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Ecotoxicity of engineered nanoparticles to aquatic invertebrates:
a brief review and recommendations for future toxicity testing
A. Baun ÆN. B. Hartmann ÆK. Grieger Æ
K. O. Kusk
Accepted: 1 April 2008 / Published online: 19 April 2008
ÓSpringer Science+Business Media, LLC 2008
Abstract Based on a literature review and an overview of
toxic effects of engineered nanoparticles in aquatic inver-
tebrates, this paper proposes a number of recommendations
for the developing field of nanoecotoxicology by high-
lighting the importance of invertebrates as sensitive and
relevant test organisms. Results show that there is a
pronounced lack of data in this field (less than 20 peer-
reviewed papers are published so far), and the most fre-
quently tested engineered nanoparticles in invertebrate tests
are C
60
, carbon nanotubes, and titanium dioxide. In addi-
tion, the majority of the studies have used Daphnia magna
as the test organism. To date, the limited number of studies
has indicated acute toxicity in the low mgl
-1
range and
higher of engineered nanoparticles to aquatic invertebrates,
although some indications of chronic toxicity and behav-
ioral changes have also been described at concentrations in
the high lgl
-1
range. Nanoparticles have also been found to
act as contaminant carriers of co-existing contaminants and
this interaction has altered the toxicity of specific chemicals
towards D. magna. We recommend that invertebrate testing
is used to advance the level of knowledge in nanoecotoxi-
cology through standardized short-term (lethality) tests with
invertebrates as a basis for investigating behaviour and
bioavailability of engineered nanoparticles in the aquatic
environment. Based on this literature review, we further
recommend that research is directed towards invertebrate
tests employing long-term low exposure with chronic
endpoints along with more research in bioaccumulation of
engineered nanoparticles in aquatic invertebrates.
Keywords Nanoparticles Nanomaterials
Nanoecotoxicology Crustaceans
Introduction
Only recently has there been an increased focus on envi-
ronment, human health and safety issues within the
production, use and release of nanomaterials (Royal Society
and Royal Academy of Engineering 2004; Oberdo
¨rster et al.
2005; Maynard 2006; Moore 2006; Nowack and Buchelli
2007; SCENIHR 2007). It is important to consider these
environmental, health and safety aspects at an early stage
of nanomaterial development and use in order to more
effectively identify and manage potential human and envi-
ronmental health impacts from nanomaterial exposure.
Although more that 400 studies exist on the toxicity of
nanomaterials in bacteria, mammalian cell lines and
mammals (Hansen et al. 2007), the scientific discipline of
nanoecotoxicology is still in its infancy (Moore 2006;
Nowack and Buchelli 2007). There are currently less than 50
open peer-reviewed ecotoxicity studies on environmentally-
relevant species. As the number of nano-scale applications is
rapidly growing with future estimates of an ever-increasing
development and use of nanomaterials (Roco 2005; Dunphy
Guzma
´n et al. 2006), an investigation into the effects on
the aquatic environment from nanomaterial exposure is
extremely important, particularly since it ultimately receives
run-off and wastewater from domestic and industrial sources
and has been targeted for some nano-scale environmental
remediation techniques (e.g., zero-valent iron nanoparticles)
(Defra 2007; Vaseashta et al. 2007).
The aim of the present paper is to give an overview of
the literature concerning toxic effects of engineered
nanoparticles (ENPs) in aquatic invertebrates, from which
A. Baun (&)N. B. Hartmann K. Grieger K. O. Kusk
Department of Environmental Engineering,
Technical University of Denmark, Miljoevej, Building 113,
2800 Kgs. Lyngby, Denmark
e-mail: anb@env.dtu.dk
123
Ecotoxicology (2008) 17:387–395
DOI 10.1007/s10646-008-0208-y
a number of recommendations are made for future nano-
ecotoxicology developments highlighting the importance
of invertebrates as sensitive and relevant test organisms.
