCw - k2Ct - V.~(C~ + Xo - Cf)
(4)
where Ce and Ceo were concentrations of 2,4-DNT in fish at time t and when phase II started, respectively. Cw was the aqueous concentration of 2,4-DNT at time t. The general solution of Cf for phase I could be expressed by Cf = kl/k2 C,v (1 - e"k2')
(5)
The general solution of Cf for phase II could be expressed by Cf = {Cfo-[klCw-Vmax(fto+Xo)]/(kz-Vn~) } e [~vmax'k2~t-t!fl + [kt Cw-V.~(Cfo+Xo)]/(k2-V.~)
(6)
where tl was the time when phase II started. The parameters in the model were obtained by using program STATGRAPHICS the uptake by whole fish. In phase I:
kt =9.895d -1, k2 -1.345d -l,
r2 =0.934
to simulate
1813 In phase II: kl =9.895dl , kz =11.907d -l,
r2 --0.871
V,~ =11.61 ld -1, Xo --4.427mg/g where r was a correlation coefficient. Same parameters were acquired to simulate uptake by muscle of fish. InphaseI:
k l = l l . 9 4 0 d l , k2=0.595d l,
In phase II: k~ =11.940d l , k2 =14.428dt,
r2=0.925 r2 =0.857
V,~, =14.369d -t, Xo =1.101mg/g Simulated curves of uptake was shown in Fig. 10 in which good fit of model curves with values measured could be observed. X0 estimated in rrK~del(4.4271.tg/g) was comparable with concentration of 2,4-DNT in fishes during the first steady state and
before the decline happened(4.47gg/g), which
means level of substrate(2,4-DNT) to activate certain enzymes. More work need to confirm and interpret the meaning of parameters K2 and Vn~ estimated in our test.
SUMMARY Unreported peaky bioconcentration curves of 2,4-DNT in carps were acquired by using semistatic system at two different aqueous concentrations. There were two major and successive peaks in curves of liver and intestine, the second peak was followed by sfight fluctuation. Two metabolites, 4A2NT and 2,4-DAT, were separated from fiver and identified when concentration of 2,4-DNT in liver declined. It was the first time for these metabolites of 2,4-DNT by aquatic organism were separated and identified, the peaks were derr~mstrated to be the results of metabofis~ These results provided new experimental supports for research on QSAR of chemicals to fishes, such as influences of metabolism of nitroaromatics to LCs0 and BCFs(9,18). Based on our earlier researches on 2,6-DNT(12), NB(19), and work on 2,4-DNT, a conclusion was got that there were two reasons for the occurrence of peaky bioconcentration curves of whole fish: 1. Metabolism that occurred in liver and intestine influenced the shapes of curves of whole fish. 2. Different chemicals had different shapes of bioconcentration curves, because they were rnetabolised in
1814 fiver andin~stineaccordingtodifferentmechanisms.
ACKNOWLEDGEMENT
This paper was funded by the National Nature Science Foundation of P.R.China.
REFERENCES 1. Gorge G.,Nagel R.,(1990), Kinetics and metabolism of ~4C-lindaneand 14C-atrazineill early life stages of zebrafish. Chenms_nhere. 21(9), 1125-1137 2. Welling W.,et a1.,(1992), Bioconcentration
kinetics
of the
organophosphorus insecticide
chlorpyrifos in guppies. Ee~oxicol. Environ. Safety, 23(1), 64-75 3. Barron M. G.,et a1.,(1993), Absorption, tissue distribution and metabolism of chlorpyrifos in channel catfish following waterborne exposure. Environ. Toxictfl. Chem., 12(8), 1469-1476 4. Hertl J., et aL,(1993), Bioconcentration and metabolism of 3,4-dichloroaniline in different life stages of guppy and zebraflsh. Chemosphere. 27(11 ), 2225-2234 5. De wolf W., et aL,(1992), Influence of biotransformation on the relationship between bioconcentration factors and octanol-water partition
coefficients. Environ. Sci. TeehnoL, 26(6),
1197-1201 6. De wolfW., et a1.,(1994), Bioconcentration kinetics of chlorinated anilines in guppy. Chemos_nhere, 28(1), 159-167 7. Barron M. G.,(1990), Bioconcentration. Environ. Sci. Teehnol., 24(1), 1612-1618 8. Deneer J. W., et aL,(1987), A QSAR study of fish toxicity data of nitroaromatic
compounds.
