PII: S0025-326X(99)00084-3

Marine Pollution Bulletin Vol. 39, Nos. 112, pp. 5461, 1999
1999 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
0025-326X/99 $ - see front matter

Importance of Internal Biotic

Concentrations in Risk Evaluations with
Aquatic Systems
D. W. CONNELL*, Y. CHAISUKSANT and J. YU
Faculty of Environmental Sciences, Grith University, Kessels Road, Nathan, Queensland 4111, Australia
Ecological risk evaluations are commonly performed
using aqueous concentrations and aqueous toxicity measurements as a starting point. However risk evaluations
could be carried out using internal biotic concentrations
and the internal lethal or sublethal concentrations. This
has several advantages. Firstly, the internal lethal and
sublethal concentrations are relatively consistent in groups
of chemicals having a similar mode of action. Thus in eld
situations the internal concentration, in sh and possibly
other biota, can be used to evaluate possible biotic eects.
Also other histopathological, biochemical, biomolecular
and physiological eects can be assessed and used with
this information to give an overall assessment. There are,
however, several limitations with this approach including
sensitivity, health, age and nutritional status of the biota
as well as a lack of data on dose/response relationships
with internal concentrations. 1999 Elsevier Science
Ltd. All rights reserved.

these factors when interpreting results. Furthermore,

toxic eects are more clearly expressed since they are
due to toxicants at the target sites thereby facilitating
identication and investigation of observed biotic concentrations and dierent modes of action of toxicants.
Joint toxicity of chemical mixtures may also be more
readily estimated (McCarty and Mackay, 1993). However, at present little information is available regarding
the internal concentration and the relation between the
internal toxic level and biological and physiological responses as well as the application of this approach to the
assessment of environmental eects.
The objective of this work was to compile information
on internal toxic levels related to biological eects,
briey review risk evaluations based on internal concentration, and to develop strategies for assessment of
lethal and sublethal adverse eects of organic chemicals
with aquatic organisms based on internal concentration.

Introduction

Biological Eects Related to Internal Residues

for Organic Chemicals

Ecological risk assessment in aquatic systems is usually

performed on the basis of toxicity testing data associated with external aqueous exposure concentrations and
models developed mostly from toxicity bioassays. The
actual or estimated environmental concentration (EEC)
of the toxicant is compared to a concentration of concern and a potential risk evaluated.
It has been suggested that risk evaluations could be
performed by comparison between measured biotic
concentrations and the internal lethal concentrations or
internal eective concentrations of concern, instead of
between ambient water concentrations and water concentrations causing toxic eects e.g. LC50 and NOEC
(McCarty and Mackay, 1993). In this approach accumulation kinetics, bioavailability, uptake from food and
eects of metabolism on bioaccumulation are taken into
account which could reduce the confounding eect of
*Corresponding author. Tel.: +07-3875-7519/20; fax: +07-38757459.

54

Lethal eects
Several terms have been used for the internal lethal
concentration (ILC) including critical body burden.
Generally, the level of internal residue is considerably
higher than that in the surrounding water, enabling
contaminant concentration to be more readily measured
with conventional techniques. The values of the internal
level at death in aquatic organisms are relatively consistent for hydrophobic organic compounds exhibiting a
similar mode of toxic action whereas the LC50 values can
vary up to three orders of magnitude depending on
various factors such as species, bioavailability, uptake
routes, non-steady state condition and toxicant biotransformation, which are dicult to evaluate accurately (McCarty and Mackay, 1993).
Table 1 indicates generalized ranges of internal lethal
concentrations for modes of toxic action for various
organic chemicals with a wide range of biotic species.
Non-polar narcotic compounds, which generally exhibit
the lowest toxic eect, have ILC values ranging from

Volume 39/Numbers 112/JanuaryDecember 1999

TABLE 1
Internal lethal and sublethal concentrations for some organic chemicals with aquatic organisms.
Chemical group
Non-polar narcotic
Polar narcotic
ACHE inhibitiona
TCDDb
a
b

Lethal range (mmol kg1 wet weight)

Sublethal range (mmol kg1 wet weight)

112
0.25
0.00258
0.00090.008

0.016
0.11
0.00010.5
0.0000010.0001

Acetylcholinesterase.
Tetrachlorodibenzodioxin.