Significance of aquatic invertebrates as test species
Invertebrates are composed of a large and very diverse
group of animals, consisting of more than 30 different
phyla, several of which include more than 1000 different
species (Ruppert et al. 2004). For instance, the largest
invertebrate phylum is the Arthropoda, consisting of more
than 1 million species of which insects and crustaceans are
the two largest groups. Since relatively few insects have
aquatic larvae, crustaceans therefore are the most numerous
and ecologically important group of invertebrates in
marine as well as fresh water ecosystems. Crustaceans also
play an important role in regulatory toxicity testing as a
part of the base set of organisms required for assessing
risks to both aquatic and terrestrial environments (EU
Commission 2003). Hence, it is not surprising that most
ecotoxicological tests on hazardous materials using inver-
tebrates have been performed with crustaceans. With
regards to ecotoxicological tests on manufactured nanom-
aterials, most tests have used the same species as test
organism, Daphnia magna. Furthermore, D. magna and
Ceriodaphnia dubia, another daphnia species, are the most
commonly used invertebrate species in regulatory chemi-
cals testing, and which are also included in several
guidelines and international standards for acute and chronic
tests (e.g. OECD, ISO). Therefore, daphnia species are an
obvious first choice for test organisms when performing
ecotoxicological tests on nanomaterials. Planktonic crusta-
ceans, like the daphnids, are generally the food and energy
link between the primary producers (algae) and secondary
consumers (fish and fish larvae) (Fig. 1). Crustaceans such
as harpacticoid copepods (e.g. Amphiascus tenuiremis)or
gammarids (e.g. Hyalella azteca) living in or on the sedi-
ment are also important food sources for fish larvae. At the
bottom of the water column, they consume settled organic
materials either as particles or as larger fragments, such as
leaves on which they scrape bacteria or eat fragments of the
leaves. Therefore, crustaceans work as shredders improving
the degradation process of organic material and nutrient
recycling. Also the annelid Lumbriculus variegatus live
from the organic fraction of the sediment. Because the
sediment is a sink for many contaminants in water eco-
systems (and probably also for aggregated nanoparticles),
sediment feeders can accumulate high contaminant con-
centrations. Mussels are filter-feeding invertebrates present
in marine as well as freshwater bodies (e.g. Elliption
complanata), where they filter large volumes of water and
Aquatic Environment
Fish
Human exposure
Sedimentation
of particles, decaying
organic matter and
excreted material
Suspended particles
Single NP ↔ Aggregated NP
Sedimentation
Re-suspension
Point sources / Accidental release /
Non-point source / Intentional release
Sediment dwelling
invertebrates
Planktonic
invertebrates
Algae
Fig. 1 Significance of
invertebrate species in the
aquatic food web and the
possible routes of
environmental exposure to
ENPs after release to the aquatic
environment. After entry of
ENPs into the aquatic
environment the suspended
particles will be taken up by
planktonic or sediment dwelling
invertebrates through different
exposure routes (i.e. direct
uptake from the water phase or
through food uptake)
388 A. Baun et al.
123
thus play an important ecological role. Their filtering
apparatus is cilia which are in close contact with the
gills. Hence, there is a short distance for particles and
contaminants sorbed to particles to pass from the water to
the blood. Thus, mussels as well as sediment living organ-
isms are relevant test organisms in studies investigating
environmental effects of manufactured nanomaterials
(Fig. 1).
Acute toxicity and uptake of ENPs in invertebrates
The pioneering studies by Lovern and Klaper (2006)
used D. magna as the test organism in a mortality study with
48 h of exposure to 35 mgl
-1
C
60
(Table 1). However,
LC
50
-values could not be determined in these studies since
50% mortality was not achieved. A higher toxicity
(LC
50
=0.8 mgl
-1
) was found for C
60
when the solvent
tetrahydrofuran (THF) in combination with solvent change
under reduced pressure was used to prepare the test solutions.
The preparation methods for solving/dispersing ENPs, and
especially the use of THF, have been questioned by several
research papers (e.g., Gharbi et al. 2005; Henry et al. 2007).
There is little doubt that the preparation methods and the
behavior of ENPs in aqueous solution are influencing the
monitored effects in these toxicity tests. However, further
research is needed to fully understand the governing mech-
anisms behind these effects as well as preparation–behavior
interactions for nanoparticle suspensions in general.