OSAR in Drug Design and Toxicology, 352-354 9. Deneer J. W., et a1.,(1987), QSARs for the toxicity and derivatives towards the guppy. A I l U ~ ,
bioconcentration factor of nitrobenzene
10, 115-129
10. Zhu Z. N.,et al., (1987), Uptake and clearance of organic contaminants by fish. Aeta Seientiae Cireumstantiae, 7(3), 339-346(in Chinese) 11. Zhao X. M., et a1.,(1994), Uptake, depuration
and
bioconcentration
compounds in carps. Environ. Chem.. 13(51),433-438(in Chinese)
of three nitroarorrkatic
1815 12. Lang P. Z., et a1.,(1996), Bioconcentration, in carps. ~ a h l ~ l ~ l ~ . ~ ,
elimination and metabolism of 2,6-dinitrotoluene
no.2, 1997(in chinese)
13. Cravedi J. P., et al., (1981),
Distribution and
elimination routes
of a naphthenic
hydrocarbon in rainbow trout. Environ. Contain. Toxleol., 26, 337-344 14. Zhu B. F., et a1.,(1996), Correlation between aryl hydrocarbon hydroxylase and accumulation and elimination of polycyclic aromatic hydrocarbon in tissues of carassius auratus. China Environ. Sei.. 16(1), 38-41 (in chinese) 15. Yu G.,et a1.,(1994), Fate and bioconcentration factor of nitropolycylic aromatic hydrocarbons in laboratory fish-water system Aeta Scientiae Circumstantiae. 14(1), 32-45 (in chinese) 16. Goodrich M. S.,et a1.,(1991), The toxicity, bioconcentration, metabolism and elimination of dioctyl solium sulfosuccinate(DSS) in rainbow trout. War. Res., 25(2), 119-124 17. Muir D.C.G.,et aL,(1981), Environn~ntal dynamics of phosphate esters. II. Uptake and bioaccumulation of 2-ethylhexyl
diphenyl
phosphate and
diphenyl phosphate by fish.
Chemosphere, 10(8), 847-855 18. Lang P.Z.,et a1.,(1995), QSAR study for the toxicity of nitroaromatics to the fathead minnow. Chemical Journal of Chinese Universities, 16(7), 1083-1087(in chinese) 19. Lang P.Z.,et al.,(1996),Bioconcentration,
elimination and metabolism of nitrobenzene in carps.
Acta Scientiae Circumstantiae, no.2, 1997(in chinese) 20. Simkins S. and Alexander M.,(1984), Models for mineralization kinetics with the variables of substrate concentration and population density. Appl. Environ. Mierobiol. 47(6), 1299-1306
Pergamon
Chemosphere,
PII: SO0456535(97)00258-O
BIOCONCENTRATION,
ELIMINATION
2,CDINITROTOLUENE
Pei-Zhen Lang*
IN CARPS(Cypriw
Yi Wangt
Xiao-Ming Zhao*
*Department of Environmental
AND METABOLISM
Dao-Bi Chen*
OF
Carpio L.)
Ning Wang*
Yun-Zheng Ding*
Science, Northeast Normal University
Changchun. P.R. China, 130024 TState Key laboratory of Environmental Aquatic Chemistry, RCEES, CAS, Beijing, P.R. China, 100085 (Received in Germany 15 August 1996; accepted 14 May 1997)
ABSTRACT Bioconcentration curves of 2,4-dinitrotoluene(2,4-DNT) in carps(whole
fish, liver, intestine and muscle)
were investigated using .semistatic system For whole fish, its curve could be described as a gentle peak which began with a rise in concentration to summit or steady state, then declined and reached lower level followed by another steady state. For liver and intestine, their curves both contained two successive peaks, with the second peak followed by slight fluctuation. Bioconcentration factors of 2,4-DNT in whole lish during the first and second steady state were 9.15 and 4.15,( 97.86 and 44.39, based on lipid content), respectively. By logarithmk plotting, two straight-lines with different slopes(3.6 and 0.1 de’) were measured for elimination. According to peaky curves of 2,4-DNT in whole fish, liver and intestine, smaller BCFs than calculated BCFs based on thle regression equations for inert chemicals, and large rate constants of elimination, biotransformation was inferred to have happened in tissues such as liver, intestine, and other tissues. Two mtabolites were separated 6om liver and identified as 4-amino-2-nitrotoluene(4A2NT) and 2,4-diamino-toluene(2,4-DAT) on HPLC, their retention times were 23.1 and 8.8 mitt, respectively. In bioconcentration test of 2,4-DNT in liver, two metabolites and 1799
1800 parent were determined at the sang tinz at intervals, higher concentrations of 4A2NT and 2,4-DAT were found when level of 2,4-DNT declined. Such results demonstrated our inference that metabolism caused the declines in bioconcentration curves. A one-compartment model was set up to simulate the bioconcentration. in which biotransformation adhered to Delayed Enzyme-Catalytic Logarithmic Kinetics. Good fit of model curves with measured values could be observed. 01997 Elsevier Science Ltd
INTRODUCTION In recent years, bioconcentration of chemicals in tishes accompanied by biotransformation was a focal point of aquatic toxicology, such as the work on lindane and atrazine(l), on organophosphorus insecticide chlorpyrifos(2,3), and on chlorinated anilines(4). In their work, besides determinations of dynamics in uptake and elimination, metabolism was studied, identifications of metabolites were carried out. Furthermore, some appraisals had been put forward to indicate the happen of metabolism during the uptake, for example, deviated BCF than calculated BCF based on regression equations for inert chemicals(5), large rate constants of elimination(6). and so on, but exceptions often happened. With regard to models of bioconcentration, the majority were in view of passive diffusion, which considered bioconcentration as a partitioning of a chemical between the exposure water and the lipid phase of the fish, and they could be characterized mathematically by one- or two-compartmnt
