ARTICLE IN PRESS

Ecotoxicology and Environmental Safety 57 (2004) 20–29

A review of lysosomal membrane stability measured by neutral red

retention: is it a workable earthworm biomarker?
C. Svendsen,a, D.J. Spurgeon,a P.K. Hankard,a and J.M. Weeksb
a
Centre for Ecology and Hydrology, Monks Wood, Abbotts Ripton, Huntingdon, Cambridgeshire PE28 2LS, UK
b
National Centre for Environmental Toxicology, WRc-NSF Ltd., Medmenham, UK
Received 4 August 2003

Abstract

The ability of biomarkers to integrate effects of chemicals on biota has lead to increased calls for their application in assessing the
status of polluted ecosystems. In tandem there has been an increase in our knowledge of the ecophysiological responses of keystone
species to pollutants, which has allowed the development of a number of promising methods. In contrast to the number of
biomarker development studies, the number of biomarker validation studies has remained limited. This paper redresses this
imbalance by drawing together data from studies that have used the earthworm lysosomal membrane stability response (measured
using the neutral red retention assay). This review first gives a short history of the biomarker’s development. Second, it sets
published applications of the technique against established criteria for a ‘‘good’’ biomarker (i.e., dose–response relationship,
sensitivity, ecological relevance, confounding factors, chemical specificity, species differences, time–response relationship,
methodological concerns, and overall public/regulator confidence, and acceptance). Discussion of the biomarker’s suitability to
each criterion is followed by an overall evaluation of its workability for routine soil quality assessment and caveats for its use.
r 2003 Elsevier Inc. All rights reserved.

Keywords: Neutral red retention; Earthworm; Workable; Validation; Soil quality

1. Introduction 1.1. Suitability criteria for biomarker use in regulatory

It is becoming increasingly apparent that chemical
analysis and the use of chemical-specific trigger
values cannot take into account issues such as
mixture toxicity and the environmental conditions
determining chemical bioavailability. The best integra-
tors of these complex effects are the exposed orga-
nisms themselves. The appropriate use of biomarkers
in sentinel organisms provides a first set of tools
with which we can measure the actual effects that
chemicals are having on biota in the field. For a
biomarker to be of maximum use for practical
monitoring applications, the measured response must
fulfil certain criteria, as discussed by Huggett et al.
(1992). For this review these criteria have been grouped
as listed below.

Corresponding author.
E-mail address: csv@ceh.ac.uk (C. Svendsen).
soil quality assessment
* Dose–response relationship
* Sensitivity
* Ecological relevance
* Confounding factors
* Chemical specificity
* Species differences
* Time–response relationship
* Methodological concerns
* Overall public/regulator confidence and acceptance
This review will critically examine the suitability of a
recently developed earthworm biomarker (lysosomal
membrane stability measured through the use of the
neutral red retention assay) for use in regulatory soil
monitoring with respect to the criteria outlined above.
The lysosomal membrane stability assay described by
Weeks and Svendsen (1996) has become one of the few
academically developed methods for which a critical
mass of work has been undertaken, which allows a full

0147-6513/$ - see front matter r 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2003.08.009
 ARTICLE IN PRESS
C. Svendsen et al. / Ecotoxicology and Environmental Safety 57 (2004) 20–29
investigation of the suitability and applicability of this
biomarker as a routine monitoring tool.
2. Background and history of the assay development
2.1. Lysosomal membrane integrity as a parameter
At the subcellular level the lysosomal system has been
identified as a particular target for the toxic effects of
contaminants (Moore, 1990), and pathological altera-
tions in lysosomes have been especially useful in the
identification of adverse environmental impacts on
marine organisms (Giamberini and Pihan, 1997; Moore,
1980; Moore et al., 1996). When marine molluscs such
as mussels are exposed to xenobiotics, one of the
characteristic pathological alterations is decreased in-
tegrity of the lysosomal membrane (Moore, 1988).
Lysosomal membrane integrity has also been shown to
decrease with increasing nonspecific stress (i.e., biotic
and abiotic) (Moore, 1985). The mechanism(s) causing
this alteration in membrane stability is not well
understood, but it may involve direct effects of
chemicals on the membrane or the increased frequency
of secondary lysosomes in toxicant-stressed cells (Mayer
et al., 1992).