Table 1summarizes the currently published studies on
invertebrate effects of ENPs as well as shows the prepa-
ration methods for obtaining the aqueous suspensions of
ENPs. The majority of the studies have used Daphnia
magna as the test organism, which is indeed relevant taking
into consideration their ecological significance and role as
key organisms in regulatory testing. Daphnids are filter-
feeders and have combs of setae on their ‘‘limbs’’ in the
trunk formed by the bivalved carapace (Ruppert et al.
2004). These combs serve as a mesh by which water is
filtered and particles caught. D. magna can filter relatively
large volumes of water compared to body size, and have
been found to feed on particles in the size range of 0.4–
40 lm (Geller and Mu
¨ller 1981; Gophen and Geller 1984)
normally consisting of algal cells but also larger bacteria
and other organic and inorganic particles. Single nano-
particles are thus smaller than the lower size limit.
However, intake of suspended nanoparticle aggregates is
seen when D. magna is exposed to TiO
2
and C
60
(Fig. 2),
indicating that the aggregates in suspension are micron-
sized rather than nano-sized. Uptake of aggregates of
nanoparticles via filtration may lead to a much higher body
concentration than the surrounding waters. Studies have
shown that C
60
nanoparticles in the digestive tract of
D. magna after a 48 h exposure were also excreted again
(Baun et al. 2008). However, the translocation of 20 nm
polystyrene nanoparticles from the digestive tract into
other parts of the daphnids found by Rosenkranz et al.
(2007) calls for a warning that even if nanoparticles are not
digested by the daphnids it is possible that a small fraction
might be taken up (Table 1). This may eventually lead to
accumulation and/or toxic effects.
Long-term effects and adhesion of ENPs to
invertebrates
Adhesion of nanoparticle aggregates to the exoskeleton of
the test organisms is frequently described for the crusta-
cean studies shown in Table 1. This is illustrated for
D. magna exposed to aqueous suspensions of nanoparticles
of TiO
2
as well as for A. tonsa exposed to C
60
in Fig. 3.
The nanoparticle aggregate adhesion may cause physical
effects and loss of mobility. Lovern et al. (2007) observed
significant changes in D. magna behaviour when exposed
to LOEC
48 h
levels of C
60
and a C
60
-derivative. Their
observations included repeated collisions with the glass
beakers, swimming in circles at the water surface, changes
in the number of hops, appendage movement, and heart
rate (Table 1). However, the reasons for these behavioral
changes are not clear and may be caused by other factors
than particle adhesion.
As shown in Table 1, only a few studies have observed
chronic or life-cycle related effects of nanoparticles to
invertebrates. Exposure of Daphnia magna to 2.5 mgl
-1
stirred C
60
was found to reduce the number of offspring
and to delay molting of the carapace (Oberdo
¨rster et al.
2006). In the thorough study by Templeton et al. (2006)
of life-stage developments in the estuarine meiobenthic
copepod Amphiascus tenuiremis, an increasing average
cumulative life-cycle mortality was observed with greater
concentrations of single-walled carbon nanotubes
(SWCNT), up to 10 mgl
-1
, where the mortality (36 ±11%)
was significantly higher compared to the control (13 ±4%).
At 10 mgl
-1
they also found that the development success
was reduced to 51% for the nauplius-to-copepodite stage and
to 34% overall for the nauplius-to-adult period. Furthermore,
the study showed a significantly lowered fertilization rate,
averaging only 64 ±13% during 35 days of exposure to
10 mgl
-1
SWCNT. Their study also showed that a highly
mobile fluorescent fraction of smaller particles were signif-
icantly more toxic than the ‘‘as prepared’’ SWCNT, and thus
caused significant effects on the development at the lowest
test concentration of 0.58 mgl
-1
(Templeton et al. 2006).
