Use of lethal body burdens to indicate species differences in susceptibility to narcotic toxicants

Chemosphere. Vol.31, No. 5, pp. 3201-3209, 1995

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Pergamon 0045-6535(95)00181-6

Copyright© 1995ElsevierScienceLtd Printedin GreatBritain.All rightsreserved 0045-6535/95 $9.50+0.00

USE OF LETHAL BODY BURDENS TO INDICATE SPECIES DIFFERENCES

IN SUSCEPTIBILITY TO NARCOTIC TOXICANTS

Annemarie P. van WezelAc, Dick T.H.M. SijmA, Willem SeinenA and Antoon Opperhuizen B.

AEnvironmental Chemistry Group, Research Institute of Toxicology, Utrecht University, P.O. Box 80058, NL3508 TB Utrecht, The Netherlands and SMarine Chemistry, National Institute for Coastal and Marine Management, The Netherlands

(Receivedin Germany24 January 1995;accepted19 July 1995) Abstract

Lethal body burdens (LBB) for 1,2- and 1,4-dihalogenated benzenes (F, CI or Br) are determined in rainbow trout of two age-classes. LBBs range from 0.3 to 2.4 mmol/kg. There are no significant differences between the two tested age-classes of rainbow trout. The rainbow trout data are compared to LBBs for 1,2- and 1,4-difluorobenzene in fathead minnow which range from 2.7 to 3.0 mmol/kg, and to LBBs of dichloro- and dibromobenzenes in guppy and fathead minnow [Sijm et al. 1993] which range from 2.7 to 8.0 mmol/kg.

Rainbow trout are more susceptible to dihalogenated bertzenes than fathead minnow. The LBB can be used as an instrument to examine the intrinsic toxicity of a chemical to a species, and to indicate the susceptibility of a species. Possible reasons for differences in susceptibility among species are discussed.

Introdu~ion

Literature data on species differences in susceptibility are mainly based on LCs0 experiments, the exposure concentration that is lethal to 50% of the population [Doherty 1983; Slooff et al. 1983; Holcombe et al. 1987; Nendza & Klein 1990; Cronin et aL 1991; Vittozzi & De Angelis 1991]. LCs0 data of individual compounds can be compared for different species. Also QSARs established for various species can be compared. For narcotic CCurrentaddress:MarineChemiltry,NationalInstitutefor Coastaland MarineManagement,Ministryof Transport, PublicWorksand Water Management, P.O. Box 20907, 2500 EX The Hague, The Netherlands

3201

3202 organic chemicals, rainbow trout generally appears to be more susceptible than species such as guppy and fatbead minnow [Doherty 1983; Slooff et al. 1983; Holcombe et al. 1987; Nendza & Klein 1990; Vittozzi & De Angelis 1991]. As has been pointed out by McCarty [1986], McCarty & Mackay [1993] and McKim & Schmieder [1991], LCs0 values express both the bioconcentration potential of a compound and its intrinsic toxicity, i.e. the toxicological potency of the chemical once inside the organism. Consequently, when LCs0 values are used it remains indistinct what the underlying nature is of differences in susceptibility to toxicants between species. As an alternative to LCs0 as a measure of toxicity, the molar concentration in an organism at lethality can be determined. This lethal body burden (LBB) or critical body residue (CBR), reflects the intrinsic toxicity of a compound but not the bioconcentration potential. The I.,BB is expected to be approximately a constant within a species for compounds with a similar mechanism of toxicity, and to be independent of exposure concentration and exposure time. Using literature data on LC50 and on bioconcentration, it has been derived [McCarty 1986] that for compounds which exert toxicity by narcosis the LBB varies from 2 to 8 mmol/kg. Meanwhile, experimental results have supported the LBB concept [Van Hoogen & Opperhuizen 1988; De Bruijn et al. 1991; McKim & Schmieder 1991; De Wolf et al. 1992; Sijm et al. 1993].

