Chemosphere 73 (2008) 1121–1128
0045-6535/$ - see front matter © 2008 Else vier Ltd. All rights reserved.
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1. Intro duc tion
Man u fac tured nanom a te ri als (NMs) such as nano par ti cles,
nano tubes, nano sheets, and nano wires have recently found broad
appli ca tions in drug deliv ery, med i cal devices, cos met ics, chem i cal
cat a lysts, opto elec tron ics, elec tron ics, and mag net ics. Due to their
spe cific size range, larger sur face-to-vol ume ratio, crys tal line struc-
ture, and high reac tiv ity potential, some NMs have been found to
be toxic to humans and other tested organ isms either upon con tact
or after per sis tent envi ron men tal expo sure. For exam ple Obe rdör-
ster (2004) and Obe rdör ster et al. (2005) inves ti gated the tox ic ity
of the NM, ful ler ene (nC60), to juve nile large mouth bass (Micr opte
rus sal mo ides) and uncov ered a sig nifi cant increase of lipid per ox-
i da tion in the brain and glu ta thi one deple tion in the gills of the
fish. Sub se quently, the acute tox ic ity of nC60 was also detected in
Daph nia and fat head min now (Zhu et al., 2006). Recently, a series
of stud ies with trout, zebrafish, zoo plank ton, and algae have dem-
on strated that the NMs tested may cause dif fer ent lev els of tox ic-
ity to these organ isms (Hund-Rinke and Simon, 2006; Lo vern and
Klaper, 2006; Grif tt et al., 2007; Henry et al., 2007; Lo vern et al.,
2007; Smith et al., 2007; Use nko et al., 2007; War heit et al., 2007;
Zhu et al., 2007).
The pro jected increase in quan ti ties and types of NMs for indus-
trial and con sumer prod ucts and ser vices could lead to sig nifi cant
release of NMs into the nat u ral envi ron ment, espe cially the water
envi ron ment. Given that dis charged, spilled, and dis posed NMs
will enter, through numer ous direct and indi rect routes, into the
water envi ron ment, much like the wide spread con tam i na tion of
dichloro-diphenyl-tri chlo ro eth ane (DDT) and poly chlo ri nated
biphe nyls (PCBs) in var i ous water bodies world wide dur ing the
last cen tury (Ve ith et al., 1979; Beek, 1999), there is an urgent need
to address sev eral crit i cal NM-asso ci ated eco tox i co log i cal and
envi ron men tal risk assess ment issues, such as tox i col ogy of NMs
to aquatic organ isms of var i ous tro phic lev els, molec u lar and cel-
lu lar mech a nisms of NM-induced tox ic ity, the bio avail abil ity and
bio ac cu mu la tion of NMs through the aquatic food chain, as well as
iden ti fi ca tion of bio mark ers for early sig nal ing of the occur rence,
dis tri bu tion, and fate of NMs in the water envi ron ment.
In this study, we eval u ated the tox ic ity of two types of widely
used NMs, Tita nium diox ide (TiO2) and quan tum dots (QDs),
using the green mic ro al gae Chla mydo monas rein hardtii as a model
sys tem. TiO2 is being inten sively used as a UV quencher and a
Toxicity assessment of manufactured nanomaterials using the unicellular green
alga Chlamydomonas reinhardtii
Jiangxin Wang a, Xuezhi Zhang b, Yongsheng Chen b, Milton Sommerfeld a, Qiang Hu a,*
a Depart ment of Applied Bio log i cal Sci ences, Ari zona State Uni ver sity, Poly tech nic Cam pus, 7001 E. Wil liams Field Road, Mesa, AZ 85212, USA
b Depart ment of Civil and Envi ron men tal Engi neer ing, Ari zona State Uni ver sity, Tempe, AZ 85287, USA
a r t i c l e i n f oa b s t r a c t
Received 21 April 2008
Received in revised form 7 July 2008
Accepted 13 July 2008
Available online 2 September 2008
With the rapid devel op ment of nano tech nol ogy, there is an increas ing risk of human and envi ron men-
tal expo sure to nano tech nol ogy-based mate ri als and prod ucts. As water resources are par tic u larly vul-
ner a ble to direct and indi rect con tam i na tion of nanomaterials (NMs), the potential tox ic ity and envi ron-
men tal impli ca tion of NMs to aquatic organ isms must be eval u ated. In this study, we assessed potential
tox ic ity of two com mer cially used NMs, tita nium diox ide (TiO2) and quan tum dots (QDs), using the uni-
cel lu lar green alga Chla mydo monas reinh art ii as a model sys tem. The response of the organ ism to NMs
was assessed at phys i o log i cal, bio chem i cal, and molec u lar genetic lev els. Growth kinet ics showed that
growth inhi bi tion occurred dur ing the first two to three days of cul ti va tion in the pres ence of TiO2 or QDs.