models, but few mechanisms of biotransformation had been introduced into the
establishments of these models(7). Nitroaromatics,
such
as
2,4-dinitrotoluene(2,4-DNT).2,6-dinitrotoluene(2,6-DNT),,~
nitrobenzene(NB), are important industrial chemicals and intermediate products, their big output and widespread applications have resulted in severe pollution in Songhua River and many other rivers. But few reports on bioconcentration, elimination and metabolism of these chemicals in fishes were found. Deneer et aL(8,9) reported BCFs of some nitroaromatics, including that of 2,4-DNT,which was 204(based on lipid content), and 1gBCF was 2.31. In his tests, semistatic system and rapid determination method were used, each test continued only 8 days, the interval was 1 or 2 days, so there were less than 10 samples for each chemical during its uptake. In his paper, Deneer et al. took a sceptical attitude in larger BCF of 2,4-DNT than calculated BCE based on regression equations for inert chemicals, but he didn’t give a reasonable explanation.
1801 Qn the basis of our earlier work on bioconcentration(l0, 11,12,19), and in the light of characteristics in the development of work on bioconcentration since 1990, the purpose of the present study was to investigate the bioconcentration, elimination and metabolism of 2,4-DNT in carps. Two tests using diirent
concentrations
of chemical in water were carried out, with interval shortened, exposure prolonged to 40 days, 15 samples acquired during each uptake. Two metabolites were found in liver and identified on HPLC. Based on the expetintal
results, n~~hanisrns of bioconcentration and metabolism were discussed, then a model was
established to simulate the uptakes.
MATERIALS
AND METHODS
Materials One year old carps(Cy@na
Curyio L., 5-18g) were supplied by Changchun Aquatic Institute. The
average lipid contents were 9.35%(100 lishes exposed to aqueous concentration at 613.51.@) and 7.08%(100 fishes exposed to aqueous concentration at 75.28@l), respectively. Dechlorinated tap water was used, the pH value averaged 7.6’0.2(n=20),
temperature was 15+1°C(n=40), the content of dissolved oxygen was
maintained at 9.51*0.40mg/l(n=lO). 2&DNT
was purchased horn Aldrich
Corp., its purity was 97%. The chemical was purified before
test. Three standards of metabolites including 2,4-diamino-toluene(2,4-DAT), 4-amino-2-nitro-toluene(4A2NT), and 2,4-dinitro-benzoic acid(S,CDNA), were synthesised in our laboratory. Their purities were tested and verified by tests of melting points, on HPLC, IR, and GC-MS. Gas chrornatography(Shimadzu, Model GC-7AG, with a 6-‘Ni electron capture detector and a CR-3A data processor) was used in chemical analysis. HPLC(Shimadzu LC-6A, with a SPDdAV IR(Matteion, ALPHA-600, USA), and GC-MS(Vc;Quattro, England) were used.
Methods Bioconcentmtion Phase
ultraviolet detector),
1802 200 fishes were exposed to 2,4-DNT which had two different aqueous concentrations, one was 613.5pgA the other was 75.3pg/l. Half of the contaminated water was replaced once a day, the lishes were fed daily with a commercial fish food. After 40 days the exposure was stopped. During the accumulation p’hase, three tishes and four water samples were periodically taken and individually analysed to determine the chemical. In test one, 2,4-DNT in liver and intestine was also determined. In test two, 2,4-DNT in muscle was also determined. Elimination Phase The elimination of 2,4-DNT by fish was studied for 8 days after 10 days’or 40 days’uptake. During the elimination phase, half of the test water was replaced once a day, three fishes were sampled and individualIy analysed at intervals. Research on Metabolism The concentrations of 2&DNT
were maintained(O.l, 1, 4mg/l) in the test. Half of the contaminated
water was replaced daily, the fishes were not fed during the test. Two fishes were periodically sampled, their livers excised at once and individually analysed for metabolites on HPLC. Chemical Analysis Water samples were analysed by extracting 23ml of water with 2ml of petroleum ether. The ether ;phase was analysed gas-chromatographically. The individual fish was killed by a blow on head and homogenised in a mortar, mixed with three times its weight of dry NazSO, and then extracted in a soxhlet apparatus with 30ml of extraction agent(acetone:petroleum=59:41, v/v) for 2 hours. The extract was cleaned up with an Al~O3-column, then concentrated before analysing on gas-chromatography. The glass column(3m~2mm, I.D.) was filled with chromosorb ~~-2250. The exci.stxl livers of fishes were homogenized with 20-25 volun~s of the same extraction agent in a glass tissue homogenizer. The homogenate was centrifuged at 900g for 5 mins, cleaned up with an &&column, then dried with a N?-stream before analysing on HPLC(DOS C- 18 reversed-phase column, wavelength 254nn-1,pH 7.0, temperature 40 C, flow rate 0.6ml/min). Standards of metabolites were determined on HPLC,IR,GC-MS.
