The importance of biotransformation in the toxicity of xenobiotics to fish. I. Toxicity and bioaccumulation of diazinon in guppy (Poecilia reticulata) and zebra fish (Brachydanio rerio)

Aquatic Toxicology, 21 ( 1991) 239-254 @ 1991 Elsevier Science Publishers B.V. All rights reserved 0166-445X/91/$3.50

239

AQTOX 00493

io

i

Johannes Keizer, Giuseppina

‘Agostino and Euciano Vittozzi

istituto Superiore di Sanitri, Department ofComparative Toxicology and Ecotoxicology%Biochemical Toxicology Unit, Rome, Italy (Received 11 January 1991; revision received 25 July 1991; accepted 10 October 1991)

The 96-h LC, value of the organophospho~s pesticide diazinon (~,~-Diethyl-~-2-isopropyl-4-methyl6pyrimidyl thiophosphate~ was about 0.8 m&l for the guppy and about 8 mg/l for the zebra fish. Both the guppy and the zebra fish metabolized diazinon. The detected metabolite was 2-isopropyl-d methyl-4-pyrimidinol (pyrimidinol). Diazoxon (O,O-diethyl-O-2-isopropyl-4-methyl-6-py~midylphosphate) could not be found as a me~bolite. After short exposures (up to 48 h) to low sublethal diazinon concentrations the body concentration of pyrimidinol was linearly dependent on the body concentration of diazinon. The ratio of pyrimidinol to diazinon body concentrations was about 5 times higher in the guppy than in the zebra fish. In both species however, the pyrimidinol level reached a maximum at concentrations corresponding to 1/4to ‘1, of the X5,, value and these levels were remarkably similar. Kinetic experiments at diazinon concentrations of 0.1 and 0.4 ppm pesticide in water showed that an apparent steady state was reached after 24 to 48 h. After longer exposures an increased accumulation of diazinon was observed concomitantly with the inhibition of pyrimidinol formation. The inhibition of the brain acetylcholinesterase reached 738 in the guppy and 83% in the zebra fish. The maximum of the inhibition was already reached when the concentration of diazinon in water was 25% of the LC50 value. The factor of bioconcentration (BCF) was dependent on the level of the pesticide in the water and varied between 39 (in the guppy, at a water concentration of 0.1 ppm) and more than 300 (in the zebra fish, at a water concentration of 4 ppm). The results indicate that the metabolism plays a key role in the bioaccumulation and in the species specific toxicity of diazinon in fish. Key words: Diazinon; Fish toxicity; Metabolism; Guppy; Zebra fish

Correspondence to: L, Vittozzi, Istituto Superiore di Sanita, Department of Comparative Toxicology and E~otoxi~oiogy, Biochemical Toxicology Unit, Viale Regina Elena 299, Rome, Italy.

INTRODUCTION

Some organophosphorus (OP) pesticides show a rather selective toxicity. The LD~,+ for mammals, usually in the range of 300 to 1,000 mg/kg can be higher by more than two orders of magnitudes than the LDSOfor birds (Z-15 mg/kg) (Machin et al., 1976). Insects are even more susceptible (Yang et al., 1971b). Within the fish class the sensitivity to OP pesticides varies considerably. Indeed in a review of results on fish (Vittozzi et al., 1991) it appeared that 15 out of 42 OP pesticides showed a marked species-dependent toxicity with LC so ratios between two species ranging from 10 to 300. The selective action of OP pesticides is mainly ascribed to the metabolic differences which occur among animal classes. The key metabolic difference which accounts for the selectivity between mammals, birds and insects, are the levels of oxonases. These enzymes, which belong to the A-esterase family, hydrolyze the phosphate ester forms of OP compounds. In line with the different toxicity of OP compounds, this enzymatic activity is high in mammals, lower in birds and absent in insects (Mackness et al., 1987; Brealey et al., 1980; Lasker et al., 1982; Walker et al., 1987b; Yang et al., 197 I a). Hepatic monooxygenase activities, which catalyze the oxidative activation of phosphorothioates to the toxic phosphate esters, seem not to play a major role for the selectivity of these pesticides. Indeed their levels in mammals, birds and insects are not very different. It is well known that fish have generally low levels of hepatic MFO enzymes (Chambers et al., 1976; Stegemann et al., 1979; Verdina et al., 1987; Funari et al., 1987); on the contrary little and contrasting information is available about the acitivity of oxonases in these species. Rainbow trout (Oncorynchus mykiss) was found to lack paraoxonase activity (Mackness et al., 1987), while trout (Salvo trutta) seemed to have an appreciable paraoxon hydrolyzing activity (Chemnitius et al., 1983). In one report no paraoxonase activity was found in liver, blood and gills of the carp (Chemnitius et al., 1983), but in another report carp had the highest hepatic diazoxonase activity among 6 fish species tested (Fujii et al., 1982). However the ability to process organophosphorus compounds has been demonstrated in a variety of fish species (Fujii et al., 1982; Hogan et al., 1972; Wallace et al., 1987). Diazinon shows species selective toxicity among fish (Vittozzi et al., 1991). It requires activation by the action of the MFO system, and although its metabolic pattern has been described in mammals and fish in vivo and in vitro (Shishido et al., 1972; Hogan et al., 1972; Janes et al., 1973), information available does not allow an interpretation of the mechanisms of toxicity in fish. Therefore we have undertaken some comparative investigations on the metabolism and toxicity of the OP pesticide diazinon in the guppy and the zebra fish, two species which proved to be endowed with very different levels of xenobiotic-metabolizing enzymes (Funari et al,, 1987; Donnarumma et al., 1988; Soldano et al., 1991). The knowledge of the major factors responsible for the species selective toxicity of this compound among fish may help

