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The significance of nanomaterial post-exposure responses in Daphnia magna standard acute immobilisation assay: Example with testing TiO2 nanoparticles
Ecotoxicology and Environmental Safety 152 (2018) 61–66
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
The significance of nanomaterial post-exposure responses in Daphnia magna standard acute immobilisation assay: Example with testing TiO2 nanoparticles
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Sara Novaka, , Anita Jemec Kokalja, Matej Hočevarb, Matjaž Godecb, Damjana Drobnea a b
Department of Biology, Biotechnical Faculty, University of Ljubljana, 111, Jamnikarjeva 101,1000 Ljubljana, Slovenia Institute of Metals and Technology, Lepi pot 11, 1000 Ljubljana, Slovenia
A R T I C L E I N F O
A B S T R A C T
Keywords: Acute toxicity test TiO2 nanoparticles Adsorption Scanning electron microscopy Delayed effects
One of the most widely used aquatic standarized tests for the toxicity screening of chemicals is the acute toxicity test with the freshwater crustacean Daphnia magna, which has also been applied in the toxicity screening of manufactured nanoparticles (NPs). However, in the case of non-soluble NPs most of the results of this test have showed no effect. The aim of the work presented here was to modify the standardized test by the least possible extent to make it more sensitive for non-soluble particles. The standard acute immobilisation assay with daphnids was modified by prolonging the exposure period and by measuring additional endpoints. Daphnids were exposed to TiO2 NPs in a standard acute test (48 h of exposure), a standard acute test (48 h of exposure) followed by 24 h recovery period in clean medium or a prolonged exposure in the NPs solutions totaling 72 h. Together with immobility, the adsorption of NPs to body surfaces was also observed as an alternative measure of the NPs effects. Our results showed almost no effect of TiO2 NPs on D. magna after the 48 h standard acute test, while immobility was increased when the exposure period to TiO2 NPs was prolonged from 48 h to 72 h. Even when daphnids were transferred to clean medium for additional 24h after 48h of exposure to TiO2 NPs the immobility increased. We conclude that by transferring the daphnids to clean medium at the end of the 48 h exposure to TiO2 NPs, the delayed effects of the tested material can be seen. This methodological step could improve the sensitivity of D. magna test as a model in nanomaterial environmental risk assessment.
1. Introduction The standarized toxicity test with D. magna (ISO 6341:2014) fulfils criteria for the testing of different chemicals. This test is also a part of a regulatory framework in different countries. Recently, the test with D. magna has been applied to the toxicity screening of manufactured nanoparticles (ISO/TR 16197:2014). However, a shortcoming of this assay is its low sensitivity for non-soluble nanoparticles (NPs) (Bondarenko et al., 2016). The most produced and broadly applied nanomaterial is titanium dioxide (TiO2), with up to 7800–38,000 t/year produced in the United States alone (Piccinno et al., 2012; Keller and Lazareva, 2014). It is used in a variety of products including sunscreens, clothing, paints, coatings, electronics, cosmetics, medicine, as well as in the environmental decontamination of air, soil, and water (Adam et al., 2015). Due to the large quantities of TiO2 NPs being produced, it can be expected in the environment (Gottschalk et al., 2009). In freshwater, TiO2 NPs have
been detected in concentrations in the range of a few mg/L following runoff from painted-house facades (Kaegi et al., 2008). In Europe, the highest levels of Ti are expected in landfill and soil (Keller and Lazareva, 2014; Coll et al., 2016). Bondarenko et al. (2016) published an extensive comparative study on 15 bioassays using a variety of NPs and found that only one test was ranking TiO2 NPs as potentially adverse due to the ‘‘entrapment’’ of algal cells into TiO2 NP agglomerates. These authors have reported a suitability of the D. magna test for NPs prone to dissolution (Ag, CuO and ZnO). No effect of TiO2 NPs on D. magna up to 100 mg/L of exposure concentration was found by several authors (Heinlaan et al., 2008; Cupi et al., 2015; Lovern and Klaper, 2006; Strigul et al., 2009; Zhu et al., 2009; Wiench et al., 2009), however, some authors are reporting variable 24 h EC50 values, ranging from 7.6 to 143.4 mg/L (Li et al., 2017). The high variability of the toxicological results is dependent on different TiO2 NP properties as well as different exposure protocols used in the studies (Li et al., 2017). In the work of Garcia et al.