Sublethal effects in the freshwater mussel Elliptio
complanata were studied by Gagne
´et al. (2008) after
exposure to CdTe quantum dots (QDs) for 24 h. Significant
Ecotoxicity of engineered nanoparticles to aquatic invertebrates 389
123
Table 1 Overview of invertebrate studies on toxicity and accumulation of engineered nanoparticles
Particle type Species Effect measured Suspension
preparation
Observations Particle size (diameter) Reference
C
60
, TiO
2
Daphnia magna Acute toxicity Solvent (THF)/
Ultrasonic
dispersion
TiO
2
:EC
50,48 h
: 5.5 mgl
-1
(THF preparation). Dose–
response relationship could not be established for
sonicated solutions. C
60
:EC
50,48 h
: 460 lgl
-1
(THF
preparation), 7.9 mgl
-1
(sonicated)
TiO
2
: 10–20 nm
C
60
: 0.72 nm
Polystyrene latex spheres
93 nm
Lovern and
Klaper
2006
SiO
2
, TiO
2
, ZnO Daphnia magna Acute toxicity Vigorous shaking Toxicity: ZnO [SiO
2
[TiO
2
; ZnO: 100% mortality
at 0.5 mgl
-1
. SiO
2
: 70% mortality at 10 mgl
-1
;
TiO
2
: 40% mortality at 20 mgl
-1
. Particle size was
found not to affect toxicity.
TiO
2
(66 nm, 950 nm and
44 mm), SiO
2
(14 nm,
930 nm, and 60 mm),
and ZnO (67 nm,
820 nm, and 44 mm)
Adams et al.
2006
TiO
2
Daphnia magna Acute toxicity Ultrasonic
dispersion
TiO
2
nanoparticles type P25 (Degussa, d =25 nm)
were found to entail a larger effect than the larger
particle type (Hombikat UV100, d =100 nm). Pre-
illumination of suspensions with simulated sunlight
was found to increase immobilization of the test
organisms.
Degussa P25: 25 nm
Hombikat UV100:
100 nm
Hund-Rinke
and Simon
2006
TiO
2
(coated) Daphnia magna Acute toxicity Not specified For TiO
2
with Si/Al coatings (90/7/1 wt% TiO
2
/Al/Si):
EC
50,48 h
[100 mgl
-1
. The TiO
2
had a crystalline
phase determination of 79% rutile and 21% anatase,
a median particle size of 140 nm in water and BET
SSA was 38.5 m
2
/g.
140 nm in water Warheit et al.
2007
TiO
2
Daphnia magna Behavioral and
physiological
changes
Solvent (THF) Exposure to 260 lgl
-1
C
60
increased heart rate of
D. magna. Exposure to both 260 lgl
-1
C
60
HxC
70
Hx as well as to 260 lgl
-1
C
60
resulted in
increased hopping frequency and increased
appendage movement. Recovery after exposure to
C
60
HxC
70
Hx, but not for C
60
. No significant effects
on any of the three parameters were found for TiO
2
.
C
60
and C
60
HxC
70
Hx:
10–20 nm in suspension
TiO
2
: 30 nm.
Lovern et al.
2007
C
60
C
60
HxC
70
Hx
C
60
Daphnia magna Modifying factor on
toxicity
accumulation of
pollutants
Prolonged stirring in
milliQ water.
Addition of 5–8 mgl
-1
C
60
increased the toxicity of
phenanthrene more than 10 times when results were
expressed as water phase concentrations. Uptake of
phenanthrene was faster with C
60
; 1.7 times higher
steady state concentrations were found, but due to
very fast clearance after transfer to clean water,
accumulation of phenanthrene was not affected by
the presence of C
60
.
Aggregate size: 2 nm to
several microns
Baun et al.
2008
SWCNT (coated) Daphnia magna Acute toxicity and
biomodification
Ultrasonic
dispersion
Daphnids were able to modify the SWCNT upon
ingestion (removing the lipid
lysophophatidylcholine coating) hereby reducing
solubility of the SWCNT. 100% mortality was
observed after 48 h at 20 mgl
-1
exposure.
1.2 nm Roberts et al.
2007
390 A. Baun et al.
123
Table 1 continued
Particle type Species Effect measured Suspension
preparation
Observations Particle size (diameter) Reference
Fluorescent
polystyrene TiO
2
Carbon black
Daphnia magna Acute toxicity and
accumulation
Not specified Fluorescent carboxylated polystyrene particles with a
size of 20 nm were observed to be taken up and
accumulated in the gastrointestinal tract of
D. magna. Particles were observed in storage oil
droplets. Molting frequency increased when
D. magna was exposed to TiO
2
(25 nm) and to
carbon black (14 nm). For carbon black an LC
50,96 h
value of 0.4 mgl
-1
was determined.