241

to improve the classification of OP compounds according to the regulations devoted to the environmental protection. MATERIALS

AND METHODS

Aninds

The guppy stocks were made up of adult females only, with an average weight of 0.6 _t 0. I5 g (mean + SD). The zebra fish were adult male and females with an average weight of 0.4fO.l g. Both the guppy and the zebra fish were purchased from Euraquarium (Bologna, Italy). Fish were acclimatized for at least 1 week at 20-22”C, with a 12:12 light:dark photoperiod. Dechlorinated tap water (pH 7.6 and conductivity 0.6 mS) was continuously supplied after air saturation and U.V.light irradiation. Fish were fed on a semisynthetic diet purchased from Piccioni (Brescia, Italy). Less than 5% mortality per week was observed in all the stocks used. Muterials

Diazinon (purity 98%) was purchased from D. Ehrenstorfer (Augsburg, R.F.T.). Diazoxon (purity 98.5%) and pyrimidinol (purity 99.7%) were a kind gift of Ciba Geigy AG (Basel, Switzerland). All other chemicals used were of analytical or HPLC grade. Toxicity tests

The toxicity of diazinon to the guppy and the zebra fish was assessed with a 96-h lethality test, performed according to EEC guidelines (semistatic procedure) (EEC, 1979). Nominal diazinon concentrations ranged from 0.1 to 1.6 mg/l (guppy) or from 1 to 16 mg/l (zebra fish), The toxicant was added as a solution in dimethylsulfoxide. The exposure medium was renewed daily. The test was carried out in triplicate, but some test concentrations were replicated only twice. The mortality was recorded daily. The EEC quality criteria were always fulfilled. The pesticide concentration was assayed at the beginning and the end of each 24-h period. The 96 h-KS0 was estimated graphically from the semi-logarithmic plot of cumulative mortality vs experimental concentrations of diazinon. Kinetics and distributionstudies

About 100 fish were exposed to diazinon at nominal concentrations of 0.1 and 0.4 mg/l for 48 h in glass tanks containing 100 1 medium. This was renewed after 8 h and 24 h. The pesticide concentration was assayed in each batch of medium before and after exposure. Dissolved oxygen, PI-L,temperature and conductivity did not

242

differ significantly from the conditions reported for the toxicity tests. For the kinetic experiments the fish were subsequently transferred to a flow-through tank, where the water was renewed co~tinuuusl~~ The pesticide concentration in the water was assayed once 4 h after the beginning of the depuration phase. Two samples of 2-4 fish (usually 3) each were taken at predete~ined exposure and depuration times as indicated in the figures. The fish were rinsed with clean water, killed by decapitation and weighed. They were imm~iately used for subsequent processing or frozen and stored at -20°C Bioaecum~latiun experiments with longer exposure periods and higher concentrations were also carried out basically with the same experimental procedure. For studies of tissue distribution, fish were collected at the end of the exposure phase. Due to the smah size of fish nut all organs and tissues could be assayed separately. The analysis of brain, abdominal organs ~except liver), liver, eggs and gills, was carried out on two pools of 50 fish each, For the analysis of the SMB fraction, which included skin, bones, muscles and kidneys, three pools of 8 fish each, were assayed, When mortalities occurred, dead fish were not used. However*,freshly died fish were assayed in a few cases and no differences with similarly exposed live fish were observed. Extraction of diazinorz from water