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Corresponding author. E-mail addresses: [email protected] (S. Novak), [email protected] (A. Jemec Kokalj), [email protected] (M. Hočevar), [email protected] (M. Godec), [email protected] (D. Drobne). https://doi.org/10.1016/j.ecoenv.2018.01.007 Received 23 August 2017; Received in revised form 11 December 2017; Accepted 3 January 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.
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drying the suspension of NPs in dilution water used for experiments at room temperature on a transparent carbon foil supported on a copper grid. Dynamic light scattering (DLS) measurements of the hydrodynamic sizes of particles in the dilution water (100 mg/L) were performed using a Fritsch Analysette 12 DynaSizer (Idar-Oberstein, Germany). The ζ-potentials of the suspended TiO2 NPs in dilution water were measured with a Brookhaven Instruments Corp., ZetaPALS (NY, USA).
(2011) and Lee et al. (2009) where the genotoxic effect of TiO2 NPs on daphnids was studied, the authors concluded that the TiO2 NPs were practically inert for D. magna. Amiano et al. (2012) also reported no effect of TiO2 NPs under darkness, while UVA irradiated TiO2 NPs did have adverse effects for D. magna. Das et al. (2013) provided evidence that the 48 h LC50 value for uncapped TiO2 NPs was 7.75 mg/L and no mortality was observed when daphnids were exposed to carboxyfunctionalized capped TiO2 NPs at concentrations up to 30 mg/L. Many researchers have suggested improvements to the D. magna test to increase its relevance and sensitivity for different NPs. Dabrunz et al. (2011) proposed a prolonged exposure of D. magna from the standard 48 h exposure period to a 72 h and 96 h exposure durations which detected effects of TiO2 not observed at shorter exposures (Dabrunz et al., 2011) (72 h EC50 = 3.8. mg/L and 96 h EC50 = 0.73 mg/L). Zhu et al. (2010) also concluded that TiO2 NPs exerted minimal toxicity to daphnia within the 48 h exposure time (NOEC less than 50 mg/L, 48 h EC50 and LC50 values greater than 100 mg/L), but when the exposure time was extended to 72 h (72 h EC50 = 1.62 mg/L; 72 h LC50 = 2.02 mg/L) adverse effects were evident. The prolonged exposures in test with daphnids were also conducted with CeO2 NPs, which were not acutely toxic at concentrations up to 10 mg/L in the terms of mortality but they caused inhibition of molting and growth of the daphnids (Gaiser et al., 2011). In the standard acute D. magna toxicity test, the assessed endpoint selected for the evaluation of the toxic impact is the “immobility” criterion, i.e. the inability of the test organisms to resume swimming within 15 s after gentle agitation. In the case of NP exposure, it is well reported that nanomaterials adsorb onto the animals body surface and appendages which could be the mode of action that causes the observed immobility (Asghari et al., 2012; Artells et al., 2013; Nowack and Bucheli, 2007; Baumann et al., 2014; Baun et al., 2008; Dabrunz et al., 2011; Gaiser et al., 2011; Li et al., 2017). In addition, adhesion of material to body surfaces may also lead to the disruption of the molting process (Asghari et al., 2012; Artells et al., 2013; Baumann et al., 2014; Baun et al., 2008; Dabrunz et al., 2011; Gaiser et al., 2011; Nowack and Bucheli, 2007). Many studies have provided evidence that ingested material filled the gut which might then interfere with digestive processes (Asghari et al., 2012; Kwon et al., 2015). A detailed ultrastructural analyses of the gut epithelium of D. magna exposed to TiO2 NPs revealed that TiO2 NPs did not pass into the epithelium cells and were present only in the gut lumen (Kwon et al., 2015). Our study tested if immobilisation of daphnids changed in a postexposure period in clean medium and whether this modification could be implemented as a Supplementary endpoint to immobilisation after the standard 48 h test with D. magna. In addition, molt frequency alterations and adsorption of the tested NPs on the animal body surface were observed. The aim of this study was to provide an alternative way to measure the effects of NPs while changing the standard tests as little as possible. Daphnids were exposed under three different scenarios: (i) standard acute test (48 h of exposure), (ii) standard acute test (48 h of exposure) followed by 24 h recovery period in clean medium and (iii) prolonged (72 h) exposure in the NP solution. In each of the exposure scenarios, immobilisation, adsorption of NPs onto the animal's body surface and retention of material in the gut were investigated. Molting of individual daphnids was followed as well.