1 to 12 mmol kg1 wet weight for a variety of organisms

including sh, earthworms, crabs, daphnids and mussels. Polar narcotics and other chemical groups listed in
Table 1, all have higher toxicities as indicated by the
ILC.
For crustacea, the ILC values are similar to those with
sh for each mode of action. A limited amount of data
are available for annelids and birds for organic compounds with dierent modes of toxic action. However,
the ILCs of chlorobenzenes with earthworms (0.14
mmol k1 ) fall in the same range as those with sh and
crustacea. This suggests that ILC values for the organic
chemicals with similar mode of toxic action are relatively consistent with several aquatic organisms.
Sublethal eects
The sublethal responses commonly evaluated include
survival, growth and reproduction reduction of biota
exposed to a range of sublethal concentrations of
chemical pollutants. Extensive investigations of sublethal eects have been carried out in terms of aqueous
exposure concentrations of chemicals while limited data
on sublethal responses associated with internal concentrations are available.
The generalized internal concentration values for
sublethal toxic eects, such as survival, growth rate reduction and reproduction inhibition of organic compounds with dierent modes of toxic action are
presented in Table 1. These values exhibit a pattern
similar to that of ILC values, that is, the internal eective concentration (IEC) for non-polar narcotics (0.016
mmol kg1 wet weight) is the highest whereas those for
compounds with specic modes of action are the lowest
representing higher toxicity.
It has been reported that the chronic residue level for
growth, survival and reproductive eects can be estimated from bioconcentration and acute toxicity data
(Call et al., 1985; McCarty et al., 1985). McCarty (1986)
reported the chronic residue estimates for these eects of
about 0.01 of the acute residues (ILC50 ) based on a
QSAR analysis for various organic chemicals. This is in
agreement with the measured chronic internal concentrations for some chlorobenzenes with sh reported by
Carlson and Kosian (1987), which were about 0.250.01
of the acute concentrations (0.583.8 mmol kg1 sh).
McCarty (1996) has calculated the critical internal

concentration for chronic toxicity response at LC0:01

from the acute toxicity curve.
The internal concentrations producing 50% growth
rate reduction (IEC50 ) for hydrophobic organic compounds, particularly narcotic chemicals, are relatively
consistent when compared to the aqueous exposure
concentration causing growth rate reduction at 50% of
the population (EC50 ). Chaisuksant et al. (1998) have
shown that internal concentrations to give 10% and
50% growth rate reductions are 0.51.6 and 8.327
mmol kg1 wet weight, respectively. Likewise, Mortimer
and Connell (1995) have observed reasonably consistent
critical internal concentrations in relation to a 50%
growth rate reduction for chlorobenzenes with juvenile
crabs.
Sublethal physiological responses of mussels (e.g.
feeding rate, respiration and excretion) have been reported to be sensitive to many toxicants and reect
major modes of toxic action (e.g. narcotic and neurotoxic eects on ciliary feeding, uncoupling of the respiratory system) (Donkin et al., 1989, 1991; Widdows and
Johnson, 1988). Integration of the sublethal physiological responses by means of physiological energetics
(scope for growth, SFG) provides a rapid quantitative
estimation of energy required for growth and reproduction. Feeding rate and SFG have been found to
inversely relate to the tissue concentrations of organic
hydrophobic compounds over a wide range of chemical
concentrations. Physiological measurements can be
performed on mussels in the laboratory and the eld,
hence providing the laboratory derived tissue concentration-response correlation to be used to evaluate responses in the eld (Widdows and Donkin, 1989).
Donkin et al. (1989, 1991) have established a relationship between log KOW and log KB of some hydrophobic
organic chemicals and sublethal toxic eects in terms of
external aqueous concentration and internal concentration of the chemicals which reduced feeding rate of
mussels by 50% (EC50 , and IEC50 , respectively). Also
Widdows, Donkin and Evans (1987) have observed an
inverse relationship between the scope for growth (SFG)
of mussels and the concentration of petroleum hydrocarbons in tissues of mussels (as log IEC) after exposure
for eight months to two oil concentrations (28 and 125
lg l1 total hydrocarbons). The SFG reduction of
mussels for chronic oil exposure was primarily due to a
decline in feeding and ingestion rate, and a decrease in
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Marine Pollution Bulletin

the food absorption eciency in the gut (Widdows et al.,

1987).