To determine diazinon in the water of the test tanks, the pesticide was extracted using 3 ml solid-phase-extraction columns (Supelclean, LC 18SPE, Supelco), conditioned with 2 ml methanol and 3 ml of tap water. As a rule 10Qml of the aquarium water were extracted. A vacuum manifold (Visiprep, VM, Supelco) was used for the extraction process. The flow of water through the column was about 1 ml/min. The columns were dried with nitrogen for about 2 h, The elution was carried out with 2 ml ethylacetate. The eluatc was dried under nitrogen (Visidry drying attachment, Supelco) and the diazinon residue was dissolved in 0.5 or I ml ethanol. The recovery of dia~inun frum tap water using this procedure was assessed [see below) by spiking some samples with ~,~5~ to 10 mg diazinon/~; it was 86 + 5% (9 replicates, mean + SD). Extractim of diuzinm and its metaboiitesj?rom$A tissue

Samples were humugenized in 25 ml of a mixture of ethylacetate~hexane ?:3 by means of a tissue homogenizer (Omnimixer, Sorvall). The extracts were filtered into 100 ml round bottom flasks; the tissue was extracted two more times with 15 ml of the extraction mixture and the extracts were collected. The solvents were removed in a rotary evaporator. The fatty residue was dissolved in one or two ml ethanol and lipids were precipitated by cooling the extract at 4OCfor at least 2 h. The supernatant was filtered through a nylon filter (Magna Nylon 66, 0.45 pm pores, Supelco). To calculate the recovery of the method, homogenates from uncon~minated fish were

243

spiked with different concentrations of diazinon and pyrimidinol and processed as described. The recovery was 87 f 5% (11 replicates, mean + SD) for diazinon and 91+ 10% (7 replicates; mean + SD) for pyrimidinol. HPLC analysis

Quantitative analysis of diazinon and its metabolites was performed by HPLC. A Perkin Elmer Series 10 Liquid Chromatograph equipped with a series 20 controller and an LC 135 detector was used. Analysis of diazinon and diazoxon was performed with methanol:water 70:30 as mobile phase, while pyrimidinol was analyzed with methanol:water 5050. The flow rate was 1 ml/minute and the injection volume was 6 ~1. Diazinon, pyrimidinol and diazoxon were identified by co-chromatography with analytical standards. The detection limits with the described procedures were 10 pg diazinon/l in water or 1 pg diazinon/g fish and 0.5 pg pyrimidinol/g fish, respectively. Assay of brain acetylcholinesterase

To assay the in vivo inhibition of ACHE by diazinon, fish were exposed to the pesticide at various concentraticns for 48 h. The exposure conditions were similar to those for the toxicity tests. Brain homogenates of at least 20 fish were used for the enzyme preparation. The preparation and the assay were carried out as described by Ellman et al. (1961). All assays were done in duplicate. The deviation of the samples from the mean was always less than 10%. Kinetic analysis

The kinetic description of the bioconcentration process was based on the simple one compartment, first order equilibrium model, commonly used in bioconcentration studies (Southworth et al., 198 1), modified to take into account the effects of metabolism. It was assumed that the elimination rate of the parent compound was made up of two components, a physical excretion rate governed by passive diffusional processes and a metabolic elimination rate governed by enzymatic processes within the organism. To describe the metabolism it was also assumed that the production of pyrimidinol represented the total metabolism of the parent compound, and was linearly dependent on the concentration of diazinon in the fish. So the bioconcentration and metabolism process are described by:

dcD

-

dt

= k,*cW-

k,$cD

V -_!?!!?*cD kM

(1)

244

and

dcP -*cD Vmax - k,+P

------I”

dt

k,

(2)

with the assumption that the uptake of pyrimidinol from water is negligible. In the equations reported, the meanings of the abbreviations are: CD = concentration of diazinon in fish; k, = rate constant for the uptake of diazinon; CW = concentration of diazmon in water, assumed to be constant; ken = rate constant for the excretion of diazinon; Vmax= maximal velocity of the metabolism, which produces pyrimidinol; kM = Michaelis constant for diazinon; CP = concentration of pyrimidinol in fish; kep = rate constant for the excretion of pyrimidinol. The appropriate integrated equations allowed the calculation of the following parameters:

v

F+

k,D = clearance (CL) (diazinon-elimination rate constant)