Polystyrene: 20 nm
TiO
2
:25nm
Carbon black: 14 nm
Rosenkranz
et al. 2007
C
60
Daphnia magna
Hyalella azteca
Copepod
Acute toxicity and
reproduction
Prolonged stirring
in milliQ water.
Exposure concentrations were too low to obtain LC
50
values. Exposure concentrations were 35 mgl
-1
(D. magna), 7 mgl
-1
(H. azteca) and 22.5 mgl
-1
(harpacticoid copepods). For D. magna mortality
and sub-lethal effects were observed even at low
concentrations (2.5 mgl
-1
), which could ultimately
cause reduced fecundity.
Aggregate size: 10–200 nm Oberdo
¨rster
et al. 2006
C
60
Daphina magna Mortality Solvent (THF)/
Prolonged
stirring
The effect of C
60
on D. magna was found to depend
strongly on suspension preparation method. LC
50,48 h
for C
60
was found to be 0.8 mgl
-1
when prepared
with solvent compared to[35 mgl
-1
when prepared
through prolonged stirring.
Aggregate size: 10–200 nm.
Not indicated whether
this is measure in this
study.
Zhu et al.
2006
CdTe—quantum
dots
Elliptio complanata Sublethal endpoints
(Immuno-
competence,
oxidative stress
and genotoxicity)
Centrifugation,
dialysis, placed in
distilled water at
pH 10 and diluted
CdTe was found to aggregate. 15% of the Cd was found
in the molecular fraction below 1 kDa. Exposure
concentrations of 1.6, 4 and 8 mgl
-1
CdTe did not
cause mortality or weakly closed shells. CdTe QDs
were found to induce oxidative stress in gills and
digestive gland tissues. Oxidative stress in the gills
was also detected when mussels were exposed to
0.5 mgl
-1
of molecular cadmium sulphate (CdSO4).
Results indicated that CdTe acts not only through
release of free Cd
2+
ions but effects were also
caused by colloidal CdTe.
80% of the
aggregates [450 nm
Gagne
´et al.
2008
SWCNT
(fractionated after
aqueous
dispersion)
Amphiascus
tenuiremis
Mortality,
development, and
reproduction in
life-cycle
bioassays
Dispersed in water
after oxidation of
SWCNT with
nitric acid
Average cumulative life-cycle mortality of
36 ±11% at 10 mgl
-1
. After 35 days exposure to
10 mgl
-1
showed a reduction of development
success of 51% for the nauplius to copepodite
window and 34% overall for the nauplius to adult
period. Though sex ratio and number of viable
offspring were not affected, a significantly lower
fertilization rate was observed.
Fluorescent nanocarbon:
length \18 nm, diameter
1 nm; Pure SWCNT:
lengths 50–100 nm,
diameters \10 nm
Templeton
et al. 2006
SWCNT/MWCNT
(
14
C labeled)
Lumbriculus
variegates
Uptake and
depuration of
nanotubes and
pyrene
Ultrasonic
dispersion
CNTs were found to not readily absorb into organism
tissues. CNTs detected in the organisms were found
to be associated with sediments remaining in the
organism guts.
MWNTs: 30–70 nm
SWCNTs: 1–2 nm
Petersen
et al. 2008
SWCNT: Single-walled carbon nanotubes, MWCNT: Multi-walled carbon nanotubes
Ecotoxicity of engineered nanoparticles to aquatic invertebrates 391
123
effects were found on hemocytes’ immuno competence
(hemocyte phagocytic activity, hemocyte viability and
hemocyte cell lysis potential) in the range of 1.6–8 mgl
-1
CdTe QDs. Oxidative stress in gills and digestive glands
were also found in this range. At 4 and 8 mgl
-1
of CdTe
QD a significant reduction in the number of DNA strand
breaks was observed and the authors interpret this as an
inhibition of the DNA repair activity (Gagne
´et al. 2008).
