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Ecotoxic Effect of Photocatalytic Active Nanoparticles (TiO2) on Algae and Daphnids (8 pp)_图文

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Photoctalytic Active Nanoparticles

Research Articles

Ecotoxic Effect of Photocatalytic Active Nanoparticles (TiO2) on Algae and Daphnids
Kerstin Hund-Rinke* and Markus Simon
Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie, P.O. Box 1260, 57377 Schmallenberg, Germany *Corresponding author (kerstin.hund-rinke@ime.fraunhofer.de)

DOI: http://dx.doi.org/10.1065/espr2006.06.311 Abstract

Background. Due to their large potential for manifold applications, the use of nanoparticles is of increasing importance. As large amounts of nanoparticles may reach the environment voluntarily or by accident, attention should be paid on the potential impacts on the environment. First studies on potential environmental effects of photocatalytic TiO2 nanoparticles have been performed on the basis of widely accepted, standardized test systems which originally had been developed for the characterization of chemicals. The methods were adapted to the special requirements of testing photocatalytic nanoparticles. Methods. Suspensions of two different nanoparticles were illuminated to induce their photocatalytic activity. For testing, the growth inhibition test with the green alga Desmodesmus subspicatus and the immobilization test with the daphnid Daphnia magna were selected and performed following the relevant guidelines (algae: ISO 8692, OECD 201, DIN 38412-33; daphnids: ISO 6341, OECD 202, DIN 38412-30). The guidelines were adapted to meet the special requirements for testing photocatalytic nanoparticles. Results. The results indicate that it is principally possible to determine the ecotoxicity of nanoparticles. It was shown that nanoparticles may have ecotoxicological effects which depend on the nature of the particles. Both products tested differ in their toxicity. Product 1 shows a clear concentration-effect curve in the test with algae (EC50: 44 mg/L). It could be proven that the observed toxicity was not caused by accompanying contaminants, since the toxic effect was comparable for the cleaned and the commercially available product. For product 2, no toxic effects were determined (maximum concentration: 50 mg/L). In the tests with daphnids, toxicity was observed for both products, although the concentration effect-curves were less pronounced. The two products differed in their toxicity; moreover, there was a difference in the toxicity of illuminated and non-illuminated products. Discussion. Both products differ in size and crystalline form, so that these parameters are assumed to contribute to the different toxicities. The concentration-effect curves for daphnids, which are less-pronounced than the curves obtained for algae, may be due to the different test organisms and/or the differing test designs. The increased toxicity of pre-illuminated particles in the tests with daphnids demonstrates that the photocatalytic activity of nanoparticles lasts for a period of time. Conclusion. The following conclusions can be drawn from the test results: (I) It is principally possible to determine the ecotoxicity of (photocatalytic) nanoparticles. Therefore, they can be assessed using methods comparable to the procedures applied for assessing soluble chemicals.

(II) Nanoparticles may exert ecotoxicological effects, which depend on the specific nanoparticle. (III) Comparable to traditional chemicals, the ecotoxicity depends on the test organisms and their physiology. (IV) The photocatalytic activity of nanoparticles lasts for a relevant period of time. Therefore, pre-illumination may be sufficient to detect a photocatalytic activity even by using test organisms which are not suitable for application in the preillumination-phase. Recommendations and Perspectives. First results are presented which indicate that the topic 'ecotoxicity and environmental effects of nanoparticles' should not be neglected. In testing photocatalytic nanoparticles, there are still many topics that need clarification or improvement, such as the cause for an observed toxicity, the improvement of the test design, the elaboration of a test battery and an assessment strategy. On the basis of optimized test systems, it will be possible to test nanoparticles systematically. If a potential risk by specific photocatalytic particles is known, a risk-benefit analysis can be performed and, if required, risk reducing measures can be taken.
Keywords: Algae; daphnids; ecotoxicity; nanoparticles; photo-

catalysis; TiO2

Introduction

The use of nanoparticles is of increasing importance, since they are suitable for manifold applications. In the field of medicine, for example, devices and vehicles have been developed in the micro and nano-scale to carry drugs to specific sites (Ducan 2003). In the scope of soil remediation, studies have been performed which demonstrate that nanoparticles, e.g. particles made from polyurethane acrylate anionomers, are suitable to desorb hydrophobic contaminants from soil particles (Tungittiplankorn et al. 2004). Metal oxides, such as Fe(II)oxides, can be used to reduce contaminants in soils (Maithreepala and Doong 2004). Some types of particles, e.g. TiO2, show photocatalytic activities resulting in the photochemical degradation of substances. This process is based on the reactive properties of electronhole pairs generated in semiconductor particles under illumination with light of an energy surpassing that of the semiconductor bandgap. The photocatalytic activity of TiO2 powder, for instance, has to be induced by UV-light. The charge carriers can reach the particle surface and react with species in solution possessing suitable redox potentials. This method has been successfully applied for the degradation of organic pollutants (Mills and Le Hunte 1997). Moreover,