1803
RESULTS Recoveries of Chemical The recovery of the fish analysis of 2,4-DNT was 92.9%(n=lO), the recovery of the determination of concentration of 2,4-DNT in the water was 92.3%(n=lO). The data presented were corrected for these recoveries. Bioconcentration Table 1 and Table 2 gave the concentrations of 2,4-DNT in whole fish during the bioconcentration tests at two different aqueous concentrations. The accumulation data in the period were shown in Fig.1 and Fig.2. The data in Table 1 were more accurate than those in Table 2, because triplicate lishes were sampled in the forrrtsr, while only one fish was sampled in the latter. If the abnormal data on 6 days in Fig.2 was got rid of, which might be resulted horn error in determination, there were many similarities between curves in Fig.1 and Fig.2, such as similar shape, similar tine required to reach steady state, similar time for decline to happen. The bioconcentration factors(BCFs) during the first and the second steady state were 9.15 and 4.15, respectively. Table 3 and Fig.3 gave the concentrations of 2,4-DNT in muscle during the exposure to aqueous concentration at 753yg/l. The peak of muscle was milder than that of whole fish, probably because of the simplicity of muscle, but it could also reflect the metabolism in tissues such as liver, intestine, and other tissues. The accumulation data of 2,4-DNT in liver and intestine during the exposure to aqueous concentration at 613.5l~~/l were presented in Table 4 and shown in Fig.4. Both curves contained two successive peaks, with the
second
peal:
followed by slight fluctuation.
Elimination In Table 5 and Table 6, Fig.5 and Fig.6, the elimination data of 2,4-DNT by fishes after exposure to different aqueous concentrations were given and shown. An expression consisting a composite exponential fnnction(C~O.98e”~~-1.31e”“) was to describe the process because the elimination curve was a biphasic course
1804
Identification of Metabolites Two major nztabolites, 4-amino-2-nitrotoluene(4A2NT)
and 2,4-diio-toluene(2,4-DAT),
were
found in liver extracts of 2 days and 15 days after the fishes had been exposed to 2,4-DNT(concentration at lmg/l), when the concentration of 2.4DNT in liver declined on 2 and 15 days. Peak(o) in Fig.9 was supposed to be 2-amino-4-nitrotoluene(2A4NT)
by comparing its retention tims on HPLC with retention tinr: values of
2A4NT under similar analysis conditions in literature. It was not identified in our test because we have not standard of 2A4NT. Charts of control of liver extract, standards of metabolites, mztabolites in liver extracts (of 2 and 15 days on HPLC were shown in Fig.7, Fig.8 and Fig.9.
DISCUSSION Bioconcentration Factors Table 7 gave the BCFs of 2,4-DNT in whole fish presented in this paper, reported by Deneer et aL(9), and calculated BCFs based on regression equations for inert chemicals. It was shown that BCFs during the first steady state presented in this paper corresponded to the calculated BCFs, while the BCFs during the second steady state was much lower than the calculated BCFs, because of the metabolism taken place after the tirst steady state. The BCFs reported by Deneer et al. should be recognised as our BCFs during the lirst steady state, for his test was continued for less than 10 days, while in our test concentration of 2,4-DNT declined from day 11. In Deneer’s test, the aqueous concentration of 2,4DNT was about 61.7mg/l(that was l/5 of its LCW), the IgBCF value he got was 2.31, which was higher than our values(l.90 or 1.99) determined, but no reasonable interpretation had been given by Deneer et al.
1805 Table 1. Data of biocoucentration test in which aqueousconcentration was 6 13.5 pg/L Standard errors are given in parentheses.