Interaction with other compounds and accumulation
The previously mentioned nanoparticle adhesion to the
exoskeleton and deposition in the gastrointestinal tract also
represents a possible route of exposure to co-existing con-
taminants sorbed to ENPs (Nowack and Buchelli 2007;
Baun et al. 2008). The influence of ENPs on toxicity and
accumulation of associated contaminants is particularly
interesting since e.g. C
60
-derivatives have been suggested
as potential drug-carriers for transporting pharmaceuticals
to specific organs or for crossing the blood-brain-barrier
(Vogelson 2001; Kreuter et al. 2002; Levi et al. 2006).
Due to their small size, nanoparticles will have a larger
surface area relative to mass and hereby potentially
increasing sorption capacity. Contaminated sediment parti-
cles have been shown to influence the health of D. magna,as
well as cause acute effects and contribute to tissue con-
centrations of contaminants (Weltens et al. 2000). Sorption
of pollutants onto nano-sized particles has been investigated
in a few studies (Cd/TiO
2
(Zhang et al. 2006), As/TiO
2
(Sun
et al. 2007), Diuron/Carbon Black (Knauer et al. 2007)).
However, only the study by Baun et al. (2008) has consid-
ered the potential carrier effect of ENPs in invertebrates
thus far. In this study, changes in D. magna mobility were
observed after exposure to the chemical compounds methyl
parathion, phenanthrene and pentachlorophenol in the
presence and absence of aqueous suspensions of C
60
nano-
particles. It was found that the toxicity of methyl parathion
was not affected by the presence of C
60
aggregates, whereas
a 1.9 times decrease in toxicity was observed for penta-
chlorophenol. For phenanthrene an 85% sorption to C
60
-
aggregates was observed, however, the toxicity of phenan-
threne was increased by 60% in the presence of C
60
aggregates. This shows that the sorbed phenanthrene was
available for the organisms (Baun et al. 2008).
Exposure pathways and modification upon uptake
As shown in Fig. 1, numerous exposure pathways are pos-
sible for the uptake of ENPs by invertebrates. Baun et al.
(2008) showed that C
60
nanoparticles may adhere to algae
which may then be ingested by filter-feeders, possibly
leading to a bioconcentration of nanoparticles and transfer
to higher trophic levels (see Fig. 1). Some ENPs may
aggregate in the aquatic environment, leaving only a minor
part of the total mass of nanoparticles in the size ranges
relevant for direct uptake throughout the water phase.
However, significant sedimentation can be expected, and
sediments should be regarded as an important sink for
ENPs discharged to the aquatic environment. Nanoparticle
aggregation is found to depend on surface charge and pH. In
the case of 6 nm iron oxyhydroxide (FeOOH) particles,
Gilbert et al. (2007) showed a pH-driven aggregation and
disaggregation with larger aggregate radius at higher pH
and vice versa. The pH in aquatic environments varies from
acidic to alkaline, while the internal pH of organisms can
significantly vary from the surrounding environment. In
the case of crustaceans, the pH of the digestive tract in
D. magna was found to vary from 6.8 in the anterior end to
7.2 at the caudal end (Hasler 1935). Thus, a change in the
pH and ionic strength in animal stomachs may lead to
changes in nanoparticle aggregation, hereby also affecting
particle uptake in the organism. Another alteration of ENPs
in organisms was demonstrated by Roberts et al. (2007)
who found that upon ingestion of 1.2 nm water-soluble lipid
coated SWCNT, daphnids were able to remove the lipid
coating and excrete the unmodified SWCNT. Although the
bioaccumulation study of phenanthrene in the presence of
C
60
carried out by Baun et al. (2008) did not show a net
accumulation, a tendency towards smaller aggregates of C
60
was observed after being excreted by D. magna.
Recommendations for future research in invertebrate
nanoecotoxicology
Based on this state-of-knowledge review of effects found in
invertebrates upon exposure to ENPs, we have the fol-
lowing recommendations for future invertebrate toxicity
testing. While some are specific to invertebrates others
may also be relevant for the emerging science of
nanoecotoxicology.