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the bactericidal mode of titanium dioxide photocatalysis and the destruction of cancer cells have been studied intensively (for example Huang et al. 2000, Mills and Le Hunte 1997, Sakai et al. 1994, Sakai et al. 1995). Their photocatalytic properties have made these particles a valuable tool in water decontamination and air purification. Furthermore, the particles are used for photocatalytic coatings to have a selfcleaning effect on the surface of various materials. Several products, such as glass coatings, are already commercially available (Mills et al. 2004). Besides the numerous advantages of nanotechnology, it is well-known that inhaled, ultrafine particles may cause a risk for human health (Borm et al. 2004). Iron oxide nanoparticles have been proposed for an increasing number of biomedical applications. Due to its toxicity, a coating is necessary. DMSA-coated maghemite nanoparticles resulted in only weak cytotoxic and no genotoxic effects (Auffan et al. 2006). The environmental impact of nanoparticles should also be considered, since large amounts of nanoparticles may reach the environment voluntarily or unintentionally. Respective studies were performed by showing the toxic impact of fullerenes on fish (Oberd?rster 2004a) and daphnids (Oberd?rster 2004b). For the notification of chemicals, ecotoxicological tests are required to provide information on respective substance properties. For this purpose, several standardized tests are available. As aquatic test organisms, for example, daphnids and algae are applied. Whereas tests with water soluble chemicals are easy to perform, the use of solvents may be necessary for testing fairly water soluble chemicals. For chemicals forming layers in aqueous solutions (e.g. mineral oils), a special procedure using water accommodated fractions (WAFs) is recommended (ASTM Standard D 6081-97 1997, CONCAWE 1992, Müller and Wenzel 2002). Information on the ecotoxicity of nanoparticles is not required so far. Therefore, no standardized procedures exist for testing environmental effects caused by photocatalytic nanoparticles, such as TiO2. To provide data on a potential impact of TiO2 on the environment, first studies have been conducted using standardized test systems that are usually applied for the characterization of chemicals. The investigations are presented in the following. The tests were carried out using the green alga Desmodesmus subspicatus and the daphnid Daphnia magna as test organisms. The existing procedures were adapted to meet the special requirements demanded for testing photocatalytic nanoparticles. For the tests, the photocatalytic activity of TiO2 powder was induced by UV-illumination, and the effects on the test organisms were determined.
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1.1

Research Articles
ured by determining fluorescence. To adapt the determination method on photocatalytically active nanoparticles, the growth test of algae was modified as follows: For every concentration applied, TiO2 was dispersed in algae test medium (prepared according to the guideline) in a 50-fold concentration. Dispersion was achieved by ultrasonic dispersion. 1 mL of the dispersion was diluted (1:50) with algae adjusted to a concentration of 10,000 cells/mL in test medium according to the guideline. To avoid separation, the mixture was continuously stirred on a magnetic stirrer. The dispersion (50 mL in a 100 mL beaker) was irradiated, in a SUNTEST CPS+ Tabletop Xenon Exposure System (Atlas, Linsengericht/Altenhasslau, Germany), with simulated sunlight (300–800 nm). The intensity of the xenon lamp and the duration of the irradiation were varied. After irradiation, sub-samples of the dispersion were transferred to 96well microtiter plates and incubated according to the guideline in a Multitrone shaker (Infors AG, Bottmingen, Switzerland) for 72 h at 20 ± 2°C [Eisentr?ger et al. 2003]. As a light source, 'OSRAM L 36W/21-840 Plus Eco' lamps were used. In each test variant, 8–12 sub-samples of 200 ?L were measured. During incubation, the plates were shaken to obtain a mixture of algae and particles. Before and after the incubation period of 72 h, the fluorescence intensity of the algae was measured using a Spectrafluor plus microtiter plate reader (Tecan, Crailsheim, Germany). All results are presented as [%] of the yield (fluorescence at the end of the test minus fluorescence at start of the test) determined in the control sample. The control varied depending on the investigated topic. NOEC (no observed effect concentrations), EC50-values and statistical significance were determined (calculation program 'ToxRat?'; Toxrat Solutions GmbH; Alsdorf, Germany). The modified test method was elaborated stepwise: (I) Elaboration of optimum irradiation conditions: Irradiation of the algae without adding TiO2 and variation of light intensity and duration (25 W, 15 min; 250 W, 30 min; 500 W, 15 min; 500 W, 30 min). Control: no irradiation of the algae. (II) Influence of preliminary irradiation: Tests with TiO2 were performed without irradiation (= control) and compared to a test with preliminary irradiation. (III) Investigation of potential shading effects: To prove if TiO2 does not cause a growth inhibition upon reduction of the light intensity in the wells during the test period lasting 72 h, a special test design was applied (Fig. 1). A suspension of algae (10,000 cells/mL) was filled in one third of the wells of a white microtiter plate (200 ?L/well). The second third of the wells was filled with a dispersion of algae and several concentrations of nanoparticles (concentrations in the wells: 0 mg/L = control; 12.5 mg/L; 25 mg/L; 50 mg/L), while the remaining wells were filled with a mixture of algae and several concentrations of the reference substance K2Cr2O7 (concentrations in the wells: 0 mg/L = control; 0.156 mg/L; 0.313 mg/L; 0.625 mg/L; 1.25 mg/L). One third of the wells of a second, transparent, microtiter plate was filled with different concentrations of