Mea sure ments of lipid per ox i da tion mea sure ment indi cated that oxi da tive stress of the cells occurred as
early as 6 h after expo sure to TiO2 or QDs. The tran scrip tional expres sion pro fil ing of four stress response
genes (sod1, gpx, cat, and ptox2) revealed that tran sient up-reg u la tion of these genes occurred in cul tures
con tain ing as low as 1.0 mg L¡1 of TiO2 or 0.1 mg L¡1 of QDs, and the max i mum tran scripts of cat, sod1,
gpx, and ptox2 occurred at 1.5, 3, 3, and 6 h, respec tively, and were pro por tional to the ini tial con cen tra-
tion of the NMs. As the cul tures con tin ued, recov ery in growth was observed and the extent of recov ery,
as indi cated by the final cell con cen tra tion, was dos age-depen dent. QDs were found to be more toxic to
Chla mydo monas cells than TiO2 under our exper i men tal con di tions.
© 2008 Else vier Ltd. All rights reserved.
Anti ox i dant enzyme
Gene expres sion
Lipid per ox i da tion
Quan tum dots
Tita nium diox ide
* Cor re spond ing author. Tel.: +1 480 727 1484; fax: +1 480 727 1236.
Email address: hu qi email@example.com (Q. Hu).
1122 J. Wang et al. / Chemosphere 73 (2008) 1121–1128
whit en ing agent in cos met ics, sun sc reens, and food prod ucts, as
well as used as an effec tive pho to cat a lyst in water and waste wa ter
treat ment (Ye ber et al., 2000; Fu bin i and Hub bard, 2003; Kubo et
al., 2005). QDs are fluo res cence-emit ting semi con duc tor nano crys-
tals that have been widely used in bio log i cal and med i cal research
(Micha let et al., 2005). The ratio nale for using C. rein hardtii was
that the entire genome of this organ ism has been sequenced (Mer-
chant et al., 2007), the gene trans for ma tion sys tem, RNA inter-
fer ence and micro array tech niques are avail able (Wu-Scharf et
al., 2000; Sch roda, 2006), it has the abil ity to grow rap idly under
photo-, mixo- and het ero-tro phic con di tions (Har ris, 1989), and it
has the sen si tiv ity and abil ity to rap idly respond to var i ous envi-
ron men tal stim uli (Lei sing er et al., 1999; As amiziu et al., 2000;
Cruz et al., 2001; Ru bi nel li et al., 2002; Mi ura et al., 2003; Yos-
hida et al., 2004). By using real-time RT-PCR cou pled with sev eral
phys i o log i cal and bio chem i cal approaches, we addressed three
spe cific ques tions related to the potential tox ic ity of the NMs to
mic ro al gae: (1) is TiO2 or QD NMs toxic to Chla mydo monas, and if
yes, what is the thresh old con cen tra tion of TiO2 or QDs at which
adverse effects can be detected at the cel lu lar or molec u lar level?
(2) does Chla mydo monas respond to TiO2 and QDs dif fer ently? In
other words, is tox ic ity of NM to Chla mydo monas NM-spe cific? and
(3) can spe cific genes or their expres sion pat terns be used as sen si-
tive, reli able bio mark ers for the pres ence of spe cific NMs?
2. Mate ri als and meth ods
2.1. Nano ma te ri al prep a ra tion
De gussa P25 TiO2 NM powder, with an aver age Bru nauer–
Emmett–Teller (BET) sur face area of 50 m2 g¡1 and an aver age par-
ti cle size of 21 nm, was used to pre pare nano par ti cle sus pen sions
for the exper i ments. Phys io chem i cal param e ters of P25 TiO2 NMs
are shown in Table 1.
Water sol u ble CdTe quan tum dots (QDs) were pur chased from
Amer i can Dye Source Inc., Can ada. Thi o gly co late (HS–CH2–COO–)
cap ping ligands are bound to the CdTe cores of QDs via thio groups.
QDs were received as red stock sus pen sions with mass con cen tra-
tion of 0.25 wt Cd % and pH from 10 to 11. The reported size of QDs
ranged from 3.5 to 4.5 nm.
2.2. Char ac ter iza tion of NMs
The con cen tra tions of NMs rang ing from 0.75 to 11.5 lM were
mea sured by Micro-Flow Imag ing sys tem (MFI, DFA 4100, Bright-
well Tech nol o gies Inc., ON, Can ada). Vol ume size dis tri bu tions of
TiO2 and QD NMs in cul ture media at dif fer ent times (0, 6, 12, 24,
48 h) were mea sured using dynamic light scattering (DLS) (90 Plus
Par ti cle Size Ana lyzer, Brook ha ven Instru ment Corp., NY, USA), and
the median diam e ters were reported.