2.2. Technique development
Prior to the development of the neutral red retention
assay, two techniques were traditionally used for
assessing the stability of the lysosomal membrane, one
cytochemical (Moore, 1976) and the other biochemical
(Baccino and Zurretti, 1975). Both techniques are based
on the measurement of the activity or amount of
lysosomal enzyme that leaks through the lysosomal
membrane after either pH or hypoosmotic shock,
respectively. Neither of these techniques operates
under normal physiological conditions when observing
the leakage from the lysosomes. Technically the neutral
red retention assay is much simpler than both the
cytochemical and the biochemical techniques and
operates under physiologically relevant in vitro
conditions.
The original neutral red assay was developed as an
in vitro cytotoxicity assay based on the use of
mammalian cells in culture but was also adapted for
ecotoxicity studies using fish cells in culture (Babich and
Borenfreund, 1990). This original assay was based on
the uptake and binding of neutral red, a cationic dye, in
the lysosomal matrix of viable cells after their incuba-
tion with toxic agents. The assay was used in a wide
range of cytotoxicity studies to determine relative acute
cytotoxicities of chemicals, structure–toxicity relation-
ships for related chemicals, metabolism-mediated cyto-
toxicity, and mixture interactions and was utilized in
21
other pharmaceutical testing. Lowe et al. (1992) devel-
oped this assay further for work with cells isolated from
organisms exposed in vivo and used it to study
lysosomal injury in isolated liver cells of fish caught in
‘‘clean’’ and contaminated sites. An assay has been
developed that quantifies the process of leakage of the
dye from the lysosomes under in vitro conditions (Lowe
and Pipe, 1994). This has been used for assessments of
contaminant-induced lysosomal membrane damage
using neutral red dye retention as a determinant in
marine mussels (Lowe et al., 1995).
On the basis of this technique, further modifications
were implemented by Svendsen and Weeks (1995) and
Weeks and Svendsen (1996) to enable the use of the
neutral red retention assay with freshwater and terres-
trial invertebrates. This further-adapted technique oper-
ates with minimum treatment stress on the cells, as both
sample preparation and observation are completed
under physiologically relevant in vitro conditions; most
importantly, it offers a greatly increased time resolution
of the response and therefore has higher sensitivity
(Svendsen and Weeks, 1995; Weeks and Svendsen,
1996). The technique is conducted on extracted cells
(coelomocytes in earthworms) obtained using a hypo-
dermic needle and syringes. Extracted cells are incu-
bated on a microscope slide in a physiological saline
solution containing 40 mg mL 1 neutral red stain. Only
lysosomes in healthy cells permanently retain the
cationic dye after initial uptake; in cells in which the
integrity of the lysosomal membrane has been impaired
by chemical exposure, the neutral red dye is able to leak
from the cell, with the result that the cell cytosol stains
red. Decreasing times for reddening of the cell cytosol
equate to increasing damage to the lysosomal mem-
brane.
3. Assessing neutral red within the context of the
biomarker suitability criteria
3.1. Dose–response relationship
The relationship between the level of chemical
exposure and the biomarker response ideally should be
clear and consistent. For practical applications in soil
quality assessment this relationship should be mono-
tonic (i.e., the response must either increase or decrease
with increasing exposure). Biomarkers, such as induced
heat shock proteins (HSPs), may have bell-shaped dose–
response curves; these are difficult to interpret in the
‘‘single-dose’’ scenario of a field site, as it is impossible
to determine from which side of the bell shape a low
response has come.
For earthworms the first dose–response curve of
neutral red retention time (NRRT) against nominal soil
chemical concentration was provided by laboratory
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exposures of Eisenia andrei to copper (20–320 mg kg 1)
in sandy loam for 28 days by Svendsen and Weeks
(1997a). Although this relationship was monotonic,
it appeared stepped, with the NRRT reduced slightly
to 40–50 min at low doses and further decreased to
less than 20 min at soil copper concentrations of
80 mg kg 1 or more. When analyzing the dose–response
curve of the NRRT against earthworm tissue concen-
trations, it was apparent that the copper tissue
concentrations in the group of earthworms with severely
decreased lysosomal membrane integrity covered a
continuous range (25–115 mg g 1) that overlapped
that of the group with only slightly decreased NRRTs
(10–60 mg g 1). Thus, severely decreased lysosomal
integrity appeared linked more to high exposure
concentrations than to the actual earthworm tissue
concentrations.