Materials and Methods
Ecotoxicological test with algae

The ecotoxicological test with the green alga Desmodesmus subspicatus was conducted following the guidelines ISO 8692, OECD 201 and DIN 38412-33. In this test, algal growth is determined during an incubation period of 72 h. In the present study, the reproduction of algae was meas-

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Upper, transparent plate

Illumination

Wells with TiO2

Wells with test medium

Upper, transparent plate Lower, white plate

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Wells with algae + TiO2

Wells with algae + K2Cr2O7

Fig. 1: Schematic view of the 'sandwich' test used to assess shading effects by TiO2

TiO2 dispersions (0 mg/L = control; 12.5 mg/mL; 25 mg/ mL; 50 mg/mL). The remaining wells were filled with the same amount of test medium, but without adding TiO2. The second, transparent, plate was located on top of the white microtiter plate filled with algae (sandwich-test). The upper plate was thoroughly positioned in a way that the wells of both plates were congruent. The wells that had been filled with TiO2 were positioned above the wells containing the algae, whereas the wells filled with test medium were placed above the wells containing TiO2/algae and K2Cr2O7,/algae, respectively. As the upper plate was transparent, the light could pass through and provide the energy required for the growth of the algae placed on the lower plate. As this plate was made of white plastic, no lateral incidence of light could occur. Therefore, potential differences in illumination could only be caused by the dispersion of TiO2.
1.2 Ecotoxicological test with daphnids

had been filled with 20 mL of dispersion fluid. 5 daphnids were added to each Petri dish. The relation of solute and number of organisms corresponded to the guidelines. As the validity criterion accepting maximally 10% immobilized daphnids in the control vessels was fulfilled, although the height of water was lower than in the usually applied small beakers, this procedure was clearly acceptable. Three to five replicates per concentration were used. The incubation was performed under the conditions required in the guideline (20 ± 2°C, 48 h; diffuse light, day/night rhythm 16 h/8 h). After an incubation period of 48 h, immobilization of the daphnids was determined.
1.3 TiO2-nanoparticles

The ecotoxicological test with the daphnid Daphnia magna was performed following the guidelines ISO 6341, OECD 202 and DIN 38412-30. Before starting the test, the dispersion was illuminated as described for the test with algae. Since daphnids are damaged by stirring, no daphnids were added to the dispersion before pre-illumination. Sub-samples were transferred to the test vessels. To increase the contact between test organisms and nanoparticles, the test was performed in Petri dishes made of glass (? 55 mm) which

Two commercially available TiO2-nanoparticles were applied: Product 1 had a particle size of 25 nm (crystalline form: mainly anatase), while the particle size of Product 2 was 100 nm (crystalline form: 100% anatase). As particles of technical grade were used, the particles applied in one of the experiments were washed to provide information on a potential contamination that might have an impact on algal growth. The particles were cleaned according to the recommendation of one of the producers, i.e. stirring 10 g of the nanoparticles in 500 mL of deionised H2O for 19 h at room temperature. In addition to the recommendation, a second cleaning step was performed. After centrifugation for 1 h at 20,000 g, the pellet was dispersed again in 500 mL of water, stirred for 24 h, centrifuged and dried at 55°C.