2.3. Algal strain and cul ture con di tions
C. rein hardtii (y1 strain) was pro vided by Dr. J. Ken neth Hoo ber
(Ari zona State Uni ver sity), and CM cul ture medium (Sue oka, 1960)
was used to grow the algae. The stan dard cul ture con di tions for
these exper i ments were: (1) 250 mL glass flasks con tain ing 100 mL
of CM medium; (2) a light inten sity of 55 lmol m¡2 s¡1, tem per a-
ture of 25 ± 0.5 °C; (3) with agi ta tion on a shaker table at a speed
of 85 rpm. Dur ing the first day of cell cul ture (ini tial cell den sity
of 10000 cells mL¡1), six con cen tra tions, 0.001, 0.01, 0.1, 1, 10,
100 mg L¡1, of TiO2 or QDs NMs were added to the flasks from an
ini tial 5000 mg L¡1 stock solu tion. The CM growth medium con tain-
ing 100 mg L¡1 TiO2 NMs was milky in color, whereas the medium
spiked with 1 mg L¡1 TiO2 NMs or a lower con cen tra tion was clear
and trans par ent. Dur ing the first day of expo sure, there were
white-green pre cip i tates observed at the bot tom of the cul tures
con tain ing 100 mg L¡1 or 10 mg L¡1 TiO2 NMs. By day four, these
cul tures turned white-green, and the pre cip i tates at the bot tom
decreased. There were no vis i ble pre cip i tates in the con trol and
treat ment cul tures with lower ini tial TiO2 con cen tra tions dur ing
the same expo sure time period.
2.4. Expo sure pro ce dure
For the gene expres sion pro fil ing test, algal cells grow ing expo-
nen tially in 250 mL glass flasks con tain ing 100 mL CM medium
were spiked with a dilu tion series of either TiO2 or QDs. Algal sam-
ples for phys io chem i cal and gene expres sion tests were taken at a
given time inter val for imme di ate anal y sis, or har vested by cen tri-
fu ga tion and stored at ¡80 °C for fur ther use.
2.5. Cell count ing
Chla mydo monas cells were counted with a hemo cy tom e ter
under a light micro scope (Wang et al., 2004).
2.6. Chlo ro phyll a fluo res cence mea sure ment
Chlo ro phyll a fluo res cence mea sure ments were applied as pre-
vi ously described (Max well and John son, 2000).
2.7. Lipid per ox i da tion
Lipid per ox i da tion was deter mined as pre vi ously described
(Hod ges et al., 1999). In brief, 50 mL sam ples were har vested and
homog e nized with liquid nitro gen using mortar and pes tle, then
resus pended in 4 mL 80% eth a nol. After cen tri fu ga tion at 3000g
for 10 min (4 °C), the super na tants were trans ferred to new micro-
tubes and 500 lL was used for mal ondi al de hyde (MDA) deter mi-
2.8. RNA iso la tion
Har vested cells (50 mL) were pul ver ized in a mortar with liquid
nitro gen and the powder was trans ferred to a micro-tube con tain-
ing 1 mL of TRI ZOL Reagent (Invit ro gen, USA). After pre cip i tated
and washed with 100% iso pro pyl alco hol and 70% eth a nol, respec-
tively, the RNA pellet was sus pended in a suit able vol ume of DEPC
water accord ing to the man u fac turer’s instruc tions.
RNA solu tions were quan ti fied by Nano Drop 3.0.0 (Cole man
Tech nol o gies Inc., USA). Ali quots were stored at ¡70 °C.
Phys io chem i cal param e ters of P25 TiO2 NMs
Phys io chem i cal param e tersValueUnit
BET sur face area
Aver age original par ti cle size
Water con tent (105 °C for 2 h)
Loss dur ing cal ci na tionb
pH (in 4% sus pen sion)
TiO2 con tentc
Al2O3 con tentc
SiO2 con tentc
Fe2O3 con tentc
HCl con tentc
Mocker, retain in sieve of 45 lmd
50 ± 15
a DIN ISO 787/XI, August, 1983.
b Cal cined at 1000 °C for 2 h.
c Base on the weight after cal ci na tion.
d DIN ISO 787/XVIII, April, 1984.