A subsequent mesocosm experiment by Svendsen and
Weeks (1997b) used the same soil type and copper
concentrations for exposure of Lumbricus rubellus.
Sampling was undertaken after 17, 40, 70, and 110
days. Dose–response relationships for the NRRT
followed the same general pattern as that seen in the
laboratory for E. andrei, except for on day 110
(December), on which low NRRTs were found in all
treatments, including controls. This coincided with
severe flooding and waterlogging of the mesocosms.
The relationship between the NRRT and earthworm
tissue concentrations (Fig. 1) showed a development in
the distribution of NRRTs from being nearly contin-
uous on day 17, to being stepped in a manner
comparable to the 28-day laboratory study with E.
andrei on day 40, to being highly stepped on day 70.
This step, in response, developed as the worms exposed
to soil copper concentrations of 20 and 40 mg kg 1
showed an increased NRRT (i.e., less cellular stress) as
exposure time increased. Concurrently, the initial over-
lap in tissue copper concentrations between worms
exposed to p40 and X80 mg kg 1 gradually disap-
peared (see Fig. 1). These changes may have been caused
either by decreased bioavailability of the copper as the
soils age or by the worms being able to adapt their metal
handling systems enough to successfully regulate copper
when exposed to the lower concentrations but not at the
higher ones. Either way, this indicates that decreased
lysosomal integrity as measured by the NRRT did not
simply reflect the internal tissue concentrations, but
rather showed how well copper regulation was handling
the current exposure.
Following the initial detailed characterization of the
monotonic dose–response relationship for copper,
similar relationships have been found for a range
of other pollutants, including metals and organic
compounds.

(a) (b)

(c) (d)
Fig. 1. Neutral red retention times (min) plotted against corresponding earthworm body copper concentrations (mg g 1 dry wt) for the earthworm
(L. rubellus) exposed to an increasing range of soil copper concentrations for 17 (a), 40 (b), 70 (c), and 110 (d) days, respectively, in a field mesocosm
experiment. The different symbols represent the different nominal exposure concentrations: control (black circle), 20 mg Cu kg 1 (open triangle, up),
40 mg Cu kg 1 (black triangle, down), 80 mg Cu kg 1 (open diamond), 160 mg Cu kg 1 (black star) and 320 mg Cu kg 1 (open circle).
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3.2. Sensitivity and ecological relevance
To best enable regulatory monitoring to detect
pollution effects before they become irreversible, the
response of the biomarker (in terms of both response
time and the ability to detect low-dose exposure) should
be sensitive enough to precede effects on key life cycle
parameters such as reproduction and growth. Such links
between the biomarker response and effects at higher
levels of organization (e.g., growth, reproduction, and
population growth) or the likelihood of such effects
occurring can be either correlative or, preferably,
mechanistic.
In the experiments described above significant effects
of copper were observed on cocoon production, weight
change, and survival at 160 and 320 mg kg 1 for E.
andrei in the laboratory (Svendsen and Weeks, 1997a)
and on weight change and survival at 320 mg kg 1 for L.
rubellus in the mesocosm study (Svendsen and Weeks,
1997b). NRRTs in these studies were significantly
reduced at 20 mg kg 1 both in the laboratory and in
the mesocosm exposures, with NRRTs severely de-
creased (less than 15–20 min) at soil copper concentra-
tions greater than 80 mg kg 1 (Fig. 1). Changes in
lysosomal integrity were thus more sensitive than the
ecological life cycle endpoints measured. The same
picture of sensitivity and relevance was shown in a
study by Scott-Fordsmand et al. (1998) in which E.
veneta were exposed to soil containing up to
1000 mg kg 1 nickel. Again, the NRRT was more
sensitive than the measured life cycle endpoints, with a
dose-dependent decrease in the NRRT that correlated
with lowered cocoon production and with effects on
survival occurring only when NRRTs decreased to less
than 15–20 min.
A further study by Scott-Fordsmand et al. (2000)
demonstrated the relevance of the NRRT results over
chemical measurement for identifying the bioavailability
of metals to earthworms. Here E. fetida was exposed to
a copper-contaminated field soil that had been aged for
70 years and freshly spiked soil of matching total copper
concentrations and pH. Measured NRRTs were again
more sensitive than life cycle endpoints, with the NRRT
reduced to 15–20 min at copper concentrations lower
than those causing changes in cocoon production,
weight, and ultimately (least sensitive) mortality. Com-
parison of the NRRT responses for the two soils
indicated differences in the toxicities of the soils
attributable to their histories. The 70-year-old field soils
were less toxic (higher NRRTs and lower life cycle
effects) than freshly spiked soils. Unexpectedly, chemical
analysis showed both soils to have similar total and
water-extractable copper concentrations and to result in
similar accumulated earthworm tissue concentrations.