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NOEC of ≥ 50 mg/L was determined. In contrast to this, the test design allowing a direct contact between algae and nanoparticles showed a clear concentration-effect curve resulting in an EC50-value of 14 mg/L and a NOEC of < 12.5 mg/L. A concentration-effect curve, with an EC50-value of 0.5 mg/L, was also obtained for the reference substance K2Cr2O7, which was within the expected range (ISO 8692). As (I), the growth of algae separated from TiO2-nanoparticles was comparable to the control and, as (II), there was a doseresponse-relationship in the wells with the reference substance K2Cr2O7, it is concluded that the concentration dependent inhibition of the fluorescence in the vessels containing a mixture of algae and TiO2 nanoparticles remaining under that of the control is unlikely to be caused by a lowered light intensity, but is due to a toxicity of TiO2.
2.1.3 Effect of different nanoparticles

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2.1

Results
Tests with algae

2.1.1 Elaboration of optimum irradiation conditions

The standardized test systems do not foresee a pre-incubation with sunlight. As the UV-sensitivity of the laboratory culture of Desmodesmus subspicatus was unknown, irradiation conditions were elaborated that do not cause a measurable adverse effect on algal growth. The results are presented in Table 1. Upon preliminary illumination with 250 W (15 min, 30 min) and 500 W (15 min), a comparable fluorescence was yielded for illuminated and non-illuminated algae (= control) in the subsequent growth period of 72 h. The conditions required in the guideline were considered. In contrast, a pre-illumination with 500 W lasting 30 min resulted in a 37%-reduction of fluorescence. The guideline requires a light intensity in the range of 6,000– 10,000 lux. Even a pre-illumination at 250 watts was far above the range mentioned in the guideline. Nevertheless, three of the four variants did not cause an effect on growth. For the following experiments, a pre-incubation of 250 watts and 30 min was selected.
Table 1: Effect of various intensities and periods of illumination on Desmodesmus subspicatus (10,000 cells/mL)

Effect of various intensities and periods of illumination on fluorescence of Desmodesmus subspicatus (10,000 cells/mL) Illumination conditions 250 watts, 15 min 250 watts, 30 min 500 watts, 15 min 500 watts, 30 min [%] of untreated control 103 110 113 63

Table 2 and Fig. 3 show the ecotoxicity of the two tested nanoparticles. The values obtained for the fluorescence (see Table 2) indicate algal growth, although the test design had been modified (preliminary illumination; nanoparticles). Furthermore, the standard deviation was in a range which was assumed to be acceptable for particle testing. At the beginning of the experiment, the standard deviation amounted to 2–5%. After a growth period of 72 h, the maximum standard deviation was 14%. Both products differ in their toxicity. Product 1 shows a clear concentration-effect curve (see Fig. 3). On the basis of the results determined for the commercially available product, an EC50-value of 44 mg/L (95% confidence interval: 30– 94 mg/L) was determined. It could be proven that the toxicity is not caused by accompanying contaminants, since toxicity did not significantly decrease after washing the product. The EC50 for the additionally cleaned product was 32 mg/L (95% confidence interval: 20–82 mg/L). In contrast to product 1, product 2 demonstrated no or at least a lower toxicity. Although significant differences to the control were determined for some concentrations, no obvious dose-response curve exists. For the commercially available product, between 80% and 100% of the fluorescence was determined in the vessels with TiO2 compared to the

2.1.2 Investigation of potential shading effects

To find out whether nanoparticles cause a shading of the algae resulting in a reduced growth, a sandwich test was performed. The test design that physically separates algae and nanoparticles showed that the presence of the particles did not cause a reduction of algal growth (Fig. 2). Independent of the concentration of the nanoparticles layered above the algae, algal growth was comparable to the control. A

Fig. 2: Algal growth in three test variants: (I) Algae and TiO2 physically separated; (II) mixture of algae and TiO2, (III) mixture of algae and the reference substance K2Cr2O7; statistical significance: *** p ≤ 0.01