J. Wang et al. / Chemosphere 73 (2008) 1121–1128 1123
2.9. Realtime RTPCR
Tran scrip tional expres sion of four genes that encode for SOD,
GPx, CAT, and PTOX2 was mea sured using a real-time RT-PCR
method (Gib son et al., 1996). First strand syn the sis was car ried out
using Taq man Reverse Tran scrip tion sys tem accord ing to man u fac-
turer’s instruc tion (Applied Bio sys tems, CA, USA). One micro gram
of total RNA was applied to a 50 lL syn the sis mix ture. Prim ers
included for ward – 59-CAA GCC TAT CCC TAG CCG AAA-39 sequence
and reverse – 59-ATC CCC ATC ACG ATG CAG TT-39 for tar get gene
18S rRNA (Gen Bank access num ber: EF682842), for ward 59-GGA
GGC TGC AGG AAA ACT GA-39 and a reverse 59-ATT TCC AGC CTG
GGC TAC CT-39 for cat2 (Gen Bank access num ber: AF016902), for-
ward 59-CGA AGC CGC ACA TGG TAT AGT-39 and reverse 59-TGC
TCC AAT CAC GAC CTA TTT G for gpx (Gen Bank access num ber:
AY051144), and for ward 59-ACC CAA GAA CAA GGC GAT A-39 and
reverse 59-CAG CGA CTC GTA CAG GTG CA-39 for ptox2 (Gen Bank
access num ber: AF494290), and for ward 59-ACG GCT CCC TGT CGA
TCG A-39 and reverse 59-CGC CAC GTC CGG CAG CGC GG-39 for sod1
(Gen Bank access num ber: DS496117). The prim ers for real-time
RT-PCR were designed to pro duce 100 to 150 bp PCR prod ucts. Plas-
mids con tain ing the tar get cDNAs were used as the tem plates for
stan dards. Real-time PCR was per formed in trip li cate on cDNA sam-
ples and neg a tive con trols on an ABI Prism 7900 sequence detec-
tion sys tem (Applied Bio sys tems, USA). In each exper i ment, a stan-
dard dilu tion series of plas mids con tain ing spe cific PCR frag ments
and 25 ng cDNA (total RNA equiv a lent) of unknown sam ples were
ampli fied in a 20 lL reac tion con tain ing SYBR Green PCR Mas ter
Mix (Applied Bio sys tems, USA). The exper i ments were repeated
at least three times. To stan dard ize the results, the rel a tive abun-
dance of 18S rRNA was also deter mined and used as the inter nal
stan dard. All cal cu la tions and sta tis ti cal anal y ses were per formed
as described in the ABI 7900 sequence detec tion sys tem User Bul le-
tin 2 (Applied Bio sys tems, USA).
2.10. Sta tis ti cal anal y sis
All exper i ments were done in trip li cate, and the results were
pre sented as mean ± stan dard devi a tion. The exper i men tal data
were ana lyzed by ANOVA using a SPSS pro gram (Sta tis ti cal Prod uct
and Ser vice Solu tions, for merly Sta tis ti cal Pack age for the Social Sci-
ences, dis trib uted by SPSS Inc. of Chi cago, Illi nois, USA). Sta tis ti cal
sig nifi cance was accepted at a level of p < 0.05.
3.1. Char ac ter iza tion of par ti cle size and par ti cle num ber of NPs
Changes in median diam e ter of the par ti cles of TiO2 and QD
NMs in buffer solu tion from 0 to 48 h are shown in Fig. 1a. Median
par ti cle size increased with time and became rel a tively sta ble after
24 h. The median par ti cle size of QD and TiO2 aggre ga tions at 48 h
were 710 and 892 nm, respec tively, which are much smaller than
that of the algae (5–10 lm). Vol ume size dis tri bu tions of TiO2 and
QDs at dif fer ent times are shown in Fig. 1b and c, respec tively. NMs
have a much smaller size, larger sur face area and sur face energy
com pared to other mate ri als, so they are prone to aggre gate
(Adams et al., 2006). Fur ther more, rel a tively high ion strength in
CM cul ture medium com press their elec tri cal dou ble layer (EDL)
which may make the elec tric repulse between par ti cles decrease,
and thus eas ier to aggre gate (War heit et al., 2007). Due to aggre-
ga tion of NMs in the algal cul ture medium, nano-scale TiO2 or QD
par ti cles were in trace amounts rel a tive to their aggre ga tion forms.
A sim i lar sit u a tion has been pre vi ously observed by oth ers (Adams
et al., 2006; War heit et al., 2007).