M

Vmax =

metabolite accumulation ratio.

hl%P

They were fitted to the experimental data for diazinon and pyrimidinol in both uptake and excretion phase by non-linear regression. The ‘reduced chi square method’ was applied to make the best fit. RESULTS

Toxicity of diuzinon to the zebraJish and the guppy

The most evident symptom of poisoning was swelling of the gills. Furthermore the fish had coordination problems and just before death they drifted on their backs at the water surface. The zebra fish showed these symptoms only prior to death whereas some guppies showed them temporarily and then recovered. Deaths occurred between 24 and 48 h exposure. The triplicate toxicity test yielded 96 h-L& values of 0.8 _+0.5 ppm for the guppy and 8 f 1 ppm for the zebra fish (mean + SD). At these external concentrations, the tissue concentration of diazinon after 48-h exposure was 85 ppm in the guppy and 1,800 ppm in the zebra fish, ~ptQke~ excretion and ~etQbo~~s~ kinetics

Both fish species absorbed diazinon rapidly from water and an apparent steady

245 B

DlAZl us/g

: 0.4 PP

FISH *

0

a

16

24

32

40

48

66

64

72

80

16

0

EXPOSURE TIME (HRS)

32

OUPPY-DIAZINON

.

QUPPY-PVRIMIDINOL

+

GUPPV-DIAZINON

*

ZEBRA

0

ZEBRA

*

ZEBRA

FISH-PYRIYIMN

64

80

96

EXPOSURE TIME (HRS)

+

FISH-DIAZINON

46

FISH-DIAZINON

. q

GUPPY-PYRIMIDINOL ZEBRA

FISH-PYRIMIMN

Fig. 1. Uptake, excretion and metabolism of diazinon in zebra fish and guppy exposed at nominal diazinon concentrations of 0.1 and 0.4 ppm. The figures show the linear regression plots, obtained applying eqs. (3) (4), (6) and (7). Experimental diazinon concentrations were (mean f SD, in mg/l): 0.07+0.01 and 0.3 1 + 0.03 (guppy) or 0.09 rtO.01 and 0.38 f 0.01 (zebra fish) respectively. All water samples taken during the depuration phase contained no detectable diazinon. All data points are means of at least two pools of 2-4 fish each.

state was reached between 24 and 48 h (Fig. 1). The only metabolite which could be detected in these samples, was pyrimidinol. After 48-h exposure at the nominal concentration of 0.1 ppm diazinon in water, the zebra fish accumulated more than 5 times as much diazinon as did the guppy, while at 0.4 ppm nominal concentration in water, diazinon accumulation in the zebra fish was 50% higher than in the other fish species. On the contrary, the accumulation of the metabolite, which was 50%higher in guppy at 0.1 ppm exposure, became three times as much as in the zebra fish at the higher water concentration. The kinetic analysis of the data of Fig. 1 showed that (Table I) at 0.1 ppm exposure level, diazinon was taken up more rapidly and eliminated more slowly in the zebra fish than in the guppy. Correspondingly, the BCF was higher in the zebra fish than in the guppy. The kinetic analysis also gave some indication that the uptake and clearance constants in the guppy and the uptake constant in the zebra fish were reduced at higher exposure levels (0.4 vs. 0.1 ppm nominal concentrations). This indication was also confirmed by the generally excellent agreement between the BCFs experimentally determined at the steady state and those calculated from the kinetic constants. As to pyrimidinol accumulation, the kinetic analysis performed with different equations and data gave very consistent results (Table II).

246

TABLE I Uptake and elimination rate constants and bioconcentration

Zebra fish

GUPPY Diaz./water (ppm) nominal k, (h-t) (initial slope) k, (h-r) (kinetic analysis) Clearance BCF,,,,,, BCFkinetic

factor af diazinon in guppy and zebra fish.