Thus, it is clear that both the NRRT and the life cycle
endpoints were able to detect differences in bioavail-
23
70
60
NRRT±SD (min)
Without Cu value
50
Y=3906x + 0.6
40 R2 = 0.65, p < 0.016
30 Without Cu value
20
Y=2481x + 16.7
10
R2 = 0.73, p < 0.003
0
-0.01 -
-0.005 0 0.005 0.01 0.015 0.02
Intrinsic rate of population increase
Fig. 2. Relationship between NRRT (mean7SD) and the intrinsic
rate of population increase in L. rubellus exposed to copper (gray
triangles) and cadmium (black squares) in the laboratory. The NRRT
was measured in adult worms, and the intrinsic rate of population
increase was calculated by integrating measurements of adult and
juvenile life cycle traits within a demographic-based model. Regres-
sions are based on the whole data set (i.e., Cu and Cd exposures), both
including and excluding the ‘‘heavily weighted,’’ highly toxic Cu
treatment.
ability between the soils that were not shown by the
chemical analysis of water-extractable copper.
To confirm the ecological relevance of NRRT in
relation to effects on population-level parameters,
Spurgeon et al. (2001) determined the relationship
between the NRRT measured for L. rubellus at a
number of copper and cadmium concentrations and the
calculated ‘‘intrinsic rate of population increase.’’ The
results showed that a decreased NRRT could be related
to lower intrinsic rates of population increase for both
metals, even when excluding the highly toxic Cu
exposure found at the highest concentration (Fig. 2).
Decreased lysosomal integrity has been shown to
occur at exposure concentrations lower than those for
effects on ecologically important endpoints such as
growth, reproduction, and survival for a range of
compounds (Svendsen, 2000). There is, as mentioned
under the Introduction, evidence of many pollutant
classes inflicting lysosomal changes and damage, yet no
direct functional link to effects at the whole-organism
level has been identified. The ecological significance of a
decreased NRRT must therefore be inferred through the
correlation of responses and the relative sensitivities of
endpoints, as demonstrated in the above section.
3.3. Confounding factors
Effects on the biomarker responses from nonchemical
factors (i.e., season, temperature, pH, sex, weight, and
handling) should be identifiable within narrow limits to
eliminate their interference with the interpretation of
any toxic effects. Alternatively, if nonchemical factors
that may cause large changes in the response patterns
exist they must be well understood so that false-
negatives and positives can be avoided.
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In a full field study, measurement of the NNRT in
worms from an area impacted by an industrial plastics
fire initially identified a pollutant gradient produced by
the runoff fire-fighting water. Repeat samplings here
also showed no differences between autumn and the
following spring (Svendsen et al., 1996).
The major biotic and abiotic factors that could affect
the NRRT of earthworms from routine sampling in field
soils are explained below:
3.3.1. Season
Repeat measurement of NRRT in worms collected
from a pollutant gradient produced by an industrial
plastics fire showed no differences between autumn and
the following spring (Svendsen et al., 1996). Although this
is the only seasonal study of field-collected worms, the
absence of any direct seasonal effects between the three
mesocosm samplings made from late summer to early
winter shown in Fig. 1a–c provides cooperating evidence.
3.3.2. Soil type and pH
Changes in pH are known to impact earthworms in
general and lysosomal integrity specifically, but such
changes are unlikely to occur rapidly in soils. In polluted
soils the effects of soil properties on the bioavailability
of the pollutant are likely to be more important to the
NRRT than any direct stress due to increased hydrogen
ion concentration or changes in soil type. Additionally,
a range of authors have conducted earthworm studies
that measured the NRRT in a wide range of soils, and
none have consistently failed to produce acceptably high
NRRTs for the control treatments in any soil.