3.2. Effect of TiO2 NMs on growth
Expo nen tially grow ing cells were inoc u lated into fresh CM
medium spiked with TiO2 NMs rang ing in ini tial con cen tra tion
from 0.001 to 100 mg L¡1 and the results are shown in Fig. 2a. As
a con trol, Chla mydo monas cells under went a short lag phase, fol-
lowed by an expo nen tial growth phase that lasted for about two
days before enter ing a sta tion ary phase, due prob a bly to light lim i ta-
tion of the cul ture. When the ini tial con cen tra tion of TiO2 NMs was
1 mg L¡1 or lower, there was little dif fer ence in growth rel a tive to
the con trol. Con sid er able reduc tion in growth occurred in cul tures
spiked with 10 mg L¡1 TiO2 NMs after two days of incu ba tion, with
the growth com pletely inhib ited on day three. Growth resumed
there af ter and by day five the cell con cen tra tion was sim i lar to
the con trol. As the ini tial con cen tra tion of TiO2 NMs increased to
100 mg L¡1, reduc tion in growth was notice able by the end of day
010203040 50 60
Particle size (nm)
Time (h) MPS (nm)
Particle size (nm)
Time (h) MPS (nm)
Fig. 1. Median par ti cle size (MPS) and vol ume size dis tri bu tion of TiO2 and QD NMs
in algal cul ture medium.
1124 J. Wang et al. / Chemosphere 73 (2008) 1121–1128
one. Sig nifi cant growth inhi bi tion (p < 0.05 vs. con trol) occurred on
days two and three, fol lowed by rapid recov ery. The final cell con-
cen tra tion was about 80% of the con trol on day five.
Cell aggre ga tion was observed in cul tures spiked with TiO2
NMs. The higher the ini tial TiO2 NMs con cen tra tion, the greater
the aggre ga tion of cells with the TiO2 NMs in the cul ture for the
first 2–3 d (Fig. 3a–c). Fig. 3a shows a homog e nous sus pen sion of
Chla mydo monas cells in the CM growth medium after three days
of cul ti va tion. In con trast, notice able cell/TiO2 NMs aggre ga tion
occurred in cul tures spiked with 100 mg L¡1 TiO2 NMs dur ing the
same time period. As the exper i ment pro ceeded, how ever, the
extent to which cell/ TiO2 NMs aggre ga tion occurred was reduced
in all the treat ment cul tures. It was con cluded that TiO2 NMs may
inhibit growth of Chla mydo monas cells when the ini tial TiO2 con-
cen tra tion was 10 mg L¡1 or higher. How ever, the growth inhi bi-
tion, to a large extent, was tem po rary, and as the cul ture was main-
tained for an addi tional period of time (i.e., 5 d), algal pro lif er a tion
resumed and the cell con cen tra tions in all treat ments reached or
was near the con trol level.
3.3. Lipid per ox i da tion
Mal ondi al de hyde (MDA), a molec u lar indi ca tor of lipid per ox i-
da tion that may impair struc ture and func tion of the cel lu lar mem-
brane sys tem, was mea sured in Chla mydo monas cells grown in the
pres ence or absence of TiO2 NMs. The pur pose of the exper i ment
was to deter mine whether growth inhi bi tion was due to oxi da tive
stress induced by TiO2 NMs, sim i lar to that caused by heavy met als
(Pinto et al., 2003). As shown Fig. 4, the MDA level was low and
remained more or less con stant in con trol cells or in the cul ture
spiked with 1 mg L¡1 TiO2 NMs dur ing a 24 h period. A sig nifi cant
increase in (p < 0.05 vs. con trol) MDA level was mea sured 6 h after
cul tures were spiked with 10 mg L¡1 TiO2 NMs and the MDA con-
tent declined some what there af ter. Fur ther increase in MDA con-
tent was mea sured in the cul tures spiked with 100 mg L¡1 with
the max i mum MDA level occur ring 12 h after treat ment, and then
decreas ing grad u ally. The growth inhi bi tion at high con cen tra tions
of TiO2 NMs (10 mg L¡1 or higher) may be related to lipid per ox i da-
tion caused by TiO2 NM-induced oxi da tive stress.
3.4. Gene expres sion pro files of anti ox i dant enzymes
If TiO2 NMs at higher con cen tra tions indeed induced cel lu lar
oxi da tive stress, result ing in lipid per ox i da tion and thus growth
inhi bi tion, then they might at the same time also affect cel lu lar
enzy matic defense activ i ties designed to cope with the stress.
Fig. 3. Light micro scopic images of Chla mydo monas cells in the absence of NMs (a)
in the pres ence of 100 mg L¡1 TiO2 (b), or 10 mg L¡1 QD NMs (c). Bar: 10 lm.
Cell numbers (10
4 cells mL-1)
Cell numbers (10
4 cells mL-1)
Fig. 2. Growth kinet ics of Chla mydo monas cells exposed to the var i ous con cen-
tra tions of TiO2 (a) or QDs NMs (b). d = con trol; s = 0.01 mg L¡1; m = 0.1 mg L¡1;
n = 1 mg L¡1; r = 10 mg L¡1; and e = 100 mg L¡1.
