3COTOXICOLOCY
AND
ENVIRONMENTAL
SAFETY
9, 32 l-326
(1985)
Joint Toxicity of Mixtures of Groups of Organic Aquatic
Pollutants to the Guppy (Poecilia reticulata)
JOOP HERMENS,
Department
PETER LEEUWANGH,’
AND
AALT
qf 1 ‘eterinary
Pharmacology.
Pharmacy,
and Toxicology.
Postho.x 80176. 3508 TD Utrecht. The Netherland,
Received
April
MUSCH
L’rziver.Cfy
of Ckrecht.
17, 1984
In this study acute lethal concentrations
(L&)
to the guppy (Poecilia
reticulata)
were
determined
for mixtures
of 4 groups of aquatic pollutants.
The groups were composed of 11
nonreactive,
nonionized
organic chemicals,
1 I chloroanilines.
I I chlorophenols,
and 9 reactive
organic halides. Earlier studies indicated that the joint toxicity within each of these groups was
concentration
additive,
probably
because of a similar mode of action. The joint toxicity
of
combinations
of one representative
from each group showed a high variance,
but generally
tended to be partially
additive to concentration
additive. This high variance is probably
caused
by the low number
of compounds
in these mixtures.
Experiments
with mixtures
of whole
groups gave more accurate results. The toxicity of a mixture of the first three groups, containing
33 well-known
aquatic pollutants, was almost completely
concentration
additive. Concentrations
of 0.04 of the individual
LCSO values contributed
to the toxicity of this mixture.
c 1985 Academic
Press, Inc.
INTRODUCTION
Rivers such as the Rhine are polluted with a large number of chemicals.
Information about joint toxicity of these complex mixtures is rather scarce. Among
the pollutants in these mixtures, several groups containing chemicals with similar
modes of action can be distinguished. The joint toxicity of mixtures, composed of
similarly acting toxicants, can be predicted theoretically with the concentration
addition model (Konemann, 1981a: Muska and Weber, 1977; Plackett and Hewlett,
1952; Sprague, 1970). Indications for similarity of mode action can be found in
structure-activity relationships (QSARs) (KGnemann, 1980).
It is the purpose of this study to determine the joint toxicity of combinations of
groups of compounds with different modes of action. Combinations were made of
four groups of chemicals, each with probably a different mode of action according
to a QSAR with 14-day LCsO as toxicity parameter and with a joint toxicity of
mixtures of the components of each group conforming to concentration addition
(seeTable 1). Joint toxicity as presented in Table 1, was evaluated with the mixture
toxicity index (MTI) as proposed by Kijnemann (198 la) (see Table 2). The
composition of the four groups is given in Table 3. In order to test the possibility
of prediction of this kind of joint action of mixtures of groups from experiments
with representatives of these different groups, the joint toxicity of combinations of
single representatives of the four groups of Table 1 was also determined.
’ Present address: Institute
for Pesticide
Research,
Marijkeweg
22, 6709 PC Wageningen.
The Netherlands.
322
HERMENS.
LEEUWANGH.
TABLE
AND MUSCH
1
ACUTELETHALITYTOTHEGUPPYOFFOURMIXTURESOFORGANICPOLLIJTANTS
Group
A Nonreactive (chlorinated)hydrocarbons
B Chloroanilines
C Chlorophenols
D Reactive organic halides
MTId
Md
llP
1.02”
0.9
50
0.96 h
1.1
1.00”
1.00’
1.0
1.0
11
11
9
uData drawn from KGnemann(1981a).
bData drawn from Hermenset al. (1984).
’ Determinedin this study.
d Definition of M and MT1 are given in Eq. (1).
‘Number of compoundsin the mixtures.
METHODS
Fourteen-day LCsO experiments with guppies (Poecilia reticdata) were carried
out as described earlier by KGnemann (I 98 lb). The mixtures were prepared in
equitoxic concentration (identical fractions of the LCsOvalues). In experiments with
chlorophenols the pH was kept at 7.3 with a buffer solution (KGnemann and
Musch, 1981). The LC5Dvalues of the single compounds are given in Table 3. The
joint toxicity was evaluated with the MTI. For equitoxic mixtures this MT1 is
defined as
MT1 = 1 - (log M/log n)
(1)
in which A4 = C c/LCsO at 50% effect in the mixture, and n = total number of
compounds in the mixture. Tests with mixtures of one compound per group were
carried out four times, each time with different representatives from the groups.