The objective of this paper is to compare LBBs of narcotic compounds for different species, in order to examine if the LBB can be used to indicate toxicological species susceptibility. The LBBs for 1,2- and 1,4-dihalogenated benzenes (F, el or Br) are determined in rainbow trout, and LBBs for 1,2- and 1,4-difluorobenzene are determined in fathead minnow. These data are compared among eachother and with LBBs of dichloro- and dibromobenzenes in guppy and fathead minnow which have been determined previously in the same laboratory [Sijm et al. 1993].

Material and Methods

Chemicals

1,2-difluorobenzene (1,2-diFBz, 98 %) and 1,4-difluorobenzene (1,4-diFBz, 99 %) were purchased from Riedel-de Hahn, 1,2-dichlorobenzene ( 1,2-diCBz, > 95 %) and 1,4-dichlorobenzene ( 1,4-diCBz, > 95 %) were obtained from BDH chemicals, 1,2-dibromobenzene ( 1,2-diBBz, > 98 %) and 1,4-dibromobenzene ( 1,4-diBBz, > 98 %) were obtained from Merck. All chemicals were used without further purification, since GC-analysis showed no impurities.

Organisms

Fathead minnows were reared in the laboratory at 25"C. The fathead minnows were six months old, weighed 1.2±0.4 g and had a fat content of approximately 5% (w/w), they were used for experiments 1-4. Experiments were performed at room temperature, after an acclimatization period of one week.

3203

Juvenile rainbow trout were obtained from a local hatchery, and acclimated in the laboratory for at least two weeks at 12"C. Nine months old rainbow trout were used for experiment 5-13, weighing 56:t: 12 g and with a fat content of 4.2+0.9%. For experiments 14-18 six months old rainbow trout were used with a weight of 165:5 g and a fat content of 5.95:1.3 %.

Water contamination A generator column was used to add the crystalline compounds 1,4-diCBz or 1,4-diBBz to water. Water was contaminated with the fluid compounds 1,2-diFBz, 1,4-diFBz, 1,2-diCBz or with 1,2-diBBz by dissolving the chemical in water at approximately 50% of the aqueous solubility, and stirring during at least one day.

Exposure Utrecht tap water was used for experiments with fathead minnow, Cu:+-free Utrecht tap water was used for the experiments with rainbow trout. The fish were acclimatized to the water used in the experiments during the acclimatization period. Fathead minnows were exposed in a static system at room temperature in 10 L aquaria. Rainbow trout were exposed in a static system at 12"C, using 30 L aquaria. Exposure concentrations at the start of the experiments are given in Table I. Oxygen concentration and temperature of the water were measured at least two times a day. The water was aerated when the oxygen dropped below 4.0 mg/L. Dead fish were removed from the aquaria as soon as possible. During day time the fish were monitored at least twice per hour.

Chemical analysis Water samples were taken regularly and were extracted with hexane. Dead fish were weighed and homogenized. Fish from experiments 5-13 were, due to their size, cut into three pieces which were extracted separately. The homogenate was extracted by heating under reflux for 1.5 hour with 50 mL of distilled water and 50 mL of hexane. For the dichloro- and dibromobenzenes the hexane layer was separated from the water layer after centrifugation. To determine the fat content gravimetrically, 10 mL of the hexane was evaporated to dryness. The remainder of the hexane extract was concentrated under nitrogen to approximately 1 mL. Clean-up followed by elution with hexane through H:SO4-silica and NaOH-silica. Water and fish samples for the dichloro- and dibromobenzenes were analyzed on a Hewlett-Packard 5880A gas chromatograph equipped with a ~3Ni electron capture detector. Helium was used as a carrier gas. A DB-5 column was used, 15 m, i.d. 0.32 mm, film thickness 0.25 #m (J&W Scientific). For the difluorobenzenes, water and fish samples were diluted and analyzed directly after extraction to diminish losses due to evaporation. Water and fish samples were analyzed for the difluorobenzenes on a Merck Hitachi L-6200 I-IPLC connected to a Merck Hitachi L-4000 UV-Detector (254 nm). Acetonitrile:water (60:40) was used

3204 as eluent, and a Cn column, 100x3.0 nun (Chrompack) was used. The recovery percentages of the extraction procedures were determined at the beginning of each experiment using clean samples with a spike of the chemical in hexane, and are given in Table I. The results were corrected for the recovery.