J. Wang et al. / Chemosphere 73 (2008) 1121–1128 1125
15 18 212427
MDA (pmol per 107 cells)
Fig. 4. MDA con cen tra tion in Chla mydo monas cells exposed to 0, 1, 10 or 100 mg L¡1
of TiO2 NMs for 24 h. d – Con trol, s – 1 mg L¡1, r – 10 mg L¡1, D – 100 mg L¡1 of
A rapid increase in sod1 tran scripts rel a tive to the con trol was
detected in cells exposed to 1 mg L¡1 TiO2 NMs for 1.5 h and the max-
i mum tran script level occurred 12 h after the treat ment (Fig. 5a).
The max i mum tran scripts were higher when cells were exposed
to 10 mg L¡1 TiO2 NMs. Fur ther increase in ini tial con cen tra tion of
TiO2 NMs to 100 mg L¡1, how ever, led to a some what decreased
max i mum tran script level. As the treat ments pro ceeded, the level
of sod1 tran scripts declined.
Tran sient up-reg u la tion of gene expres sion was also observed
with gpx, cat, and ptox2, although the exact expres sion pattern was
some what dif fer ent among the indi vid ual genes and at dif fer ent
ini tial con cen tra tions of TiO2 NMs. The max i mum tran scripts of
gpx occurred 3 h after treat ment, yet exhib ited a dose-depen dent
response: the higher the ini tial con cen tra tion of TiO2 NMs, the
greater the max i mum tran scripts (Fig. 5b). The cat gene showed a
sim i lar mRNA expres sion pattern to gpx, although the max i mum
tran scripts of cat occurred 1.5 h after the treat ments (Fig. 5c).
Con versely, a rapid up-reg u la tion of ptox2 tran scripts occurred
at 1 mg L¡1 TiO2 NMs and the max i mum of ptox2 tran scripts
decreased as the ini tial TiO2 con cen tra tion increased to 10 mg L¡1
to 100 mg L¡1 under the same exper i men tal con di tions (Fig. 5d).
3.5. Tran scrip tional expres sion of pho to syn thetic and carot en oid
bio syn the sis genes
In order to deter mine whether TiO2 NMs exerted any major
effect on the func tion of the chlo ro plast, in par tic u lar pho to syn the-
sis and carot en oid bio syn the sis, we mea sured the tran scrip tional
expres sion of psbA (a nucleus gene encod ing D1 pro tein in pho-
to sys tem II reac tion cen ter com plexes), rbcS (a ribu lose-1, 5-bis-
phos phate car box yl ase/oxy gen ase small sub unit gene), and pds (a
gene encod ing phy to ene desat ur ase involved in carot en oid bio syn-
the sis) in the pres ence or absence of TiO2 NMs. Our preliminary
results indi cated that these genes were not altered at the tran scrip-
tion level in the pres ence of dif fer ent con cen tra tions of TiO2 NMs
tested (data not shown).
3.6. Chlo ro phyll a fluo res cence mea sure ments
Pho to syn thetic response of Chla mydo monas to TiO2 NMs was
also inves ti gated using a chlo ro phyll a fluo res cence method. TiO2
NMs were added to Chla mydo monas cul tures with a final con cen-
tra tion of 1, 10, or 100 mg L¡1 and the cul tures were main tained
for 48 h. Dur ing this time period, a num ber of chlo ro phyll a fluo-
res cence param e ters were mea sured as a func tion of time. The
param e ters included effec tive PS II quan tum yield (Y, II), quan tum
yield of non-reg u lated energy dis si pa tion (Y, NO), quan tum yield
of reg u lated energy dis si pa tion (Y, NPQ), elec tron trans port rate
(ETR), non-pho to chem i cal quench ing (NPQ), coef cient of pho to-
chem i cal quench ing (qL), and max i mal PS II quan tum yield (Fv/
Fm). For these param e ters, no sig nifi cant dif fer ence (p > 0.05) was
observed among the dif fer ent treat ments (data not shown). It was
con cluded that TiO2 NMs exerted little impact on pho to syn the sis
of Chla mydo monas under our exper i men tal con di tions.
3.7. Effects of QDs on growth and gene expres sion
We also inves ti gated the effect of QDs on growth of Chla mydo
monas cells. As shown in Fig. 2b, growth inhi bi tion was observed
in cul tures con tain ing 1 mg L¡1 or a higher level of QDs. Clearly,
Chla mydo monas cells were more sen si tive to QDs than to TiO2 NMs
with 10 mg L¡1 QDs severely inhib it ing cell growth. Like TiO2 NMs,
Relative mRNA level
Relative mRNA level
Relative mRNA level
Relative mRNA level
Fig. 5. Tran scrip tional expres sion pro files of the genes in Chla mydo monas exposed
to 0, 1, 10 or 100 mg L¡1 of TiO2 NMs for 12 h. (a), sod1, (b), gpx, (c), cat, (d), ptox2.