Also, the experiments with combinations of the groups were carried out four times.
The compositions of the groups of compounds are given in Table 3.
RESULTS AND
DISCUSSION
The results of the experiments with mixtures of groups are given in Table 4.
Joint toxicity of the mixtures appears to be not much less than concentration
TABLE
2
MIXTURE TOXICITY SCALES
MT1
MTI<O
MTI=O
O<MTI<l
MT1 = I
MTI>
1
Classificationfor toxicity of mixtures (possibletypes of joint
action)
Antagonism
No addition (independentaction. r = +l)b
Partial addition
Concentration addition (simplesimilar action)
Supraaddition (potentiation of the toxic actionsof one or more
of the compoundsin the mixture)
’ MT1 calculated after K6nemann (198 la).
b Positive correlation
between susceptibilities
chemicals in a mixture.
of the individual
organisms
to the single
TABL.E
COMPOSITION
OF THE GROUPS
OF CHEMICALS
3
TOGETHER
WITH
THE
LC5,, VALUES
log LCSO’
No.
Group
1
2
3
4
5
6
7
8
9
10
11
A: Nonreactive (chlorinated)
1,3-Dichlorobenzene
1,2,3-Trichlorobenzene
Monochlorobenzene
1.2,3,4-Tetrachlorobenzene
Benzene
Pentachlorobenzene
Toluene
2,4-Dichlorotoluene
nr-Xylene
4Chlorotoluene
Chloroform
Group
hydrocarbons”
1.70
1.11
2.23
0.57
2.9 1
-0.15
2.87
1.46
2.55
1.67
2.93
B: Chloroanilines”
12
13
14
15
16
17
18
19
20
21
22
2-Chloroaniline
3,5-Dichloroaniline
2.3,4-Trichloroaniline
3,4-Dichloroaniline
Aniline
3-Chloroaniline
4-Chloroaniline
2,5-Dichloroaniline
2,4-Dichloroaniline
2.4,5-Trichloroaniline
2.3,4,5-Tetrachloroaniline
1.69
1.38
0.85
1.59
3.13
2.02
2.31
1.01
1.59
I .oo
0.19
23
24
25
26
27
28
29
30
31
32
33
Group C: Chlorophenols’
3-Chlorophenol
2,4-dichlorophenol
3,4,5-TrichlorophenoI
2,3,5,6-Tetrachlorophenol
Phenol
2-Chlorophenol
3,5-Dichlorophenol
2,3,5-Trichlorophenol
2,3,6-Trichlorophenol
2.3,4,5-Tetrachlorophenol
Pentachlorophenol
1.70
1.41
0.76
0.77
2.50
1.94
1.22
0.90
1.41
0.52
0.15
34
35
36
37
38
39
40
41
42
Group D: Reactive organic halidesd
Allylchloride
1,4-Dichloro-2-butene
Chloroacetone
Benzylchloride
2.3-Dichloropropene
a,&-Dichloro-m-xylene
1-Chloro-2,4-dinitrobenzene
2.4-a-Trichlorotoluene
Hexachlorobutadiene
0 LCSo
b LCso
’ LC5,,
d LC5,,
’ LCsO
values taken
values taken
values taken
values to be
in pmol/liter.
from Konemann ( 198 1b).
from Hermens et al. ( 1984).
from Konemann and Musch (198 1).
published elsewhere.
323
1.20
-0.16
0.88
0.49
1.01
-0.16
-0.19
0.08
-0.20
334
HERMENS, LEEUWANGH.