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Table 2: Fluorescence of algae at the beginning of the growth tests with two different nanoparticles and after 72 h (mean value of 8–12 sub-samples of mixture, commonly illuminated per concentration prior to the growth test)
Fluorescence of algae at the beginning of the growth tests with two different nanoparticles and after 72 h (mean value of 7–8 sub-samples of mixture, commonly illuminated per concentration prior to the growth test) Concentration [mg/L] – 3.1 6.25 12.5 25 50 0h 1672 ± 33 – – – – – Control 72 h 27117 ± 2562 – – – – – Product 1 (commercially available) 0h – 1736 ± 94 1701 ± 57 1717 ± 54 1761 ± 52 1776 ± 77 72 h – 26090 ± 3654 24118 ± 2447 20246 ± 1389 16569 ± 858 14870 ± 1851 Product 1 (additionally cleaned) 0h – 1643 ± 39 1648 ± 43 1680 ± 53 1677 ± 38 1677 ± 55 72 h – 29664 ± 2605 28264 ± 2854 22582 ± 1843 14369 ± 275 12202 ± 490 Product 2 (commercially available) 0h – 1692 ± 42 1743 ± 49 1772 ± 35 1788 ± 62 1819 ± 64 72 h – 24203 ± 3024 27050 ± 2563 24312 ± 2070 22400 ± 2465 22349 ± 1373 Product 2 (additionally cleaned) 0h – 1625 ± 55 1649 ± 53 1688 ± 39 1716 ± 31 1715 ± 49 72 h – 23663 ± 2501 20795 ± 2400 23377 ±1709 23260 ± 1556 18571 ± 1752

Fig. 3: Effect of two different TiO2-nanoparticles; significance: * 0.05 ≥ p > 0.01; ** 0.01 ≥ p > 0.001; *** p ≥ 0.001

control. No EC50 could be calculated. For product 2 as well, cleaning did not result in a decreased toxicity.
2.1.4 Influence of preliminary irradiation

To determine the effect caused by the initial pre-illumination period, illuminated particles and particles that had not

been illuminated before the main test were tested in parallel. Pre-illumination caused no additional effect (Fig. 4). As the effect of irradiated particles may be more or less pronounced (12.5 mg/L versus 25 mg/L, 50 mg/L) compared with non-irradiated particles, differences seem to be due to technical reasons and biological variability.

Fig. 4: Effect of pre-illumination on the toxicity of TiO2 (particle size: 25 nm) in the growth test with algae ; significance: *** p ≤ 0.01

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therefore, were divided into two phases, a pre-illumination period and a test period. Pre-illumination aims to induce the photocatalytic activity of TiO2. In the test with algae, the test organisms could be added to the test medium already in this phase of the experiment. No damage occurred caused by the light, but the algae can be damaged by the photocatalytic activity. It was assumed that adverse effects would be even more pronounced during the following test period, when the algae should reproduce. No difference in growth reduction was observed for the tests, regardless of whether preliminary illumination took place or not. This may be explained by the following two reasons: I) The portion of relevant wavelengths of the lamp applied during the incubation period of 72 h was sufficient to induce and maintain a photocatalytic activity or, II), the measured toxic effect was caused by the TiO2 itself and not by a photocatalytic effect. It is evident from the obtained results that TiO2 nanoparticles may affect algae. Therefore, the above effects were not differentiated. For the smaller (25 nm) product, an EC50 of about 40 mg/L was determined for algae. So far, it is unknown which concentrations of nanoparticles, such as 25 nm of TiO2, may occur in the environment. Therefore, an environmentally relevant assessment of the results by comparing the predicted environmental concentration (PEC) and the effect concentration is not possible. According to the EU Directive on classification, packaging and labelling of dangerous substances (Council Directive 67/548/EEC), a toxicity in the range of 10–100 mg/L combined with no biodegradability has to be classified as 'harmful to aquatic organisms; and may cause long-term adverse effects in the aquatic environment'. So far, this directive is not applied to nanoparticles. However, by transferring the classification to this substance group for a first assessment, a harmful effect has to be stated for the smaller one of the two tested particles. In contrast to the tests with algae, it is not possible to add daphnids to the TiO2-mixtures before pre-illumination, since stirring of the mixtures, which is required to ensure a homogenous activation of the particles, would severely damage the test organisms. Therefore, the daphnids were added to the test system after the pre-illumination period. The results showed the following: (I) In the tests without pre-illumination, the immobilisation rates were lower than after pre-illumination. This indicates that the light source which is usually applied in the test with daphnids had either no potential at all or a negligible potential for inducing the photocatalytic activity of TiO2. (II) Toxicity increases due to photocatalytic exposure. (III) As daphnids which were added to the test solution after pre-illumination were immobilized to a higher extent than daphnids in mixtures that had not been illuminated at all, it can be concluded that the photocatalytic activity of TiO2-nanoparticles lasts for a relevant period of time. Therefore, the presented method offers the possibility of performing experiments with test organisms which are not suitable for pre-illumination because they are damaged by the light intensity or by the stirring or shaking.