0.5
1.61(0.42)
0.43(0.03)
3.7(0.98)
1.5
4.65(2.62)
0.51(0.05)
9.1(5.14)
3.0
4.61(1.20)
osqo.03)
9.2(2.39)
5.0
4.12(1.17)
0.54(0.00)
7.6(2.70)
8.0
4.33(0.47)
0.44(0.07)
9.9(1.09)
9.0
4.95(0.62)
0.45(0.07)
10.0 4.13(0.30)
0.51(0.04)
8.1(1.59)
11.0 2.51(0.53)
0.46(0.02)
5.5(1.16)
12.0 1.45(0.21)
0.34(0.02)
4.3(0.62)
14.0 2.90(0.87)
0.43(0.03)
6.8(2.02)
ll.o(l.41)
16.0 1.46(0.08)
0.42(0.03)
3.qO.18)
19.0 l.lg(O.13)
0.28(0.04)
4.2(0.48)
24.0 1.13(0.19)
0.53(0.08)
2.6(0.37)
29.0 1.21(0.22)
0.34(0.11)
3.5(0.66)
40.0 3.53(0.28)
0.24(0.03)
14.7(1.86)
4
01
5
10
f5
10
25
30
5!j
time(d) Fig 1 c, of 2.4DNT in fish vs
a) data of 3 Mm.
timeduring
b) data of 4 water samples.
concenuation was 613.5pg/L
Table 2.
Data of bioconcentrationtest in which
t
t
car
Cd)
(&did
(d) C-h&)
1 2
1.271 1.340
8 11
1.447 2.036
24 29
4 6
1.382 0.299
15 19
0.799 0.682
39
a) data of 1 tish.
C”f
Table 3. Data of 2,4-DNT in muscle of test in which aqueousconcentration was 75.28ugA
aqueousconcentration was 75.28pg’L t
uptake inwhich
C”r
6.Q (P.&T)
t
C”
t (4
cg,
t
cm
QG.‘.@
(4
Q.&g)
W
(wk,
0.773 0.728
1 2
0.858 0.917
8 11
1.700 1.318
24 29
0.804 0996
0.879
4 6
1.376 1.274
15 19
0.885 1.162
39
0.923
a) data of 1 fish.
&
20
IO
30
43
20
10
time(d)
tit=(d)
Fig.2 Gof 2,4-DNT in fish samples vs tirre during
Fig.3 c, of 2,4-DNT in muscle of
uptake test in
fish vs tin-e during uptake test in
which aqueousconcentrationwas
which aqueous concentrationwas
maintajnedat 75.28mg/L
maintainedat 75.28mgk
Table4.
Data of2,4DNT
in liverandintestine of
biioncentration test in which aqueousconcentrationwas 613.5pgA. Standarderrors were given in parentheses. t(d)
~4Mk)
0.5
61.434(0.366)
1.0
87.825(1.075) 61.660(1.100)
E 215 3.0 5.0 6.0 8.0
11.0 12.0 15.0
48.218(0.800) 35.300(8.800) 24X96(3.996) 61&X(0.284)
117.29Q9.210)
ca dnr(P&) 76.18Ot3.620)
107.034(17.034) 53.397(5.503) 48.34q10.719) 47.310(7.coO) 53.334(16.334)
73.615(14.715) 93.610(12.820)
43.848(2X89)
34.560(2.360)
41.146(6.021)
32.091(3.480)
32.067(0.000) 20.036(0.000) 38.689(0.390) 36.692(5.166)
24.134(0.X16) 43.966(16.434) 43.299(1.629) 36.060(0.690) 26.780(2.608) 30.400(7.400) 27.9C0(0.6!IO) 25.85qO.525) 27.667(6.800)
19.0 23.0 27.0 31.0 35.0 38.0
30.400(7.400) 25.878(1.902) 30.%2(3.391)
40.0
31.31l(4.022)
19.511(0.833)
ajdata of 2 livers (intestines)of 2 fishes
I20
2280 3 $40
f2D
g .B 80
I
u
40
0
25
f0
IS
20 *(d)
25
30
Fig.4 Ch and L during the uptake in which1 aqueousconcentxition was maintained at 613.5pg/l
1807 Table 5. Data of elimination by fish after biincentration
Table 6. Data of elimination by fish after biocmcentration test in which the
test in which the
aqueousconcentration was 75.28pgA.
aqueousconcentration was 613SpgA Standarderrors were given in parentheses
0 4 8 11
2.537( 1.050) 1.391(1.208) 1.02qO.499) 1.433(0.386)
24 48 96 192
0.241(0.055) 0.233(0.048) 0.170(0.065j 0.124(0.076)
0 8 24
0.810 0.582 0.127
48 % 192
0.085 0.062 0.014
a) data of 1 fish.
a) data of 2 lishes.
48
tii(W Fig.5 G of 2,4-DNT in 6sh during elimination vs tirm(after exposure to 613.5pfll).
76
f44
tim$hrj Fig.6 Cf of 2,4-DNT in fish during elimination vs tim$after exposure to 75.28/.@).
192
0
0
4
4
12
m
tikne(min)
16 Uy
36 32
36
ppy
8
A
20
time(mw
of~&diks
Ii 28
hi ZWZi
24
of 2 and 15 days on HPLC.