Rel a tive mRNA lev els were nor mal ized with 18S rRNA as the inter nal stan dard.
d = 0 mg L¡1 (con trol); s = 1 mg L¡1, r = 10 mg L¡1, D = 100 mg L¡1 of TiO2 NMs.
1126 J. Wang et al. / Chemosphere 73 (2008) 1121–1128
QDs induced cell aggre ga tion in a dose-depen dent man ner (Fig.
3c). As the cul ture pro ceeded, the extent of cell aggre gates was
reduced. Tran scrip tional expres sion of the stress response genes,
i.e., sod1 and cat was inves ti gated under these con di tions and the
preliminary results revealed that the mRNA lev els of these two
genes were sig nifi cantly enhanced by QDs at con cen tra tion as low
as 0.1 mg L¡1 after 3 h treat ment (Fig. 6).
4. Dis cus sion
The median effec tive con cen tra tions (EC50) of 10 mg L¡1 and
5 mg L¡1 for TiO2 and QD NMs, respec tively, resulted in a 50% reduc-
tion in growth rate of Chla mydo monas within 72 h com pared to the
con trol. These EC50 val ues were com pa ra ble to that of other algal
spe cies tested in the pres ence of NMs. For exam ple, a 72 h EC50 of
16–26 mg L¡1 was reported for the green alga Pseud okirch neri el la
sub cap i ta ta in the pres ence of TiO2 NMs (War heit et al., 2007), and
a EC50 value of 14 mg L¡1 and a NOEC value of <12.5 mg L¡1 were cal-
cu lated for another green alga Des modes mus sub spic a tus exposed
to TiO2 NM (Hund-Rinke and Simon, 2006).
The recov ery of growth inhi bi tion took place in cul tures three
days after the TiO2 NM treat ments. The aggre ga tion of NMs in
the CM cul ture medium might have decreased its tox ic ity to the
cells. Indeed, the dynamic light scattering (DLS) anal y sis of the
par ti cle size dis tri bu tions of TiO2 NMs in cul ture media revealed
a pro gres sive for ma tion and accu mu la tion of NM aggre gates over
the time period (Fig. 1). The NM aggre ga tion may decrease active
sur face sites, and thus reduce the tox ic ity to Chla mydo monas cells.
A decrease in pho to cat a lytic activ ity of TiO2 NMs over the cul ture
period may be another reason for the reduced inhib i tory effect on
algal growth. Also, plau si ble is the detox i fi ca tion of TiO2 NM by
Chla mydo monas whereby cel lu lar secre tion of cer tain mac ro mol e-
cules such as poly sac cha rides which may affect the active sites of
the NMs can not be excluded at this point.
Lipid per ox i da tion is a man i fes ta tion of free rad i cal activ ity in
bio log i cal sys tems (Se va nian and Ur sin i, 2000). A dose-depen dent
increase in the max i mum MDA con tent for the first 12 h was clearly
indic a tive of cel lu lar lipid per ox i da tion induced by TiO2 NMs. The
level-off or decrease in lipid per ox i da tion after 12 h expo sure may
have resulted either from increas ing anti ox i dant defense capac ity
(Moore, 2006) or due to aggre ga tion of TiO2 NMs and/or the pos si-
ble bi omo di fi ca tion of TiO2 NMs by the cells. The increase of the
anti ox i dant enzymes, as indi cated at the tran scrip tional or pro tein
level could have led to scav eng ing of the ROS pro duced by TiO2
NMs, thereby directly or indi rectly reduc ing lipid per ox i da tion
(Afaq et al., 1998).
The lipid per ox i da tion and growth inhi bi tion caused by TiO2 NMs
stim u lated the up-reg u la tion of the genes known to be involved in
anti ox i dant reac tions. The molec u lar response of Chla mydo monas
cells to TiO2 NMs was sim i lar to NM-induced stress responses in
red blood cells. Car bon nano tubes were shown to induce oxi da tive
stress responses in kerat i no cytes after in vitro expo sure (Shve do va
et al., 2003). The dif fer ent tran scrip tional expres sion pat terns of
selected genes (cat, gpx, sod1 and ptox2) obtained from Chla mydo
monas cells in response to TiO2 NMs may indi cate the com plex
tox i co log i cal mech a nisms of action of TiO2 NMs on algal cells
and/or reflect com plex gene reg u la tion sys tems in response to
var i ous types and/or con cen tra tions of NMs. Pre vi ously, Ding and
col leagues (2005) employed a gene expres sion array tech nique to
inves ti gate multi-wall car bon nano-onion- and multi-wall car bon
nano tube-induced alter na tions in gene expres sion of human skin
fibro blasts and also observed dis tinct qual i ta tive and quan ti ta tive
dif fer ences in gene expres sion pro files in response to the dif fer ent
types and con cen tra tions of NMs.