Approaches to Risk Assessment Using Internal

Concentrations
McCarty and Mackay (1993) have proposed an approach using the internal lethal concentration and
mode of toxic action of hydrophobic organic chemicals
in risk assessment as described in Fig. 1. Chemical
pollutants from various sources, including agricultural,
municipal, industrial and domestic wastes accumulate

in the tissues of sh and other aquatic biota. These

internal levels of chemicals can be determined by
chemical analysis and, in general, related to their
modes of toxic action. The measured IC from the eld
and/or the estimated IC obtained from bioaccumulation data are then compared with the reference ILC for
each mode of action. In this approach, a nal judgement of likelihood of adverse biological eects of the
toxicants on aquatic organisms can be made based on
the consistency of the ILC of chemicals and their
possible modes of toxic action (McCarty and Mackay,
1993).

Fig. 1 Diagrammatic representation of the use of the constant lethal

concentration and mode of toxic action in environment risk
assessment (from McCarty and Mackay, 1993).

56

Volume 39/Numbers 112/JanuaryDecember 1999

Van Wezel (1995) has also proposed the use of internal concentrations in risk assessment of mixtures of
environmental contaminants, particularly narcotic organic chemicals, for lethal eects with sh. It has been
reported that the joint toxicity of a mixture of narcotic
industrial organic chemicals in aquatic organisms follows a concentration addition model at the lethal response level (K
onemann, 1981; Deneer et al., 1988a,b;
Hermens and Leeuwangh, 1982; Hermens et al., 1984,
1985). In addition, it has been suggested that the internal
concentration for narcotic chemicals in a mixture is
likely to be additive (McCarty et al., 1992; Van Wezel,
1995). Van Wezel (1995) found that 1,2-dichlorobenzene
and 1,4-dichlorobenzene in mixtures tested with the
guppy appeared to contribute to the joint toxicity in
terms of concentration addition based on the internal
lethal concentration. It is likely that other narcotic organic chemicals will also exhibit concentration additivity
with the ILC due to their similar modes of action (Van

Wezel, 1995). Furthermore, variation of exposure concentrations, extra uptake from food, limited bioavailability and metabolism of parent compounds are not
critical factors in evaluating the ILC of mixtures. The
use of ILC in risk assessment of chemical mixtures in
eld conditions, as proposed by Van Wezel (1995), is
represented in Fig. 2.
Widdows and Donkin (1989) have described the use
of combined tissue residue and sublethal physiological
energetic measurements of mussels (Mytilus edulis) in
the assessment of environmental contamination. Physiological measurements can be performed on mussels in
the laboratory and the eld, hence providing the laboratory derived tissue concentration-response relationship which can be used to evaluate responses estimated
in the eld (Widdows and Donkin, 1989). Scope for
growth (SFG) with the mussels has been found to inversely relate to the tissue concentrations of organic hydrophobic compounds over a range of chemical

Fig. 2 Diagrammatic representation of proposed use of ILC in risk

assessment with sh populations exposed to mixtures of environmental contaminants in eld conditions (from Van Wezel,
1995).

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Marine Pollution Bulletin

concentrations (Donkin et al., 1989, 1991). These tissue

concentration-response relationships can be used for
other related compounds in eld studies.
Organisms are collected from the eld contaminated
with chemical toxicants and analysed for the internal
toxicant concentrations. The SFG for the organisms can
then be predicted from the observed IEC using the
SFGlog IEC relationship. If an observed SFG reduction in the eld studies is found to be greater than predicted this would be possible due to action from
additional environmental contaminants, and consequently, further chemical pollutant analysis of biotic
tissue and environment needs to be performed (Widdows and Donkin, 1989).