- Charts -7
12
B
32
\
\C
extiacts
C
onday
40
36
40
onday
Fig.7 Chart of control of liver extract on HPLC. B o
8
4
-2 24. 4:
2A4NT)
4
12
time(ti)
8
16
20
24
28
Fig. 10 Fitted bioconcentration curves of 2.4-DNT in cam.
2
3.
5-
6-
0: unidentifie4l n-etabolite(maybe
o
F ig. 8 Charts of standardsof 2 metabolites on HPLC.
1~
7t
8
c: 2,4-DW
B: 4A2NT
A: 2,4-DAT
0
32
36
40
1809 Table 7. Comparison of BCFs determined and BCFs calculated.
Det. a by author
BCF
Det. a by Deneer et al.(9)
Calc. b
first equil.¢ second equil, c 9.15
4.15
(Calc. b based on lipid content) BCF
97.86
44.39
204
lgBCF
1.99
1.65
2.31
1.90d : 1.99
a) Det. was determined values, b) Calc. was calculated values. c) equil, was equilibrium, d) calculated values obtained from equation of Wolf(5):lgBCF=l.061gKow0.20. e) calculated values obtained from equation of Deneer et al.(8): lgBCF=0.961gKow+0.09
Identification of Metabolites Resuks showed that higher concentrations of two metabolites in livers occurred at the time of 2 days and 15 days during the uptake concentration of 2,4-DNT in water was maintained at lmg/1. The same metabolites were not found in two other tests on biodegration of different aqueous concentrations(0.1 and 4mg/1), probably because of the unfitness of these concentrations for stimulating certain enzymes. The mechanism of metabolism was reduction step by step, but transitional and reactive metabolites such as nitroso-compounds were not found in the tests. The irreversible protein binding of these reactive metabolites might cause greater toxicity to fish.
The enzymes for reduction still need
further work to be identified, however, some researches(ll,12,13) showed that aryl hydrocarbon hydroxylase, nitro-reducing enzymes, and intestinal microflora might be of some importance.
Bioconcentration Curves of Whole Fish, Liver and Intestine It was obvious that peaky bioconcentration curves of liver and intestine influenced the shapes of
1810 curves of whole fish, and the peaks in Fig.2 looked more like those of liver and intestine. Based on our work(12,19) and some reported work(10,14), a relationship among concentrations in whole fish and in
its all tissues was suggested as Equ. 1, namely, the concentration of 2,4-DNT in whole fish might be the weighted averages of concentrations in its all tissues.
Cf,t = ~Ci,t = Ch.t + C~t + C~t + ...
(I)
i
where Cct and C~t were concentrations of cbemical in whole fish and in any tissues at time t, respectively. While C~.t, C~,, and C~t were concentrations in liver, intestine, and muscle at time t(expressed in mg/g whole fish weight), respectively. For example, several parameters such as Cf.,, C~.t, C ~ were calculated according to data in Table 1 and Table 4 in order to check the relationship. After 6 days' uptake, Ck,6 was l17.29mg/g, C~6 was 93.61mg/g, while W~(weight of liver) was 0.29g, W~(weight of intestine) was 0.26g, so Mk.6(amount of 2,4-DNT in liver) was 0.034mg, M~.6(amount of 2,4-DNT in intestine) was 0.024mg. Since the Wf(weight of whole fish) was 15g, the C~.6 and C~6 in Equ.1 were 2.27 and 1.60ro~,/g, respectively, the sum was 3.87mg/g. By con~arison, Cc6 determined in Table 1 was 4.20mg/g, the proportion of 3.87 to 4.20 was 0.90. The calculation was rough because the fish samples were not identical, but it supported the soundness of the relationship in Equ. 1.