Table I: Experimental conditions and LBBs

Exp.

Compound

n')

COLICb)

Rec %°)

LBBd)

Tdeath`)

Fathead minnows 1

1,2-diFBz

5

460

91±15

2.7±1.3

0.8-142

2

1,2-diFBz

4

282

915:15

2.7±0.4

6.3-167

3

1,4-dilWBz

5

426

79+2

2.9+1.2

0.3-23

4

1,4-diFBz

6

328

795:2

3.05:0.8

0.5-46

Nine-months old rainbow trout 5

1,2-diFBz

4

189

99±11

0.98±0.47

7.0-21

6

1,2-diFBz

4

132

99 ± 11

0.65 ±0.05

7.0-21

7

1,4-diFBz

4

136

96±13

0.38+0.15

0.6-1.6

8

1,4-diFBz

4

64.0

96+13

0.29±0.01

3.3-4.0

9

1,4-diCBz

4

25.5

66±13

0.32±0.12

0.8-1.2

10

1,4-diCBz

4

6.35

66+13

0.47+0.23

2.3-20

11

1,2-diBBz

4

111

70±11

1.8±0.6

0.8-1.7

12

1,2-diBBz

4

57.0

70±11

1.95:0.7

1.7-3.6

13

1,4-diBBz

4

54.4

51+7

0.87+0.31

0.7-1.4

Six-months old rainbow trout 14

1,2-diCBz

4

40.9

75 ±0.5

0.94+0.09

3.0-4.7

15

1,2-diCBz

4

16.2

75 ±0.5

1.0±0.3

3.5-8.2

16

1,2-diBBz

4

107

115+18

0.57+0.13

0.4-0.6

17

1,2-diBBz

4

74.5

115-t-18

2.4±1.1

1.3-5.5

18

1,4-diBBz

4

20.2

61 -t-4

0.78±0.23

2.9-4.8

'~: number of fish b): water concentration at the start of the experiment 0~mol/L) °): recovery percentage of the extraction procedure a): LBB (mmol/kg wet weight) °): time of death (h)

3205

Statistics The software of NCSS (Hintze, Utah) was used. To discriminate between the separate experiments or between groups of experiments an unweighed means ANOVA was used with a < 0 . 0 5 , after which a Duncan's range test followed.

Results and discussion

Sublethal effects Sublethal responses after exposure to the dihalogenated henzenes were similar for the two fish species used in the experiments, as well as for the different compounds used. After the fish were exposed for a while, their initial brief response was to swim in an unoriented way. Thereafter they lost equilibrium. Turning around their axis they fell downwards. This behaviour was alternated with swimming. Then, the fish came to lie down at one side at the bottom of the aquaria, they were often curved. The ventilation frequency decreased. This 'immobility phase' could last for several hours. During this process, the fish darkened. The fish were considered death when they did not move either their mouth or their opercula.