3.3.3. Soil moisture and temperature
Both of these factors can vary rapidly in field soils and
may affect epigeic and endogeic species. The 110-day
sampling of the mesocosm exposure by Svendsen and
Weeks (1997b) in Fig. 1 showed that waterlogging of the
soil caused a decreased NRRT even in the control
treatments. Extreme soil moisture conditions (water-
logging or drought) are, however, easily avoided, as they
are obvious at the time of sampling. Temperature
extremes (recent or current) are less obvious when
sampling in the field. Svendsen (2000) examined the
effects of exposure temperature on measured NRRTs in
E. veneta exposed to copper (0–270 mg kg 1) at three
temperatures (15 C, 20 C, and 25 C). This temperature
range represented the outer thermal limits for optimal
reproduction in this worm. No effect of temperature on
either the control NRRTs or the shape and position of
the dose–response curve were found.
3.4. Chemical specificity
Biomarkers are divided into nonspecific and specific
responses on the basis of the range of chemicals to which
they respond. Nonspecific biomarkers that respond to a
broad range of pollutant classes will be most useful in
screening surveys in which the types of toxicants that
may be present are unknown. Specific biomarkers with a
high sensitivity to a particular pollutant class or a given
pollutant (e.g., acetylcholine esterase and d-aminolevu-
linic acid dehydratase) will be more useful for linking
observed effects to specific exposures.
The examples above have shown clear monotonic
dose–response relationships between NRRT and cop-
per, cadmium, nickel, and the complex mixture resulting
from a plastics fire. In most cases, decreases in NRRT to
less than 15 min accompanied observable effects on life
cycle parameters. In further investigations of the
chemical specificity of NRRT, E. andrei were exposed
to four metals and two fungicides (copper, cadmium,
zinc, lead, carbenmdazim and iprodione) and the
response profiles and the relative sensitivity of the
NRR, and the life cycle endpoints were compared
(Svendsen, 2000). On the basis of comparisons of the
NRRT EC75 (i.e., the dose decreasing the NRRT to
15 min) and of the reproduction EC50 to the LC50, the
six compounds separated into three groups based on the
level of sublethal toxicity exhibited.
3.4.1. Cadmium (high sublethal toxicity)
NRR EC75 was 120 times lower than LC50.
Reproduction EC50 was 12 times lower than LC50.
3.4.2. Copper, zinc, lead, and carbendazim (moderate
sublethal toxicity)
NRR EC75 was 30–40 times lower than LC50.
Reproduction EC50 was 4–10 times lower than LC50.
3.4.3. Iprodione (low sublethal toxicity)
NRR EC75 was circa equivalent to LC50.
Reproduction EC50 was circa equivalent to LC50.
The sensitivity of the lysosomal integrity to this range
of pollutants thus reflected the comparative sublethal
toxicity of these compounds.
Further work has demonstrated that the earthworm
NRRT is responsive to a wide range of chemicals.
Stubberud (1998) demonstrated clear dose–response
relationships for the NRRT for mercury and cadmium
of E. veneta in water- and soil-based exposures. The
assay has been successfully used for complex energetic
compounds, such as the explosives TNT, RDX, and
HMX, under both laboratory and field conditions
(Robidoux et al., 2002). Exposure of E. andrei to the
polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene
lowered the NRRT to less than 12 min at 20 mg kg 1
and 7 min at 100 mg kg 1 (Eason et al., 1999). There
were, however, no other observable toxic effects during
the 28-day exposure, which may be explained partly by
the mode of toxic action of this compound (carcino-
genic). Exposure of E. andrei to either pure or 50%
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diluted (with clean soil) gas site soil contaminated
primarily with a mixture of PAHs gave similarly
decreased NRRTs (7 and 5 min, respectively), with only
the pure gas site soil causing significant weight loss and
mortality (Eason et al., 1999).
For the pesticides, chloropyrifos (an organopho-
sphate) decreased the NRRT to 10 min at a soil
concentration of 125 mg kg 1 (this concentration also
caused weight loss) and to 5 min at 250 mg kg 1, at
which high mortality occurred (Eason et al., 1999).
These results compare well with the NRRT and
sublethal effects from field rates of chloropyrifos found
in laboratory and semifield trials with Aporrectodea
caliginosa by Booth et al. (2001) Booth and O’Halloran
(2001). These studies found both the NRRT and the
sublethal endpoints to be similarly sensitive to field rates
of another organophosphate, diazinon. To date the only
compound tested that did not to affect the NRRT is the
carbamate methiocarb in a mesocosm exposure of L.
terrestris, in which acetylcholeinesterase inhibition was
observed, confirming that exposure did take place
(Grafton, 1995). Thus, although all other pollutants
tested (representing a wide variety of modes of action
including the same as that of the carbamate) have given
clear dose–response relationships that in most cases
reflected the sublethal toxicity of the pollutants, there
are compounds that do not affect the NRRT.