2.2

Tests with daphnids

Further to the comprehensive investigations with algae, several tests were conducted with daphnids. Whereas 30 min of illumination at 250 W did not cause an immobilization of the daphnids (data not shown) in the presence of TiO2, toxicity was observed for both products (Fig. 5). In the control, maximally 10% of the daphnids were immobilized at the end of the test. This confirms that the validity criteria required in the guidelines were fulfilled, although a comparatively thin layer of liquid was applied in comparison with the method described in the guideline. As in the tests with algae, a lower toxicity was observed for the product possessing a larger particle size. However, clear concentrationeffect curves cannot be deducted for either of the products. Furthermore, high standard deviations were determined.

Fig. 5: Immobilization of daphnids by TiO2; significance: * 0.1 > p ≤ 0.5

The toxicity of the particles that had been illuminated before the test seems to be higher compared to the particles which had not been pre-illuminated, since immobilized daphnids were observed in a higher number of pre-illuminated samples. For product 1, the number of concentrations at which immobilised daphnids were determined increased from two to five, as a result of pre-illumination. In the tests with product 2, immobilised daphnids were determined at four concentrations in pre-illuminated samples, whereas immobilization was observed for one concentration only in the samples which had not been pre-illuminated.
3 Discussion

The photocatalytic activity of TiO2 is induced by ultraviolet light. Therefore, it is advisable to perform ecotoxicological tests using UV-irradiation. Devices simulating sunlight including the UV-portion are commercially available. They are applied to investigate the stability of products towards sunlight or to determine photodegradation according to the OECD-guideline 'Phototransformation of Chemicals on Soil Surfaces' (Draft 2002). In the present study, a SUNTEST CPS+ (Atlas) was applied to ensure a constant illumination with the relevant wavelengths of < 400 nm in all experiments by automatically correcting a reduced light intensity. Preliminary illumination experiments caused a complete immobilization of daphnids and the death of algae. The tests,

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The applied TiO2-nanoparticles differ in their toxicity as well as in size, crystalline form and pore structure. Product 1 is characterized by a particle size of 25 nm and a crystalline form mainly consisting of anatase; product 2 has a particle size of 100 nm, and a crystalline form consisting of 100% anatase. Particle size and pore structure influence the specific surface area. Moreover, it is assumed that nanoparticles formed aggregates in the dispersions used in the tests. Therefore, the determined effects cannot be directly linked to the undispersed nanoparticles. The size of the aggregates has not been determined yet. So far, it is unclear which properties contribute to the toxicity. Furthermore, nothing is known about the mode of action and concerning whether the particles exhibit their toxic effect at the surface of the organisms or whether they are incorporated. At least for daphnids, incorporation can be assumed, although it must be confirmed by more comprehensive investigations. In contrast to the tests with algae, no concentration-effect curves were determined in the tests with daphnids, although effects were observed. Furthermore, the standard deviations were more pronounced. This may be due to the different test organisms and/or differences in the test designs. In the test with algae, shaking of the mixtures during the incubation period minimized a sedimentation of particles and algae. Even if particles and algae sediment to a minor extent, they remain in direct contact so that toxic effects can occur. In the test with daphnids, the mixtures were not shaken, in order to avoid physical damage of the organisms. Consequently, the particles sediment after some hours. In contrast to algae, daphnids do not sediment due to their mobility, so that there is less contact between organisms and particles. On the other hand, a constant current of water is produced by the movements of the thoracic appendages of the daphnids. Small particles of less than 50 microns in diameter are filtered out of the water by fine setae located on the thoracic legs and are moved to the mouth. It is generally assumed that particles of a suitable size are ingested without any selective mechanism. In the presented test design, the determined toxicity is the result of two antagonistic processes, a reduced toxicity due to sedimentation of the particles with resulting separation of organisms and particles, and an increased toxicity caused by fast movements of the thoracic appendages, which results in an active transport of the particles to the mouth and subsequent ingestion or by blocking of the gills. It is assumed that a concentration-effect curve could not be established due to the unequal dispersal of the nanoparticles and the rather accidental contact between organisms and test product. This may also be the reason for the comparatively high standard deviation. A periodical dispersion of the particles during the incubation period did not improve the results (data not shown). Flow-through systems may be a suitable test device. The smaller effect observed for product 2 may be explained by the larger particle size, the higher weight and by an increased tendency for sedimentation.