0.1

0.4

0.1

0.4

13.6

13.9

18.3

17.6

9.7 + 3.6 0.21+ 0.12 39& 5 46f31

23.3 -i_ 2.9 0.13& 0.03 168 +20 179 +47

4.5 Jro.4 0.08 +0.01 59 &6 56 +8

13.6 k3.8 0.11*0.07 86 &IO 123 f85

The experimentally dete~ined diazinon con~ntrations in water were 0.07~O.OI or a.31 +0.03 for the ex~riments with guppy and 0.09~0.01 or 0.37+0.01 For the excrements with zebra fish (mean rt SD of all assays of diazinon concentration in water). The values of the kinetic constants were calculated from the application of the appropriate equation to the uptake and depuration phases. The steady state BCF was calculated from the mean steady state concentrations of diazinon in fish and exposure medium.

TABLE II Values of the metabolite accumulation ratio (~~~~/(~~,~~~~) for py~midinol in guppy and zebra fish. Data sets

Zebra fish

GUPPY h-’

Uptake data Diazinon/water (ppm, nominal): 0.1 0.4 Excretinn data Diazinon~water (ppm, nominal): 0.1 0.4 Uptake and excretion data Mean t_ SD

0.87 It;0.04 0.82&0.07

0.13~0.07 0.16+0.01

0.80~0.03 0.71+0.06 0.69 + 0.04

0.12&0.02 0.3320.04 0.13+0.02

0.78 LO.08

0.17f0.09 -

The experimentally determined diazinon concentrations in water were 0.07+0.01 or 0.31+0.03 for the experiments with guppy and 0.09f0.01 or 0.37+0.01 for the experiments with zebra fish (mean -i_ SD of all assays of diazinon concentration in water). Different exposure times up to 48 h were used. Values for the a~umulation ratio are means f SE calculated from the results obtained by applying the appropriate equations to the uptake data (eq. (7)), excretion data (eq. (8)) and both uptake and excretion data (eq. (6)).

247

0

20

40

80

80

1oK)

Fig. 2. Diazinon uptake (A) and metabolism (II) in guppy during 96 h exposures at 0.4 and 0.8 ppm diazinon in water. All data points are means + SD of at least 3 pools with 3-4 fish each. Experimental diazinon concentrations were {mean + SD, in mg/l): and 0.32 rf: 0.06 and 0.63 1: 0.07, respectively.

EJects of higher concentrations and longer exposure times

When stronger treatments were performed, either by extending the exposure period and/or by increasing the levels of diazinon in the water, important kinetic changes were evident. In the guppy it appeared that at 0.8 ppm exposure level (Fig. 2a), corresponding to the LC50, the tissue concentration of diazinon tended to attain a steady state between 12 and 24 hours, but subsequently began to increase again in surviving animals for at least 4 days. After this time the body level was 5-6 times above the 24 h value. Moreover, at this exposure level, the tissue concentration of pyrimidinol peaked at about one day of exposure (Fig. 2b). It appeared also that the steady states of diazinon and pyrimidinol, settled stern 24 and 96 h at lower exposure concentrations were transient. Indeed tk; tissue concentrations of diazinon measured after 14 days were about 2-3 times higher than the values at 48 h exposure (Fig. 3a). Contrary to this the ;i;sue levels of pyrimidinol after exposure for 14 days were markedly lower than after two days. For comparison, tissue levels of diazinon and py~midinol were measured also in the zebra fish at equitoxic exposure levels, namely 1,2,4,8 ppm after 2 and 14 days of exposure (Fig. 3b). In this fish too, it appeared that tissue concentrations ofdiazinon increased more than expected from the low-dose, short term experiments. The py-

248

A FlSH (vg/g fish)

IAZINON

PYRIMIDINOL

(clg/g fish)

DMZINON I

l6 I

PYRIMIDINOL

16 14

14 80

12

60

12

1600

10

10

8

8

6

5

4

4

600

2

2

0

0 12.6

DIAZINON/WATER DlAZINON12 day4 =

PYRIMIDINOL’P

a

DIAZINON/WATER

(%LC6Oj DlAZINONIl4

day4 m

60

26

12.6

100

60

26

DIAZINONIZ

day0

PYRIMIOINOLIl4

m

daya

m

deye

PYRIYIDINOLIP

dayam

100

(%LC60) DIAZINONIl4

daya

PVAIUIDINOLIl4

days

Fig. 3. Diazinon and pyrimidinol tissue levels after 2 days and 14 days exposure of guppy (A) and zebra fish (B) to equitoxic concentrations of diazinon. All data points are means of at least 2 pools with 2-4 fish each.