3.5. Species differences
Fundamental differences in metabolic characteristics
between species may lead to differences in the biomarker
responses obtained (see Øien and Stenersen, 1984). As
these differences are not always as logical and as
obvious as might be expected, they should be investi-
gated before a new species is employed in regulatory
testing.
The fact that the studies cited so far in this review
include examples of the successful application of the
neutral red retention assay in several species of earth-
worm (E. andrei, L. rubellus, E. veneta, E. fetida, and A.
caliginosa) suggests that the assay may have cross-
species applicability, but only one study has directly
compared the effects on several species. Spurgeon et al.
(2000) exposed L. rubellus, E. fetida, A. caliginosa, and
L. terrestris to zinc for 28 days in the laboratory. Dose–
response relationships were found for the NRRT,
cocoon production, and survival, and although the
EC50 and LC50 values varied between species, the
relative sensitivities of the endpoints were always the
same within each species (NRRT4cocoon produc-
tion4survival). Thus, although the NRRT EC50 values
for the four species varied greatly (170–2000 mg kg 1
Zn), they did reflect species differences in sublethal
sensitivity to zinc (L. rubellusDA. caliginosa4L. terres-
tris4E. fetida).
25
3.6. Time–response relationship
This aspect includes two issues: (i) the time it takes
from the onset of exposure until a response is manifest
and (ii) the time the response persists after exposure
ends. Both issues have a significant impact on the
possible interpretation of a biomarker response. Re-
sponse times vary widely, from almost instantaneous
(e.g., stress proteins) to years (e.g., cancerous lesions).
Depending on the objective(s) of a study, rapid or slow
response times may be desirable (e.g., early warning or
the demonstration of a prolonged exposure). The
question of the persistence of biomarker responses
involves both transience and reversibility and has
implications for the scale of temporal integration that
a given biomarker can provide. Dependent on the degree
of time dependency of a biomarker response, conclu-
sions could span from ‘‘There has or has not been a
pulse exposure within the last 4 weeks (transient),’’ to
‘‘There is no current exposure or previous exposure has
stopped (reversible, but not transient),’’ to ‘‘Exposure
has occurred at some point in time and may or may not
still be present (permanent).’’
The speed of onset of the response from the initiation
of exposure is hard to address from the literature, as
most studies measure NRRT after several weeks.
Experience during experimentation and quality control,
however, suggests that exposure to soil copper for only
12–24 h, decreased the NRRT to a level comparable to
that of a full 28-day exposure (Personal observation).
Transience can reflect that the measured response is
part of a first-defense system that is induced for only a
short time or that an adaptative response by the
organism to the exposure over time masks the measured
biomarker response. To date this has not proved a
problem in any of the medium- and long-term contin-
uous exposures available in the literature regardless of
the exposure chemical. Also, the 70- (Svendsen and
Weeks, 1997b) and 180-day (Olesen, 1998) mesocosm
exposures proved that the decreased lysosomal integrity
was not transient. Persistence of the NRRT response
was also observed in 3-year exposures of E. fetida to
cadmium, lead, and zinc in the laboratory by Reinecke
and Reinecke (1999). This study suggests that, at least
initially, within-generation persistence of a decreased
NRRT is likely under continuous exposure. This,
however, leaves the question of adaptation at the
subcellular level. This issue is particularly relevant in
the quality assessment of field soil exposures that have
often been present for decades.
Multigeneration studies are the only way to address
the possible effects of true genetic adaptation. However,
studies of this nature involving the measurement of a
NRRT are nonexistent, and the only way to realistically
assess potential effects from adaptation over generations
of exposure is to work at field sites with medium- and
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26 C. Svendsen et al. / Ecotoxicology and Environmental Safety 57 (2004) 20–29
long-term contamination. In one such study, L. rubellus
were collected from locations at two sites where metal
pollution had been present for 70 (Avonmouth Smelter,
Bristol, UK) and 400 years (Shipham Mine, SW
England) (Svendsen, 2000). The measured NRRTs were
below 10 min, except for those worms collected from the
site furthest (10 km) from the Avonmouth Smelter. The
presence of a NRRT response in these field-collected
worms suggests that the effects on lysosomal integrity
persisted over generations.