Photoctalytic Active Nanoparticles

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Conclusion

The present study considers two products with particle sizes of 25 nm and 100 nm. As a result of future technical development, the size of industrially applied nanoparticles will further decrease. Considering that the smaller nanoparticles turned out to be more toxic in the present tests, it cannot be excluded that the overall ecotoxicological potential of nanoparticles will increase in future. From the results obtained in the present study, it can be concluded that: (I) It is principally possible to determine the ecotoxicity of nanoparticles. (II) Nanoparticles may exert ecotoxicological effects which depend on the properties of the specific nanoparticle. (III) Comparable to 'traditional' chemicals, the ecotoxicity depends on the test organisms and their physiology. (IV) The photocatalytic activity of nanoparticles lasts for a relevant period of time. Therefore, pre-illumination may be sufficient to detect a photocatalytic activity even by using test organisms which are not suitable for application in the pre-illumination-phase.
5 Recommendations and Perspectives

First results were presented which indicate that the issue 'ecotoxicity and environmental effects of nanoparticles' should not be neglected. There are still many topics that need clarification or improvement. Some of these are: (I) The cause of the toxicity: Does the specific surface area play a role, is the crystalline form dominating or are aggregates formed the size of which might be the most important factor in the test system? (II) Further improvement of the test design (e.g. stability of the dispersions; identification of optimum light sources for inducing photocatalytic activity, continuous illumination in the test without causing damage of the organisms through the light source) (III) Elaboration of a test battery suitable for the assessment of nanoparticles (IV) Assessment of the results. On the basis of optimized test systems, nanoparticles can be tested systematically. If a potential risk by specific (photocatalytically active) nanoparticles is known, a risk-benefit analysis can be performed and, if necessary, risk reducing measures can be conducted. The potential benefits of nanotechnology are large – but so are the perceived risks, which must be addressed early in time. The precautionary principle should not be used to stop research related to nanotechnology and nanoparticles. Instead, a sound balance between further development of nanotechnology and the necessary research should be strived for, to identify potential hazards in order to develop a scientifically defensible database for the purpose of risk assessment (Roco 2005, Oberd?rster et al. 2005, Nel et al. 2006). Considering the observed potential ecotoxicity of nanoparticles and taking into account the known low stability of some commercially available products (Mills et al. 2004), the stability of photocatalytic coatings used in the environment is of high priority.

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For a comprehensive assessment of nanomaterials, information on the whole product-life cycle is necessary (Krug 2005). This includes, besides information on human health, environmental effects, including fate, transport, transformation, bio-availability and bioaccumulation, and applies for aquatic as well as for terrestrial environments. First studies on the distribution and leaching of nanoparticles in porous media are already available (Lecoanet et al. 2004, Lecoanet and Wiesner 2004). Information on the accumulation and potential ecotoxicological impact of nanoparticles on the terrestrial environment is still lacking. Investigations on this matter are postulated.
Acknowledgement. The authors would like to thank Ricarda N?ker and Katja Mock for their excellent technical assistance. The investigations were part of a joint research (Fraunhofer Photocatalysis Network, consisting of eight Fraunhofer Institutes) sponsored by the FraunhoferGesellschaft. The task of the Fraunhofer Institute for Molecular Biology and Applied Ecology was to work on the environmental aspects.