PYRIMIDINOL

(pg/g)

r-

0

20

80

40

80

DIAZINON

100

120

140

0

600

(vg/g)

concentr./exp.tlme

* - *-

0.1-0.4

ppm/O-48

0.8 ppfn 6,12 hr

hre y -O-

1600

1000

DIAZINON

2000

&j/g)

ooncentr./exp.time 0.1-0.4

ppmll4

day8

0.8 ppm 24,4fJ. Q6 hr

-*K

l-8 ppm, 48 hrs

’

O.l-O.*pm,O-48

hro

l-4 ppm, 14 days

Fig. 4. Dependence of tissue pyrimidinol concentration on tissue diaainon concentration in guppy (A) and zebra fish (B). Data points are from experiments performed with different experimental conditions as indicated. Data points are means of at least two samples with 2-4 fish each.

249

rimidinol tissue levels after 14 days maximum tissue levels were obtained In comparison with the guppy, it pyrimidinol was reached when body nearly 20 times higher.

of exposure were lower than after 2 days and at 2 ppm diazinon in the water. appeared that a strikingly similar tissue level of concentration of diazinon in the zebra fish was

The p_vri~idin~~ to d~u~in~nbody concentrationratio The pyrimidinol body levels were linearly dependent on the diazinon body burden up to 0.4 ppm concentration in the water and 48 h exposure. The plot of Fig. 4 is the graphical description of eq. (5) and indicates that the metabolite accumulation ratio was constant under the experimental conditions indicated above. Tn the guppy it was 5 times higher than in the zebra fish. When exposures were prolonged or treatment levels increased, the ~yrimidino~ accumulation ratio decreased in both species. Distributionof diuzinon~ndpyr~~idinu~in$sh The results of the tissue distribution studies are shown in Fig. Sa and b. The distribution of diazinon was different in the two species. The highest concentrations of

I5

A

z 8. ug/g TISSUE I

I

FE3

60 70 60 60 40 30 20 10 0

@MB

L

B

E

Q

A0

ORGANS MADNON

m

PYRIMDINOL

M

SMB

L

B

E

G

AQ

M

ORGANS DIAZINON

m

PYRIMIDINOL

Pig. 5. Body distribution of diazinon and pyrimidinol in guppy (A) and zebra fish (B) after 48 h exposure to 0.4 mg/l dia~non (nominal con~ntration}. SNB: skin, bones, muscles; L: liver; B: brain; E, eggs; 6: gills; A0 a~omina1 organs, inner body fat, M: mean, whole fish; all values are means of at least two organ pools. The maximal deviation from the mean was smaller than 15%.

250

diazinon in the zebra fish were found in abdominal organs (not including liver), gills and in the SMB fraction. In the guppy, diazinon was most concentrated in abdominal extrahepatic organs. Diazinon was found in the liver of the zebra fish at concentrations somewhat higher than in the corresponding organ of the guppy, while the most pronounced difference in diazinon concentration occurred between the SMB and the gills fractions of the two fish. This difference amounted to about 4 times. The qualitative distribution of pyrimidinol was markedly similar in the two species but higher levels were observed in the organs of the guppy in accordance with the higher pyrimidinol levels already observed in the whole-body kinetic studies. Inhibition of brain acetylcholinesterase in vivo The ACHE activity in the brain of the guppy was 60% inhibited at 0.1 ppm nominal water concentration of diazinon. At 0.2 ppm, inhibition reached 73% and no further increase in AC E inhibition took place at higher concentrations. The zebra fish enzyme was inhibited 45% and 80% at 0.4 ppm and at 2 ppm diazinon in the water. Exposure to higher concentrations of diazinon did not result in higher inhibition. ACHE inhibition was assessed after 48-h exposure, but was already maximal after 24 h. DISCUSSION