The potential for recovery of the NRRT response is
based on new coelomocytes being produced at all times
and their not being exposed if exposure is abated.
Recovery has not been specifically addressed; however,
that it is possible can in part be inferred from the results
discussed earlier under dose–response relationships for
the worms exposed to 20 and 40 mg Cu kg 1 and shown
in Fig. 1. This suggests that in cases in which exposure
ceases or is eased (remediation or mitigation), the
NRRT will recover as new coelomocytes replace
previously damaged cells and can be used to monitor
improvements in soil status.
3.7. Methodological concerns
For a biomarker measurement to be acceptable and
workable in any routine monitoring scheme, it must
meet practical and methodological concerns relating to
the precision, quality control, and cost of the analysis.
The best biomarkers will be reproducible, technically
undemanding, and of low cost in terms of technical
sophistication, training, and staff time.
As set out under Section 2, the technique is based on
visual observation. Undertaken by a trained operator
the collection and preparation of coelomocytes on
microscope slides yield several hundred cells per slide,
and once the technique becomes routine each counting
interval (1–2 min) accounts for the ‘‘stained/unstained
status’’ of 100 cells or more. Hence, the manual scanning
and counting of the cells should represent a statistically
sound account of how the cells are responding in terms
of neutral red retention. Because it is visually based, the
assay is open to criticisms of subjectivity due to a
possible unintentional bias (or worse, intentional bias)
during the scanning of the slide for either stained or
unstained cells. The operator-based selection of cells
was isolated in the study by Reinecke and Reinecke
(1999) when a computer-linked motorized microscope
stage was employed. This allowed the same set of
randomly selected cells to be revisited and assigned
‘‘stained’’ or ‘‘unstained’’ at each counting interval. The
approach gave results comparable with those obtained
using the normal ‘‘manual’’ scanning of the microscope
slide.
The determination of when a cell is stained is,
however, still dependent on the operator; and the process
from initially leakage to full stain can take a couple
of minutes. Hence, some training of new operators
is required. New and experienced operators should
observe aliquots of the same samples in order to
calibrate their differentiation between unstained and
stained cells to ensure comparability between observed
NRRTs.
The only way to remove the operator factor
completely is to develop a completely automated version
of the method, which to date has proved difficult.
The best theoretical option for such automation
relies on advanced equipment, e.g., a flow cytometer,
which would defy the simplicity of the assay. That
the only equipment needed for the current assay is a
light-microscope and a pipette is one of its major
attractions, as it is easy to train operators and to
implement the technique in most laboratories. The best
way to promote trust in the assay and counter any
criticism of operator bias is to prepare and count
randomized, coded samples in a ‘‘blind’’ setup in which
the operator does not know the origin of the separate
samples. This would remove any potential for any
‘‘intentional bias’’ and would randomize potential
operator effects.
4. ‘‘Overall public/regulator confidence and acceptance’’
For any biological effect assessment to be adopted as
routine in the assessment of contaminated soil the
confidence of stakeholders (the public, industry, and
regulators) in the approach is essential. Results must be
trustworthy to the stakeholders, but more importantly
they must understand (and believe in) the advantages
and more direct relevance of properly applied biological
effect monitoring over, say, the measurement of total
chemical levels. Initially, methodological reliability must
be proven through rigorous scientific trials. [For the
neutral red retention assay, the body of evidence built
up through both the international literature and
commissioned work has led to the Environment Agency
of England and Wales recommending and currently
trialing the NRRT as a potential method for ecological
risk assessment (Crane and Byrns, 2001; Spurgeon et al.,
2002).] Thereafter, gaining a general public acceptance
of ecological effect-based assessments is not the largest
problem, as the principle of direct effect measurement of
biota differs little from the general environmental
monitoring with which people are already familiar
(e.g., nest counts for storks). The much larger hurdle
for the actual regulatory application of these techniques
in ecological effect and risk assessment is that both
regulators and industry must understand and, impor-
tantly, agree on their limitations and the way in which
they should be applied. Only good communication of
the scientific rationales for the greater relevance of and
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C. Svendsen et al. / Ecotoxicology and Environmental Safety 57 (2004) 20–29
equal reliability of these techniques over the currently
used total chemistry data and obtain achieve this. The
paradox is that although it is easy for both parties to
measure total chemical data, which provides a number
that can be taken to court, it is very hard to justify this
as scientifically relevant. This can lead to expensive
action being enforced where there may be no ecological
effects or risks and no action being taken in case of
substantial risk. In contrast, the biological assay, with
its directly measured effects, could have a substantial
part to play in general monitoring and problem
identification.