Peaky Curves Suggested Metabolism During the Uptake Several inferences had been drawn from our results including peaks in bioconcentration curves of whole fish, liver and intestine, and the identification of metabolites as well. The peaks of liver and intestine represented the metabolism taken place in such tissues. The peaks of whole fish resulted from influences of peaks in its tissues, especially the biotransformation occurred in liver and intestine, as described in Equ. 1. There were two successive peaks both in Fig.2 and in Fig.4, the resemblance was obvious. Lang(12)reported that bioconcentration curves of 2,6-DNT in carps could be regarded as platform roughly, but its data before 4 days fluctuated violently not that error in experiment, but that its uptake curves in liver and intestine also had peaks as those in the present paper, so we drew an inference that metabolism of 2,6-DNT existed too. Gorge and Nagel(1) reported the bioconcentration and
1811 metabolism of lindane and atrazine in early life stages of zebrafish. The uptake curve of atrazine could be described well as a platform, while curve of lindane was similar to that of 2,6-DNT in shape. It was reported that 50% of lindane had been transformed into 12 metabolites within 40 hours, while only 4% of atrazine had been metabolised into 4 metabolites. Yu(15) reported that uptake curve of 2nitrofluorene in grass carp looked like a peak in which level of chemical declined to the half of the smrmfit after 96 hours' uptake. It was supposed that metabolism resulted in the decline. To sum up, we held that happens of metabolism during the uptake could be recognised from three aspects: the peaky bioconcentration curves of chemicals such as NB(19),2-nitrofluorene(15) and 2,4-DNT in whole fish; the peaky curves of chemicals in liver and intestine such as 2,4-DNT, 2,6-DNT(12), dioctyl solium sulfosuccinate(DSS)(16), and 2-ethylhexyl diphenyl phosphate(EHDP)(17); and platforms which had violent fluctuations in curves of chemicals in whole fish, such as 2,6-DNT(12) and lindane(1). Though metabolism had been verified, curves of some chemicals, such as atrazine(1) and chlorinated anilines(4), could still be described well as platforms, probably because of the srmll rates of transforrmtions. In a word, peaky bioconcentration curves of chemicals in whole fish, liver and intestine might suggest metabolism, considering other appraisals including deviated BCF than calculated BCF based on regression equations for inert chemicals, and large rate constants of elimination.
One-Compartment Bioconcentration Model Reflecting Metabolism A tentative idea was put forward in view of mechanism of bioconcentration accompanied with metabolism. During the first steady state, rates of accumulation and elimination(excretion of 2,4-DNT) balanced each other. After this period of accumulation, certain enzymes were activated to metabolise 2,4-DNT into several metabolitcs, so the level of 2,4-DNT declined quickly from the first steady state to the second, which represented another balance between rates of accumulation and elimination(excretion and metabolism of 2,4-DNT). As indicated above, the curve should he divided into two phase, the first from the beginning of uptake to the first steady state, the second from the decline of concentration to the second steady state. A one-compartment bioconcentration model, in which biotransformation adhered to Delayed EnzymeCatalytic Logarithmic Kinetics(20) reflecting metabolism was designed to describe the uptake.
1812
k2
k2, Vnu,x , Xo
phase I: kz=kw
phase II: kz=kr,2+km
where k . k2 were rate constants of accumulation and elimination, respectively. While kp~, kvz_were rate constants of excretion of parent(2,4-DNT) in phase I and phase II, respectively, k~ was rate constant of metabolism V.~x was the maximal transformation rate, and Xo was level of substrate to activate certain enzymes. Process of phase I was described by dCJdt = 0
(2)
dCddt = klCw - kzCf
(3)
Process of phase II was described by dC,,/dt = 0 dCJdt = klCw - k2Ct - V.~(C~ + Xo - Cf)
(4)
where Ce and Ceo were concentrations of 2,4-DNT in fish at time t and when phase II started, respectively. Cw was the aqueous concentration of 2,4-DNT at time t. The general solution of Cf for phase I could be expressed by Cf = kl/k2 C,v (1 - e"k2')
(5)
The general solution of Cf for phase II could be expressed by Cf = {Cfo-[klCw-Vmax(fto+Xo)]/(kz-Vn~) } e [~vmax'k2~t-t!fl + [kt Cw-V.~(Cfo+Xo)]/(k2-V.~)
(6)
where tl was the time when phase II started. The parameters in the model were obtained by using program STATGRAPHICS the uptake by whole fish. In phase I:
kt =9.895d -1, k2 -1.345d -l,
r2 =0.934
to simulate
1813 In phase II: kl =9.895dl , kz =11.907d -l,
r2 --0.871
V,~ =11.61 ld -1, Xo --4.427mg/g where r was a correlation coefficient. Same parameters were acquired to simulate uptake by muscle of fish. InphaseI:
k l = l l . 9 4 0 d l , k2=0.595d l,
In phase II: k~ =11.940d l , k2 =14.428dt,
r2=0.925 r2 =0.857
V,~, =14.369d -t, Xo =1.101mg/g Simulated curves of uptake was shown in Fig. 10 in which good fit of model curves with values measured could be observed. X0 estimated in rrK~del(4.4271.tg/g) was comparable with concentration of 2,4-DNT in fishes during the first steady state and
before the decline happened(4.47gg/g), which
means level of substrate(2,4-DNT) to activate certain enzymes. More work need to confirm and interpret the meaning of parameters K2 and Vn~ estimated in our test.