Diazinon is about 10 times more toxic for the guppy than for the zebra fish. Similar species-dependent toxicity has also been observed by other workers, who demonstrated differences of nearly one order of magnitude in the 24 h LC50 values of diazinon for topmouth gudgeon and rainbow trout (Kanazawa, 1981; Sakai et al., 1971). On the other hand, the BCF of diazinon in the zebra fish is higher than in the guppy (Table I). The BCF values measured after 48 h exposure are similar to the values calculated from the Pow (Sastry et al., 1982) and to othe;experimentally determined BCFs (Kanazawa, 1975; Kanazawa, 1981). With low diazinon concentrations in the water (0.02 ppm) it was reported a BCF of 120 in carp and of 64 in rainbow trout (Seguchi et al., 1981) and the difference was attributed to the more efficient metabolism of diazinon observed in rainbow trout. The time expected for compounds like diazinon to reach a steady state is about 50 h (Kristensen and Nyholm, 1987). However, our data (Fig. la, lb, 3a and 3b) indicate that in both the guppy and the zebra fish, the level of diazinon, reached within this time period is not a steady state. ue to the different bioaccumulation, the susceptibility difference of the t,wo species is even more striking if the tissue levels are considered. Indeed the 48 h body burden of diazinon at equitoxic exposure levels is 20 to 50 times higher in the zebra fish than in the guppy (Fig. 3a and 3b). In sharp contrast with this evidence, the tissue concentrations of pyrimidinol at

equitoxic exposure levels are comparable. The maximal levels, attained at 25 to 50% of the LC50 values, were remarkably similar in both species (Fig. 3a and b). Another interesting feature of the kinetics of diazinon and pyrimidinol is the time-dependent decrease of the metabolite accumulation ratio (Fig. 4a and b). concomitantly with the decrease in the py~midinol level, diazinon accumulation started again to increase after a transient trend to remain steady. Although the timing of the process was not evident in our data, the same phenomenon was present at all exposure levels in the guppy and in the zebra fish, as shown by the time-related changes in the body levels of diazinon and pyrimidinol (Fig. 3a and b). It is well known that glutathione-S-transferases, monooxygenases and oxonases are involved in the metabolism of organophosphorus pesticides (Shishido et al., 1972, Motoyama et al., 1977, Chemnitius et al., 1983, Wallace et al., 1987). The observed decrease of pyrimidinol body levels may therefore be explained by an induction of glutathione-~-transferases acting on diazinon or by an inhibition of either diazinon monooxygenase or diazoxonase; an increase of the excretion rate of py~midinol, afforded by induction of enzymatic conjugation is in principle also possible. The inhibitory action of thioates on the monooxygenase activity has already been observed in vitro but not in vivo. It has been attributed to the fact that the reaction intermediate partitions among several pathways producing, besides the oxons, alcoholic moieties and very reactive sulfur and oxygen radicals (De Matteis, 1974; A

B

96 1OW 8

Wg

fish

%

Wg fiah2000

100*-------4

I

- ail eo-:

DIAZINQN/

ER

DIAZINCNUWATER

(%LCSO)

48 hr valuu~r, -

DIAZINON

-* - ACWVITY

(%LCSO)

48 hr vuhlea

(II@/&

-e

PVRIMIQ~WOl. 4%)

-

ACHE f+)