AND MUSCH
TABLE 4
RESULTSOF 14-DAY LCs,, EXPERIMENT WITH
GUPPIES OF MIXTURES OF GROUPS
Combination
Group A-group B
n”
Mb
MTI’
22
1.2
1.2
0.94
0.94
0.87
0.87
1.5
1.5
Group B-group C
22
1.2
1.2
0.94
0.97
0.94
1.0
1.oo
1.1
Group A-group C
22
1.3
1.5
1.2
1.4
0.92
0.87
0.94
0.89
Group A-group D
18
1.3
1.4
1.1
0.91
1.3
Group A-group Bgroup C
33
1.5
1.3
1.2
1.2
0.88
0.97
0.91
0.88
0.92
0.95
0.95
’ Total number of compoundsin mixtures.
’ Each combination wastestedfour times.The
definition of M is given in Eq. (1).
’ MT1 calculatedwith Eq. (1).
additive. Such a relatively high joint toxicity was also found in experiments with
mixtures of chemicals with diverse modes of action, and can be explained in several ways:
(i) Mortality can be related to the disturbance of several biological systems.
Toxicants with different modes of action can lead to disturbance of the same system
(a system may be affected at more than one site). Therefore completely independent
actions between the toxicants, which will result in a lower joint toxicity, are unlikely
in experiments with mortality as overall criterion of effect.
(ii) The joint toxicity between chemicals within each group is concentration
additive. This phenomenon enhances the toxicity of combinations of these groups.
(iii) Although different QSARs can be calculated for the four groups, this does
not mean that the chemicals have completely different modes of action. If the
compounds from the different groups have common toxic actions, their combined
effect will be more in the direction of concentration addition.
In the mixture of 33 compounds, the combination of nonreactive (chlorinated)
hydrocarbons, the chloroanilines and the chlorophenols, concentrations of 0.04 of
the LCsa values do contribute to the toxicity of the mixture, and the joint toxicity
TOXICITY
OF AQUATIC
POLLUTANTS
TABLE
RESULTS OF 14DAY
325
TO THE GUPPY
5
LCs,, EXPERIMENTS WITHGUPPIES
OFMIXVJRES COMPOSED
OF ONECOMPOUND
PERGROUP
Combination”
Al”
Group
I-12
2-13
3-14
4-15
Group
l-23
2-24
3-25
4-26
Group
l-12-23
3- 13-24
3- 14-25
4-15-26
MTl’
Combination”
A-group B
1.3
0.7
0.9
1.2
0.62
1.51
1.15
0.14
12-24
13-23
14-26
15-25
A-group C
1.2
1.5
1.2
1.4
0.74
0.43
0.74
0.51
l-34
2-35
3-36
4-37
A-group
B-group
1.5
1.5
1.6
1.4
Mb
Group
Group
B-group
1.4
1.5
1.5
1.5
MTI’
C
0.51
0.42
0.42
0.42
A-group D
1.1
1.8
0.8
0.9
0.86
0.15
1.33
1.15
C
0.63
0.63
0.51
0.69
” Numbers correspond with those from Table 3.
’ Definitions of hf and MT1 are given in Eq. (I).
is almost completely concentration additive (MT1 = 0.93 + 0.02). This means that
in mixtures of this kind of aquatic pollutants, also at concentrations far below the
unobserved lethal concentrations, the joint effect is much higher than is to be
expected from the influence based on a single compound.
The results of the experiments with mixtures composed of one compound per
group are given in Table 5. The joint toxicity of most of the mixtures is partially
additive (0 < MT1 < 1). Within a combination. for instance between groups A and
B, the MT1 values show a high variance (MT1 varies from 0.62 to 1.51). A possible
explanation for this high variance is that the results of mixture toxicity experiments
with only a few compounds are less reliable compared to mixtures of many
chemicals (Kiinemann, 1981a). Because of this high variance in the experiments
with mixtures of two or three chemicals, a comparison of the joint toxicity of
combinations of one representative from each group with the joint toxicity of
mixtures of whole groups is not to be recommended.
ACKNOWLEDGMENTS
This work was supported by the Department of Housing, Physical Planning. and Environment, The
Netherlands. We thank Prof. Dr. van Genderen and Dr. H. KBnemann for their stimulating discussion
and comments on the manuscript.
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AND
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