A Proposed Risk Assessment Strategy for

Organic Chemicals with Aquatic Organisms
During this current work, a strategy for assessment of
lethal and sublethal eects of environmental contaminants with aquatic organisms was developed and is illustrated in Fig. 3. Organisms sampled from the eld
population at dierent sampling sites and dierent seasons are analysed for chemical contaminants. The estimated concentrations in biotic tissues, together with
investigations of blood chemistry, biochemical and
biomolecular alterations, histopathological conditions,
reproduction, growth and bone development, can be
additionally used to predict lethal and sublethal eects
elicited from organisms in the population of concern.
Then a comparison of the measured internal concentration with the internal lethal concentration obtained in
the laboratory can be carried out for risk assessment. A
judgement based on the consistency of the ILC50 for
lethal eect assessment is also needed. The onset of the
lethal eect in a biotic population can be described when
the tolerance distribution of individual organisms
among the population to chemical exposure is known.
The tolerance distribution of a biotic population to a
toxicant can be estimated in the laboratory from the
range of measured internal lethal concentrations obtained by direct exposure to the toxicant or by modelling
based on tolerance distributions from acute toxicity
bioassays as proposed by Breck (1988). It has been
suggested that the lethal eect is directly associated with
the internal concentration and the mean ILC50 is equal
to the lethal internal concentration in the average organism (Hickie et al., 1995; McCarty, 1996). According
to the ILC50 approach, the lethal eect will be observed
when the internal concentration of a toxicant reaches a
critical value and the internal lethal concentration is
presumably a surrogate for the toxic (lethal) level at the
target site in the organisms. The values of ILC50 for
hydrophobic organic chemicals, particularly narcotic
compounds, have been found to be reasonably constant
among sh species if the exposure time is relatively
short, as indicated in Table 1. Also, the ILC50 values for
58

narcotic chemicals with dierent kinds of organisms

including sh, crustacea and annelids, fall in the same
range of about 0.110 mmol kg1 wet weight. Hence, the
dierence in species sensitivity is unlikely to create a
high uncertainty level of extrapolation when the ILC50
approach is applied in risk analysis as compared to the
acute toxicity (LC50 ) approach. Furthermore, the ILC
values for a range of exposure concentrations of organic
compounds with similar modes of toxic action are found
to be reasonably consistent.
In setting acceptable exposure concentrations, we
have set the ILC at 1% of internal level causing lethal
response in an organism (0.01) as the maximum acceptable internal toxic level or no eect ILC (ILC1 ). The
maximum allowable aqueous toxicant concentration (in
molarity) can then be derived from the following equation:
Maximum allowable aqueous toxicant concentration
MATC ILC1 =KB ;

where KB is the bioconcentration factor of the toxicant

for the test organisms.
In assessment of the lethal toxic eect of environmental pollutants on organisms captured in the eld for
risk characterization, the observed internal level measured by chemical analysis is compared to the ILC1 as
well as ILC50 which has the general ranges indicated in
Table 1. This comparison not only reects the likelihood
of a lethal response of the organisms but can reveal information on the possible type of contaminants. Furthermore, under ideal conditions the toxicity eect in
relation to time measures based on internal concentration can be determined:
ILC50 MATC:KB 1 ek 2t ;
where t is exposure period, and k2 is a clearance rate
constant for the toxicant which can be estimated experimentally or calculated. The duration of exposure
may be revealed by assuming continuous consistent exposure for the life of the organism. Additionally, the
percentage of the population of the organisms of concern anticipated to exhibit the lethal response can be
achieved from the correlation between percent mortality
and log ILC as previously mentioned.
To achieve a more complete and realistic evaluation
of lethal eect based on ILC50 , confounding factors including lipid content, non-steady condition, population
density within and between species, seasonal factors,
nutritional and health status of the organisms in the eld
have to be considered. Pulse and uctuating conditions
are examples of non-steady state exposure which can be
dened as one or more isolated and short exposures
(Hickie et al., 1995). Fluctuating exposure is a continuous exposure to varying levels of a chemical contaminant (Breck, 1988). It has been found that the internal
lethal concentration determined from a pulse-exposure
toxicity model, suggested by Hickie et al. (1995), seems

Volume 39/Numbers 112/JanuaryDecember 1999

Fig. 3 A proposed strategy for assessment of lethal and sublethal effects of chemical pollutants with aquatic organisms.

to give a reasonable approximation of the lethal eect

with sh. However, this model can only be applied to
organic compounds that partition following a onecompartment rst-order kinetic model and assuming
exposure time does not have a major eect over short
time periods (Hickie et al., 1995).
For environmental chemical mixtures, the measured
internal concentration of the component chemicals in
the mixture could be summed in terms of the fraction of

the individual ILC50 as obtained in the laboratory. This

assumes that the individual chemicals in the ecosystem
exist in low concentrations and their mode of toxic action contributes to the toxicity of the mixture following
concentration additivity.
Sublethal eects of environmental contaminants on
aquatic organisms can be assessed based on internal
concentration and other biomarkers such as histopathological alterations, biomolecular changes, biochemical
59