Research Articles
ISO 8692 (2004-10-00): Water quality – Freshwater algal growth inhibition test with unicellular green algae Klupinski T, Chin Y-P, Traina S (2004): Abiotic degradation of Pentachloronitrobenzene by Fe(II): Reactions on Goethite and iron oxide nanoparticles. Environ Sci Technol 38, 4353–4360 Krug HF (2005): Auswirkungen nanotechnologischer Entwicklungen auf die Umwelt. UWSF – Z Umweltchem ?kotox 17, 223–230. Lecoanet HF, Wiesner MR (2004): Velocity effects on fullerene and oxide nanoparticle deposition in porous media. Environ Sci Technol 38, 4377–4382 Leconet HF, Bottero JY, Wiesner MR (2004): Laboratory assessment of the mobility of nanomaterials in porous media. Environ Sci Technol 38, 5164–5169 Maithreepala RA, Doong R (2004): Synergistic Effect of Copper Ion on the Reductive Dechlorination of Carbon Tetrachloride by Surface-Bound Fe(II) Associated with Goethite. Environ Sci Technol 38, 260–268 Mills A, Le Hunte St (1997): An overview of semiconductor photocatalysis. Journal of Photochemistry and Photobiology A: Chemistry 108, 1–35 Mills A, Lepre A, Elliott N, Bhopal S, Parkin I, O'Neill S (2004): Characterisation of the photocatalyst Pilkington ActiveTM: A reference film photocatalyst? J Photochem Photobiol A: Chemistry 160, 213–224 Müller J, Wenzel A (2002): Ecotoxicological testing of gas oils (Daphnia magna test). Part I and II. Deutsche Wissenschaftliche Gesellschaft für Erd?l, Erdgas und Kohle e.V., Hamburg, Febr. 2002, ISBN 3-931850-92-7 Nel A, Xia T, M?dler L, Li N (2006): Toxic potential of materials at the nanolevel. Science 211, 622–624 Oberd?rster E (2004a): Manufactured nanomaterials (Fullerenes, C60) induce oxidative stress in the brain of juvenile largemouth bass. Environ Health Perspect 112, 1058–1062 Oberd?rster E (2004b): Toxicity of nC60 fullerenes to two aquatic species: Daphnia and largemouth bass [abstract]. In: 227th American Chemical Society National Meeting, 27 March–1 April 2004, Anaheim, CA. Washington, CE: American Chemical Society, IEC 21 Oberd?rster G, Oberd?rster E, Oberd?rster J (2005): Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles. Envrion Health Perspect 113, 823–839 OECD 201 (1984): OECD Guideline for Testing of Chemicals. 'Alga, growth inhibition test' OECD 202 (1984): OECD Guideline for Testing of Chemicals. 'Daphnia sp., acute immobilisation test and reproduction test' Roco MC (2005): Environmentally responsible development of nanotechnology. Environ Sci Technol 39, 106A–112A Sakai H, Baba R, Hashimoto K, Kubota Y, Fuijishima A (1995): Selective killing of a single cancerous T24 cell with TiO2 semiconducting microelectrode under irradiation. Chemistry Letters 185–186 Sakai H, Ito E, Cai R-X, Yoshioka T, Kubota Y, Hashimoto K, Fuijishima A (1994): Intracellular Ca2+ concentration change of T24 cell under irradiation in the presence of TiO2 ultrafine particles. Biochimica et Biophysica Acta 1201, 259–265 Tungittiplakorn W, Lion LW, Colhen C, Kim J-Y (2004): Engineered polymeric nanoparticles for soil remediation. Environ Sci Technol 38, 1605–1610
Received: March 2nd, 2006 Accepted: June 21st, 2006 OnlineFirst: June 22nd, 2006

References ASTM Standard D 6081 – 97 (1997): Aquatic Toxicity Testing of Lubricants: Sample Preparation and Results Interpretation. January 10, 1997 Auffan M, Decome L, Rose J, Orsiere T, De Meo M, Briosis V, Chaneac C, Olivi L, Berge-Iefranc J-L, Botta A, Wiesner MR, Botteroo J-Y (2006): In vitro interactions between DMSA-coated maghemite nanoparticles and human fibroblasts: A physiochemical and cyto-genotoxical study. Environ Sci Technol ASAP article 10.1021 Borm PJA, Schins RPF, Albrecht A (2004): Inhaled particles and lung cancer-paradigms and risk assessment. Int J Cancer 110, 3–14 CONCAWE (1992): Ecotoxicological testing of petroleum products: Test methodology. Report No. 92/56. CONCAWE, Madouplein 1, 1210 Brussels, Belgium COUNCIL DIRECTIVE 67/548/EEC: COUNCIL DIRECTIVE 67/ 548/EEC of 27 June 1967 on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances DIN 38412-30 (1989): German standard methods for the examination of water, waste water and sludge; bio-assays (group L), determining the tolerance of Daphnia to the toxicity of waste water by way of a dilution series (L 30) DIN 38412-33 (1991): German standard methods for the examination of water, waste water and sludge; bio-assays (group L); determining the tolerance of green algae to the toxicity of waste water (Scenedesmus chlorophyll fluorescence test) by way of dilution series (L 33) Duncan R (2003): The dawning era of polymer therapeutics. Nature Reviews 2, 347–360 Eisentr?ger A, Dott W, Klein J, Hahn S (2003): Comparative studies on algal toxicity testing using fluorometric microplate and Erlenmeyer flask growth-inhibition assays. Ecotoxicol Environ Saf 54, 346–54 Huang Z, Maness P-C, Blake D, Wolfrum E, Smolinski S, Jacoby W (2000): Bactericidal mode of titanium dioxide photocatalysis. J Photochem Photobiol A: Chemistry 130, 163–170 ISO 6341 (1996-04-00): Water quality – Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) – Acute toxicity test

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ESPR – Environ Sci & Pollut Res 2006 (OnlineFirst)



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