The ptox2 gene was sen si tive to the pres ence of TiO2 NMs. A
low con cen tra tion (61 mg L¡1) of TiO2 NMs stim u lated the tran-
sient up-reg u la tion of gene expres sion. How ever, fur ther increase
in ini tial con cen tra tion of TiO2 NMs had less effect on the max-
i mum tran scripts of ptox2. We also noticed little alter na tion on
tran scrip tional expres sion of the pho to syn thetic genes (e.g., psaA
and rbcS) and the ca rote no gen e sis gene (e.g., pds) and little var-
i a tion in chlo ro phyll a fluo res cence param e ters at the ele vated
con cen tra tions of TiO2 NMs. It appeared that when the ini tial
con cen tra tion of TiO2 NMs was increased, light inten sity, may
have been a fac tor. The milky color of the growth medium con-
tain ing 10–100 mg L¡1 TiO2 NMs blocked con sid er able amounts of
inci dent light avail able for indi vid ual cells in the cul ture, which
might have caused light-enhanced pho to ox i da tive stress in the
pres ence of TiO2 NMs. Given that SOD1, CAT2, GPx and PTOX2
inves ti gated in this study were located in mito chon dria, per ox-
i some, cyto plasm and chlo ro plast, respec tively, we spec u lated
the main tar get sites of TiO2 NMs to Chla mydo monas cells were
mito chon dria, per ox i some, and cyto plasm, and to less extent the
chlo ro plast.
QD NMs (mg L-1)
Relative mRNA level
Relative mRNA level
Relative mRNA level
Fig. 6. The max i mum tran script level of genes encod ing sod1 (a), gpx (b), and cat
(c) in Chla mydo monas cells exposed to var i ous con cen tra tions of QDs for 3 h. ¤ Indi-
cates the sta tis ti cally sig nifi cant dif fer ence (p < 0.05).
J. Wang et al. / Chemosphere 73 (2008) 1121–1128 1127
QD NMs were shown to be more toxic to Chla mydo monas than
TiO2 NMs at the same ini tial con cen tra tion lev els. As a result,
1 mg L¡1 QD NMs caused a notice able growth inhi bi tion and a
lower con cen tra tion (e.g., 0.1 mg L¡1) stim u lated lipid per ox i da tion
and gene expres sion. It remains to be deter mined whether the
mech a nisms for the tox i co log i cal effects of QD and TiO2 NMs on
Chla mydo monas are the same or dif fer ent.
When growth inhi bi tion was used as a param e ter to assess the
potential impact of NMs on Chla mydo monas, the min i mum con-
cen tra tion of 10 mg L¡1 TiO2 or 1 mg L¡1 QD NMs were required to
inhibit growth after 24 h treat ment. In con trast, at the molec u lar
level, up-reg u la tion of the anti ox i dant genes cat and gpx to reach
the max i mum tran script level was detected 1.5 h and 3 h, respec-
tively, after expo sure of the cells to one-tenth of the NMs con cen-
tra tion that caused notice able growth inhi bi tion. There fore, the
real-time RT-PCR-based gene expres sion anal y sis can be used as
a more rapid, sen si tive, yet quan ti ta tive tech nique for eval u a tion
of NMs on algae and perhaps other living aquatic organ isms. As
such, gene expres sion of cat and gpx by real-time RT-PCR can be
potential can di dates for bio mark ers of TiO2 and QD NMs tox ic ity
in the aquatic envi ron ment. Because the genes (cat, gpx, and sod1)
tested in this study may react to other sources of oxi da tive stress
in the envi ron ment, they may not be use ful as spe cific bio mark ers
for NMs. How ever, with the proper con trol they can be applied as
com ple men tary bio mark ers to pre dict the risk caused by the exist-
ing or emerg ing NMs under lab o ra tory con di tions or in the nat u ral
aquatic envi ron ment.
5. Con clu sion
This study uti lized the green alga, C. rein hardtii, as an organ-
is mal bi o in di ca tor and selected gene expres sion bio mark ers for
detec tion of NMs (TiO2 and QDs) under lab o ra tory con di tions. It
sug gested that both growth kinet ics and molec u lar bio mark ers can
be applied for assess ment of envi ron men tal nano tox ic ity. The tox-
ic ity of the QDs to C. rein hardtii was ca. 10-fold greater than that of
TiO2 NMs, indic a tive of dif fer ent, yet unknown tox i co log i cal mech-
Acknowl edg ments
The authors acknowl edge the research fund ing (Grant #
RD833327) from the US Envi ron men tal Pro tec tion Agency, as well
as the 90 Plus Par ti cle Size Ana lyzer pro vided by Ira A. Ful ton
School of Engi neer ing at Ari zona State Uni ver sity.
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