1. Insight in fate and bioavailability of ENPs in the
aquatic environment should be gained with standard-
ized short-term (lethality) invertebrate tests as starting
points. While these tests are needed for regulatory
purposes, they may have an added value as test
systems for studying fate and availability of ENPs in
aqueous suspensions/solutions along with biological
effects on the test species. The standardized tests have
the advantage that there is an extensive experience
knowledge base from chemical testing and the use of
fully-defined synthetic media enable systematic mea-
surements and modeling (e.g. Kusk and Wollenberger
392 A. Baun et al.
123
1999). This allows for both testing mechanisms for
ENP toxicity and uptake as well as for interactions
with already-existing contaminants in the aquatic
environment. Based on this literature review, it is
however apparent that the link to in situ characteriza-
tion techniques (i.e. in the aqueous solution and
animals before, during, and after incubation) need to
be strengthened. Also the intake by invertebrates,
translocation and adhesion of ENPs to the animal
surfaces needs to be investigated more closely. Several
studies found that D. magna filter the ENPs from the
water and the digestion tract becomes completely filled
with nanoparticles. At the same time aggregates of
ENPs adhere to the outer surfaces of the animals.
2. Long-term low exposure invertebrate tests focused on
chronic endpoints are needed. It was found that only
very few studies have so far investigated chronic
endpoints in invertebrates. Upon suspension in water,
the chemical composition, speciation and aggregation
of ENPs can change for example due to the method
of preparation, concentration and dilution, and media
composition. As the bioavailability of ENPs is
expected to be related to speciation and aggregation
state, it is not straight forward to predict whether
increased concentrations of ENPs in aqueous
suspensions will lead to higher organism exposure
concentrations in tests with long incubation periods.
The smaller crustacean species are especially well-
suited for chronic studies since the duration of their
life cycle is short compared to larger crustaceans and
to fish. Whole life cycle tests with daphnids and
marine copepods can be performed in three weeks,
which makes them less labor demanding than most
other chronic tests. At the same time they have proved
themselves to be sensitive to a variety of metals and
xenobiotic organic chemicals. Furthermore, changes in
aggregation state and translocation in organisms upon
uptake are key issues which can be studied with these
organisms in long-term low exposure tests. However,
as stated above there is also a need for in situ
Fig. 2 Daphnia magna ingest
nanoparticles from aqueous
suspension as indicated by the
presence of C
60
(A) and TiO
2
(B) in the digestive track
(arrows). (A)D. magna after
48 h exposure to an aqueous
suspension of 3 mgl
-1
C
60
(99.9%, Sigma-Aldrich)
prepared by prolonged stirring.
(B)D. magna after 48 h
exposure to an aqueous
suspension of 40.5 mgl
-1
TiO
2
(P25, Degussa) prepared by
sonication
Fig. 3 Adhesion of nanoparticles to the exoskeleton and antennae of
Daphnia magna exposed to aqueous suspensions of 40 mgl
-1
TiO
2
(A,B) and the marine copepod Acartia tonsa exposed to 0.5 mgl
-1
C
60
(C). Suspensions were prepared as described in Fig. 2. Media for
D. magna and A. tonsa was the synthetic M7—salted up to 2% for
A. tonsa (Kusk and Wollenberger 1999). Scale bars correspond to
200 lm
Ecotoxicity of engineered nanoparticles to aquatic invertebrates 393
123
characterization techniques to be able to quantify and
characterize the exposure to nanomaterials in labora-
tory studies as well as in the field.
3. Strengthen the focus on bioaccumulation studies of
ENPs in invertebrates. The presently used ENPs are
expected to persist in the aquatic environment––
metals/metaloxides are by definition non-degradable
and rapid degradation of carbon based ENPs is not
expected due to their chemical structure. Even though
emissions of ENPs to the aquatic environment may be
low, the low degradability combined with feeding
traits and habits of many invertebrates (filter feeders,
shredders, sediment dwelling) calls for research on the
bioaccumulation behaviour of ENPs in invertebrates.
For aquatic organisms in general, including inverte-
brates, there are very few studies which have addressed
this issue. In this respect, the potential vector function
of ENPs for co-existing contaminants and the use of
sediment tests should be emphasized. Sediments are
identified as a main sink of ENPs in the aquatic
environment due to the expected aggregation upon
discharge.
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