+

YORULtTV

-* - ACYiVtTV

M

OIAZINON (ucl/@ &SUE (%I

+

PVRWID1NOL

+

~~~ITV

i%l 6%)

Fig, 6. Summa~ plot of diazinon accumulation, pyrimidinol body levels, lethality and ACHE inhibition after 48 h exposure to equitoxic diazinon levels for guppy (A) and zebra fish (l-3).

252

et al., 19’75).Our data strongly support that the inhibition of the monooxygenase is responsible for the pyrimidinol decrease. Indeed, the enhancement of diazinon accumulation occurs concomitantly with the lowering of pyrimidinol levels (Figs. 2, 3 and 6a). This behavior also rules out the induction of diazinon conjugation by glutathion-S-transferases, since in this case a lowering of diazinon levels would have been observed, in spite of a constant exposure level. Moreover, if the hydrolysis and not the MFO were inhibited, an accumulation of diazoxon would result, with consequent inhibition of ACHE. This contrasts with the data, showing that no additional inhibition of ACNE occurred in the presence of decreased pyrimidinol tissue levels (Fig. 6). Furthermore diazoxon was never found in our experiments. Under the experimental conditions for which the pyrimidinol body levels are linearly dependent on diazinon tissue con~ntration (Fig. 4a and b) it may be argued, from the eq. (5) that the metabolic rate constant ( Vnrax/k~)as well as the excretory constant (kep)for pyrimidinol are not dependent on the exposure conditions. The different metabolite accumulation ratios observed in the two species (Table II) account at least in part for the different bioaccumulation of diazinon. It is likely that the metabolic rate constant (~~~~/~~) is the major determinant of this difference. However, the difference in the diazinon bioaccumulation may also be due to different uptake and excretion rates (Table I). In addition, it appears that the uptake rate constants for the two species are dependent on the diazinon concentration in the water. This observation is strengthens by the excellent agreement between the experimentally determined BCFs and the BCFs calculated from the kinetic constants. The inhibition of ACHE, which is afforded by the oxons, is considered the critical alteration which mediates the toxicity of the OPs. In our experiments, ACHE inhibition reached the maximal values already at a concentration corresponding to 25% of the E&j0 value in both species. This is in accordance with other results which showed that the AC E!activity of the guppy was inhibited to more than 80% by very low sublethal concentrations of chlorpyrifos (van der We1et al., 1989). Moreover exposure of guppies to 0.4 ppm or of the zebra fish to 4 ppm, which already resulted in maximal inhibition did not cause lethal events up to 14 days exposure except for those occurring among guppies within 24 and 48 h (Fig. 6a and b). It is well known that ACHE inhibition in particular areas of the brain may be critical for the survival of the exposed animal. Although our measurement of ACHE inhibition is not representative of the inhibition of these critical areas, our data suggest that also other mechanisms of toxicity may be relevant.

CONCLUSIONS

The differences of toxicity and bioaccumulation of diazinon in the zebra fish and the guppy are related mainly to its oxidative biotransformation. With longer exposure times the effect of the absorption and excretion kinetics on bioaccumulation of

253

diazinon should become rncrre and more important because of the ~~hjb~t~unof diazinon metabolism. In these in vivo experiments it was not possible to assess the quantitative differences of meta~lism through the direct measure of the e~rnati~ parameters and of the level of the metabolic intermediates; in vitro investigations are in progress to reach this goal. The oxidative transfo~a~un of dia~ino~ is related to the unset of deferent toxic events. Diazoxon can inhibit ACHE, and this may explain the lethal effects of diazinon. However, the other metabolites can produce other potentially harmful effects as indicated by the inhibition of the monooxygena~ activity. Finally from the point of view of the regulations for environmental protection, results of tests of accumulation and toxicity of OPs, carried out with only one species, can be misleading because of metabolic differences between species. ACKNGWLEDSEMENTS

The authors thank Prof. Colin i-LWalker for helpful discussion. The authors thank also Ciba Geigy for the kind supply of pure diazinon metaboI&es. This work is part of the doctorate thesis of 3. Keizer, Johannes Gutenberg-Universitgt Mainz. This wurk has been partially funded by the National Research Council of Italy - Targeted Project ‘Prevention and Control of Disease Factors’; Subproject ‘Environmental Quality and Health’, contract no. 91.00275.PF 41. REFERENCES Brealey, C.J., C.H, Walker and B.C. Baldwin, 1980. A-esterase activities in relation to tbe differential toxicity of~irjmiphos-methyl to birds and mammals. Festic. fci. I t, 546-554. Chambers, J.E. and J.D. Yarbrough, 1976. Xenobiotic biotransformation systems in fish, Comp. B&hem. Physiol, 55C, 77-84. Chemnitius, J.M., H. Loseh, K, Loseh and R. Zech, 1983. ~rgan~phosphate detoxi~ati~~ hydrofases in different vertebrate species. Comp. Biochem. Physioi 76C, 85-93, De Matteis, F., 1974. Covalent binding of sulphur to microsomes and loss of cytochrome P-450 during the oxidative desulfuration of several chemicals. Mol. Pharmacol. IO, 849-854. Donna~mma, L. and L. Vittoui, 1988. Xenobiotie metabolizing enzyme systems in test fish III. Comparative studies of liver cytosolic glutathion-S-transferases. Ecotoxicol. Environm. Safety 16, 180-186. EEC (European Economic Community) 1979. Directive 79/831. Annex V, Part C, 51.1. ENV/286/80. a:fO. Ellman, G.L., K.D. Courtney, V. Andres, Jr. and R.M. Featherstone, 1961. A new and rapid calorimetric determination of acetylcholinesterase activity, Biochem, Pharmacol. 7,88. Fujii, Y. and S. Asaka, 1982. Metabolism ofdiazinon and diazoxon in fish liver preparations. Bull. Environ. Contam. Toxicot. 29,455-460. Funari, E., A. Zoppini, A, Verdina, G. De Angelis and L. Vittozzi, 1987. Xenobiotic-metabolizing enzyme systems in test fish-l. Comparative studies of liver micorosomal monooxygenases. Ecotoxic. Environ-

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