Marine Pollution Bulletin

and physiological changes. After sampling of the organisms of concern from eld sites, the organisms are
examined for general condition before undergoing laboratory tests of blood for enzyme activities, lipid, protein and carbohydrate contents, tissues for lipid analysis,
bone for vertebral quality and development, and liver
for detoxicating enzyme activities, DNA damage and
histopathological estimations. General condition examinations include a measurement of total length and
weight of individual organisms and observation of the
general condition of the organisms such as presence or
absence of skin disease, body and/or mouth infection,
and internal and external parasites (Adams et al., 1990).
The measured length and weight of the organisms can be
used to estimate a condition factor and growth rate reduction.
Growth rate reduction can be revealed by measuring
total weight and length of organisms at dierent exposure periods in a eld study area. It has been found that
length is less sensitive than weight of an organism exposed to sublethal level of a toxicant (Mayer et al.,
1986). However, a length change is relatively less variable than weight change and it has been used in the
prediction of growth rate reduction of aquatic organisms (e.g. Deneer et al., 1988a; Mayer et al., 1986;
Mortimer and Connell, 1995).
Growth rate of some aquatic organisms having an
external skeleton, such as mussels that cannot be easily
and accurately assessed using length or weight measurements as carried out with sh, can be estimated in
terms of Scope for Growth (SFG) as discussed previously. Sublethal physiological responses, such as feeding
rate, respiration and excretion rates, have been found to
be sensitive to a variety of chemical pollutants. Integration of these sublethal responses by means of physiological energetic measures, such as SFG, provides a
rapid quantitative assessment of energy available for
growth and reproduction processes of the sampled organisms.
Growth rate reduction elicited by organisms exposed
to sublethal levels of a toxicant can be assessed using
internal concentrations producing 50% growth rate reduction (IEC50 ), which are relatively consistent compared to external exposure concentration causing
growth response (EC50 ). Similar to the ILC approach,
10% of the IEC50 was considered to be the maximum
toxicant level causing growth rate reduction that organisms can tolerate with undetectable growth eects. A
maximum acceptable aquatic exposure concentration
for growth eects could be obtained using the relationship presented in Eq. (1).
Histopathologic alterations in sh tissues, particularly
liver and brain, are morphologic expressions which integrate biochemical and physiological responses of an
organism when exposed to a chemical pollutant. These
alterations have been found to provide valuable information on sublethal eects of certain chemicals (e.g.
pesticides) on aquatic organisms (Arnold et al., 1996;
60

Braunbeck and V
olkl, 1991; Braunbeck et al., 1990;
Cormier and Racine, 1990; Hinton and Lauren, 1990;
Hinton et al., 1992). The internal concentrations related
to these histological changes allowing dose/response
relationships to be established have not been subject to
signicant research.

Conclusions
Risk assessment can be carried out by comparison of
measured biotic concentrations with the internal concentrations of concern derived from bioconcentration
and acute and chronic toxicity data. For lethal eects a
judgement can be made on the consistency ILC50 values.
The maximum acceptable aqueous toxicant concentration at a no lethal eect level (ILC1 ) and ILC50 at different times can be estimated from the bioconcentration
rst-order kinetic model. With sublethal eects the internal concentration causing 50% growth rate reduction, which is relatively constant, can be used to
determined a maximum acceptable exposure level for
growth eects in eld conditions. Furthermore, histopathological, biochemical, biomolecular and physiological alterations in biota exposed to toxicants can be used
in conjunction with internal concentrations to describe
and assess lethal and sublethal eects of toxicants.
Limitations of using these approaches as tools in assessment of adverse eects arise from various confounding factors including susceptibility, sensitivity, sex,
age, nutritional and health status of biota, chemical
route and type, and seasonal variation. Additionally,
sensitivity and specicity of analytical methods in detecting these responses and limited data on dose-eect
relationships for these alterations also limit the use of
these biomarkers in ecological risk assessment. However, the internal concentration is less inuenced by the
confounding factors, and dose-response relationships
for internal concentrations of toxicants can be quantied more accurately.
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(1990) Application of bioindicators in assessing the health of sh
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