LBB LBBs for the various experiments are given in Table I. In Table I also the time between the beginning of the exposure and the moment the fish were found dead (time of death) is given. Most LBBs are determined at two different exposure concentrations. The LBBs of all individual experiments listed in Table I were tested on significant differences by ANOVA. The LBBs measured in fathead minnow (experiment 1-4) differ significantly (F>0.0000) from the other experiments, except from the experiments in which the LBB of 1,2-diBBz was determined in rainbow trout (experiments 11, 12 and 17). There are no significant differences between the LBBs measured in fathead minnow, neither in the LRBs measured in rainbow trout for all compounds except 1,2-diBBz. For all tested chemicals, except 1,2-diBBz, different exposure concentrations and resulting different times of death, do not influence the height of the LBB. For 1,2-diBBz, however, a strong correlation was found between time of death and LBB in the rainbow trout of six months (Figure la). Such a time-dependence of the I.,BB was not found in the rainbow trout of nine months (Figure Ib). In Figure II the time of death is plotted against the LBB of rainbow trout for all compounds except 1,2-diBBz. The different fish populations were compared, with the impficit assumption that the intrinsic toxicity of the compounds used is identical. Three different groups of experiments are distinguished, i.e. group I (experiments 1-4) in which the LBB of difluorobenzenes is determined in fathead minnow, group II (experiments 5-13) in which the LBB of dihalogenated benzenes is determined in rainbow trout with an age of nine months, and group HI (experiments 14-18) where the LBB of dihalogenated benzenes is measured in six-months old rainbow trout. By

3206

A

B

3.0

5

2.4 o

~ 3

1.8 E

~ 1.2 1

0,6 ~e

00.0

i

i

i

i

1.1

2.2

3,3

4,4

I

0.0

5.5

0.0

I

I

I

i

i

0.8

1.6

2,4

3.2

4.0

time (h)

time (h)

Figure I: Relationship between time of death (h) and L,BB (mmol/kg) of 1,2-diBBz for a) rainbow trout of six months old and b) rainbow trout with an age of nine months.

1.50

1.20 A

~0.90

A A

0

E vE m no 0.60

A

O oLI

•

..I

|

a

D

o

G

•

0.30

0.00

0

I

B

I

I

1

2

I

3 time (h)

I

I

4

5

• 1,4-¢IFBz D 1,4-diCBz • 1,4-diBBz ~, 1,2-diCBz O 1,4-dlBBz RT,9moh~s RT, gmor~s RT,91Ytoft'dll RT,6monlM RT, Smonths

Figure II: Relationship between time of death and LBB for all compounds except 1,2-diBBz for rainbow trout (RT).

3207 means of ANOVA the LBBs of the three groups of fish were compared. The LBBs measured in group I are found to differ significantly (P>0.000) from the LBBs measured in the both other groups. No significant differences are found between the groups 1/and IlL

Rainbow trout are more susceptible to dihalogenated benzenes than fatheaa minnow, they have lower LBBs. These differences in susceptibility point to a higher intrinsic toxicity of the dihalogenated benzenes in rainbow trout than in fathead minnow. There are no significant differences in susceptibility between the two tested age-classes of rainbow trout. The LBB can be used to indicate species differences in susceptibility to toxicants.

Comparison with literature data

Several authors determined LBBs of narcotic organic chemicals. In most of the work small-sized fish species were used such as guppy and fathead minnow. In acute continuous-flow tests guppies were exposed to 1,2,3-tri, 1,2,3,4-tetra, or pentachiorobenzene until death [Van Hoogen & Opperhuiz~n 1988]. Lethal effects were found at a body burden of 2.0 to 2.5 mmol/kg fish. Guppies were exposed to bromophos and fenthion [De Bruijn et al. 1991]. The behaviour of these fish was similar to the behaviour of fish exposed to narcotic chemicals. The LBB found for bromophos was 5.00+ 1.10 mmol/kg, and for fenthion an LBB of 11.3+4.2 mmol/kg was found. The high values of these LBBs may be explained by the high lipid content of the fish used in the latter study [De Bruijn et aL 1991]. Guppies were exposed to various concentrations of 2,3,4,5-tetrachioroaniline [De Wolf et al. 1992]. The LBB of fish that died within 48 hours was 1.8 + 1.0 mmol/kg, the LBB of fish that died after this period up to 14 days was 0.7+0.5 mmol/kg. The decrease in LBB with time was explained by a shift from narcosis to a more specific mode of action [-De Wolf et al. 1992]. Sijm et al. [1993] measured the LBB of 1,2-diCBz, 1,4-diCBz, 1,2-diBBz and 1,4-diBBz in fathead minnow and in guppy. These data are listed in Table il. There were no significant differences in the susceptibility of fathead minnow and guppy for these compounds. The LBBs found for dichloro- and dibromo-benzenes in fathead minnow are comparable to those that were found in the present study for difluorobenzenes in fathead minnow (Tables I