5. Evaluation summary
In this review, evidence from published literature on
the suitability of the NRRT assay as a monitoring tool
for assessing the effects of pollutants in soils has been set
out with respect to each of the criteria listed under the
Introduction. How the assay meets each criterion is
summarized below, with the weight of evidence and
fitness of purpose for each criterion indicated by the
number of check marks [|], ranging from 1 (poor) to 5
(excellent).
5.1. Dose–response relationship [|||||]
There is substantial evidence that the dose–response
curve of lysosomal integrity (by NRRT) monotonically
decreases with increasing chemical exposure. Variation
in the response is low enough to allow the differentiation
of slight, intermediate, and severe exposures.
5.2. Sensitivity [||||] and ecological relevance
[|||]
Lysosomal integrity has been shown generally to
be more sensitive than life cycle endpoints such
as growth, reproduction, and survival and generally
reflects the sublethal toxicity of a pollutant. To date,
ecological relevance has been demonstrated through
only correlation and not mechanistic causality. These
correlations, however, are numerous and of convincing
quality. Hence, a NRRT of less than 10–15 min should
be a cause for concern and further investigation, as such
a NRRT has been shown to coincide with life cycle
effects.
5.3. Confounding factors [||]
Confounding factors have so far not proved a serious
problem in the laboratory. Confounding effects will,
however, always be possible because lysosomal integrity
is sensitive to biotic and abiotic stressors, as well as
pollutants. In the field, an effect of acute waterlogging
in the control and low-exposure-dose mesocosms has
27
been found by Svendsen and Weeks (1997b) (Fig. 1).
Sampling under such obviously stressing conditions
should, of course, be carried out with caution.
5.4. Chemical specificity [||||]
Decreased lysosomal integrity has proved measurable
through reduced NRRTs for a wide range of pollutants
representing groups such as metals, organophosphates,
PAHs, and mixtures. So far only one compound (the
carbamate methiocarb) has failed to cause a positive
response. This does, however, show that care must be
taken to establish an effect on lysosomal integrity when
novel compounds are investigated for the first time.
5.5. Species differences [|||]
Technically the assay has been successful in all
earthworm species on which it has been applied.
Although the NRRT EC50 values have varied between
species, they all reflected the general species differences
in sublethal sensitivity.
5.6. Time–response relationship [|||]
After onset of significant exposure, the NRRT will
rapidly decrease (i.e., a response occurs quickly, in
o24 h). No signs of transience (either short term or
adaptation linked) of the response magnitude have been
observed. Long-term (multigeneration) persistence has
been demonstrated, as has the ability for recovery after
the cessation of exposure.
5.7. Methodological concerns [|||]
In its current format the technique is simple to
introduce in a laboratory, demanding only a light
microscope, pipettes, and a small amount of basic
training. There are issues of potential subjectivity as the
assay is based on visual observation, but if samples are
analyzed using a randomized blind setup this is more a
theoretical issue than a real, practical concern.
5.8. Overall public/regulator confidence [||]
This is the biggest remaining hurdle, but scientific
proof exists and all that is needed is proper commu-
nication to the right people. Experience with other
biological methods (bird of prey eggshell monitoring of
organochlorine insecticides, mussel watch) suggests that
if the benefits in terms of relevance and ease of
interpretation can be demonstrated, then the value of
biological assessment beyond traditional measurements
of total chemistry will be recognized.
 ARTICLE IN PRESS
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Further reading
Robidoux, Y.P., Svendsen, C., Hawari, J., Thiboutot, S., Ampleman,
G., Weeks, J.M., Sunahara, G.I., 2003a. Use of sub-cellular and
cellular biomarkers to assess 2,4,6-trinitotoluene (TNT) exposure
in earthworms—applicability under field conditions in mesocosm
experiments using site species and laboratory worms. Biomarkers,
submitted for publication.
G., Weeks, J.M., Sunahara, G.I., 2003b. Use of sub-cellular
and cellular biomarkers to assess 2,4,6-trinitotoluene (TNT)
exposure in earthworms—links to effect assessment in labo-
ratory studies using Eisenia andrei. Biomarkers, submitted for
publication.