SUMMARY Unreported peaky bioconcentration curves of 2,4-DNT in carps were acquired by using semistatic system at two different aqueous concentrations. There were two major and successive peaks in curves of liver and intestine, the second peak was followed by sfight fluctuation. Two metabolites, 4A2NT and 2,4-DAT, were separated from fiver and identified when concentration of 2,4-DNT in liver declined. It was the first time for these metabolites of 2,4-DNT by aquatic organism were separated and identified, the peaks were derr~mstrated to be the results of metabofis~ These results provided new experimental supports for research on QSAR of chemicals to fishes, such as influences of metabolism of nitroaromatics to LCs0 and BCFs(9,18). Based on our earlier researches on 2,6-DNT(12), NB(19), and work on 2,4-DNT, a conclusion was got that there were two reasons for the occurrence of peaky bioconcentration curves of whole fish: 1. Metabolism that occurred in liver and intestine influenced the shapes of curves of whole fish. 2. Different chemicals had different shapes of bioconcentration curves, because they were rnetabolised in
1814 fiver andin~stineaccordingtodifferentmechanisms.
ACKNOWLEDGEMENT
This paper was funded by the National Nature Science Foundation of P.R.China.
REFERENCES 1. Gorge G.,Nagel R.,(1990), Kinetics and metabolism of ~4C-lindaneand 14C-atrazineill early life stages of zebrafish. Chenms_nhere. 21(9), 1125-1137 2. Welling W.,et a1.,(1992), Bioconcentration
kinetics
of the
organophosphorus insecticide
chlorpyrifos in guppies. Ee~oxicol. Environ. Safety, 23(1), 64-75 3. Barron M. G.,et a1.,(1993), Absorption, tissue distribution and metabolism of chlorpyrifos in channel catfish following waterborne exposure. Environ. Toxictfl. Chem., 12(8), 1469-1476 4. Hertl J., et aL,(1993), Bioconcentration and metabolism of 3,4-dichloroaniline in different life stages of guppy and zebraflsh. Chemosphere. 27(11 ), 2225-2234 5. De wolf W., et aL,(1992), Influence of biotransformation on the relationship between bioconcentration factors and octanol-water partition
coefficients. Environ. Sci. TeehnoL, 26(6),
1197-1201 6. De wolfW., et a1.,(1994), Bioconcentration kinetics of chlorinated anilines in guppy. Chemos_nhere, 28(1), 159-167 7. Barron M. G.,(1990), Bioconcentration. Environ. Sci. Teehnol., 24(1), 1612-1618 8. Deneer J. W., et aL,(1987), A QSAR study of fish toxicity data of nitroaromatic
compounds.
OSAR in Drug Design and Toxicology, 352-354 9. Deneer J. W., et a1.,(1987), QSARs for the toxicity and derivatives towards the guppy. A I l U ~ ,
bioconcentration factor of nitrobenzene
10, 115-129
10. Zhu Z. N.,et al., (1987), Uptake and clearance of organic contaminants by fish. Aeta Seientiae Cireumstantiae, 7(3), 339-346(in Chinese) 11. Zhao X. M., et a1.,(1994), Uptake, depuration
and
bioconcentration
compounds in carps. Environ. Chem.. 13(51),433-438(in Chinese)
of three nitroarorrkatic
1815 12. Lang P. Z., et a1.,(1996), Bioconcentration, in carps. ~ a h l ~ l ~ l ~ . ~ ,
elimination and metabolism of 2,6-dinitrotoluene
no.2, 1997(in chinese)
13. Cravedi J. P., et al., (1981),
Distribution and
elimination routes
of a naphthenic
hydrocarbon in rainbow trout. Environ. Contain. Toxleol., 26, 337-344 14. Zhu B. F., et a1.,(1996), Correlation between aryl hydrocarbon hydroxylase and accumulation and elimination of polycyclic aromatic hydrocarbon in tissues of carassius auratus. China Environ. Sei.. 16(1), 38-41 (in chinese) 15. Yu G.,et a1.,(1994), Fate and bioconcentration factor of nitropolycylic aromatic hydrocarbons in laboratory fish-water system Aeta Scientiae Circumstantiae. 14(1), 32-45 (in chinese) 16. Goodrich M. S.,et a1.,(1991), The toxicity, bioconcentration, metabolism and elimination of dioctyl solium sulfosuccinate(DSS) in rainbow trout. War. Res., 25(2), 119-124 17. Muir D.C.G.,et aL,(1981), Environn~ntal dynamics of phosphate esters. II. Uptake and bioaccumulation of 2-ethylhexyl
diphenyl
phosphate and
diphenyl phosphate by fish.
Chemosphere, 10(8), 847-855 18. Lang P.Z.,et a1.,(1995), QSAR study for the toxicity of nitroaromatics to the fathead minnow. Chemical Journal of Chinese Universities, 16(7), 1083-1087(in chinese) 19. Lang P.Z.,et al.,(1996),Bioconcentration,
elimination and metabolism of nitrobenzene in carps.
Acta Scientiae Circumstantiae, no.2, 1997(in chinese) 20. Simkins S. and Alexander M.,(1984), Models for mineralization kinetics with the variables of substrate concentration and population density. Appl. Environ. Mierobiol. 47(6), 1299-1306