Table II: Lethal body burdens of dibromo- and diehlorobenzenes in guppy and fathead minnow [Sijm et al., '93] Compound

LBB in guppy (mmol/kg)

LBB in fathead minnow (mmol/kg)

1,2-diCBz

2.7-1-0.9

4.9+ 1.6

1,4-diCBz

8.0+1.8

4.3-1-1.1

1,2-diBBz

2.4+0.6

2.4+ 1.2

1,4-diBBz

6.0+1.9

3.4+2.3

3208 and I/). The same authors measured a LBB of 28+8.7 mmol/kg in guppy for 1,4-diFBz. Since the exposure concentration of the guppies was very high (1.1"103 t~mol/L) a rapid ongoing accumulation in the dead fish may help to explain the high LBB. Therefore, we do not take this value into further consideration. Very few data have been obtained in the literature for LBBs of narcotic organic chemicals in rainbow trout. McKim and Sehmieder [1991] reported LBBs determined in large rainbow trout (0.6-1 kg) for 14 chemicals with several modes of action. Two chemicals classified as narcotics were examined: trieaine methanesulfonate (M5222) and oetanol. The LBBs found were 1.71 mmol/kg for MS222 (n=2) and 1.645:1.25 mmol/kg for oetanol (n--3).

From the literature data described above, and from the data from the present study, it can again be concluded that there are no differences in susceptibility between guppy and fathead minnow, but that rainbow trout is more susceptible to narcotic organic compounds. Differences in bioconcentration kinetics cannot on their own explain the different LCS0 values that are found for narcotic compounds in small fish species such as guppy and fathead minnow and in rainbow trout [Doherty 1983; Slooff et al. 1983; Holcombe et al. 1987; Nendza & Klein 1990; Vittozzi & De Angelis 1991]. These differences are at least in part explained by a higher intrinsic toxicity of the narcotic chemicals in the rainbow trout compared to that in guppy and fathead minnow.

Origins of the difference in intrinsic toxicity between the species

The question remains what the origin of the difference between the species in intrinsic toxicity of the dihalogenated benzenes is. Fat percentage cannot be an explanation in the present study, since the fat percentage of the species used was comparable. It is speculated that the composition of the storage lipids and the membrane lipids differs in the rainbow trout in comparison to the fathead minnow and the guppy. This may be caused by species differences per se, and by the different temperatures where the species live [e.g. Henderson & Tocher 1987; Cossins et al. 1977]. The lipid composition determines among others the internal distribution of the chemical at equilibrium and thereby the concentration at the target site. It is generally believed that the membrane is the target site for the narcotic chemicals [Overton 1901; Seeman 1972; Janoff et al. 1981; Cascorbi & Ahlers 1989; Janes et al. 1992; Tamura et al. 1991]. A change in membrane lipid composition may also change the susceptibility of the membrane for

disturbtion by narcotic chemicals.

Conclusion

It is concluded that the LBB can be used as an instrument to detect species differences in susceptibility. Rainbow trout are more susceptible to dihalogenated benzenes than fathead minnow, i.e. rainbow trout have a lower I..BB than fathead minnow. There are no significant differences in susceptibility between the two tested age-groups of rainbow trout. For all tested chemicals, except 1,2-diBBz, different exposure concentrations, and resulting dif-

3209 ferent times o f death, do n o t influence the magnitude o f the LBB.

Acknowledgement The authors want to express their gratitude to the Dutch Organization of Sciantifie Research (N.W.O.) for their financial support.

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