2.2. Test organism Adult water fleas (Daphnia magna Straus 1820) were obtained from the National Institute of Chemical Physics and Biophysics, Tallinn, Estonia. Daphnids were held in 4 L aquariums containing 2 L of modified M4 media at a constant room temperature of 21 ± 1 °C and the 16:8 h light/dark photoperiod. Daphnids were fed a diet of the pure algae Spirulina platensis dry powder (PET, Dajana professional, Czech republic) corresponding to 0.15 mg carbon/daphnia per day. 2.3. Toxicity tests with Daphnia magna All acute toxicity tests were carried out according to EN ISO 6341:2014. Each experiment was carried out in three parallels. Neonates less than 24 h old were transferred into the glass test containers with increasing concentrations of nanoparticles. Tested concentrations tested were 1, 10 and 100 mg/L TiO2 NPs. In the standard acute tests also two lower concentrations were tested, 0.1 mg/L and 0.5 mg/L, however as there was no effect on immobility these two concentrations were not tested in prolonged experiments. Controls containing only ISO dilution water (defined in EN ISO 6341: 2014) without NPs were included in all experiments. In individual experiment 20 animals for each test concentration were divided in four groups of 5 animals. For 5 animals 10 mL of test solution was provided. For the observation of adsorption of NPs on the surface of animals with scanning electron microscope (SEM) some additional animals were exposed in each group in one of the experiments. In one additional experiment daphnids (20 animals per group in two parallels) were exposed only to natural organic matter (NOM) with concentrations 10 and 100 mg/L in dilution water in order to observe the differences between body absorption of NOM and NPs. This additional control was included to investigate if the extent of NPs adsorption is similar as you would expect in the natural environment by NOM. The exposure time to NPs was 48 h and after that the animals were transferred to ISO dilution water for 24 h to depurate the gut or transferred to freshly prepared NPs suspensions to extend the exposure for additional 24 h (Fig. 1). In one additional experiment daphnids were exposed individually in the microtiter plates (12 well) in order to observe the molting. In that one well individual animal was exposed in 2 mL of suspension or ISO dilution water. 40 pre-fed neonates per exposure group were exposed individually to TiO2 NPs for 48 h, and then 20 animals per group were transferred to ISO dilution water for 24 h or to freshly prepared NPs suspensions for 24 h. The presence of exuvia in each well was observed in several time points using stereomicroscope. During the experiment all test containers were maintained in a temperature controlled chamber (21 ± 1 °C) on 16:8 h light/dark photoperiod. After exposures daphnids were inspected for immobility and the results were presented as the percentage of immobile animals for each concentration. The sensitivity of the laboratory cultures was determined with exposing the animals for 24 h to potassium dichromate (K2Cr2O7) in dilution water according to ISO 6341:2014.
2. Material and methods 2.1. Nanoparticle suspensions characterization We received the tested TiO2 NPs as a pigment and we are not allowed to disclose the name of the producer. According to the manufacturer the TiO2 NPs were in anatase form, with crystallite size ~5 nm and without coating. Transmission electron microscopy (TEM) of all suspensions in dilution water were done using a JEOL 2100 (Tokyo, Japan), operated at 200 kV. The specimens for TEM were prepared by
2.4. Adsorbtion of NPs on daphnids body surface and gut retention After the TiO2 NPs and NOM exposure daphnids were chemically fixated and prepared for observations of adsorbed material on their 62
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Fig. 1. Scheme of the experimental set-up. Daphnids were exposed in group when immobility, NPs body absorbtion and gut retention were observed. When molting of daphnids was investigated daphnids were exposed individually in 12-well plates. As no effect of TiO2 NPs was observed, the test was prolonged for additional 24 h in fresh TiO2 NPs suspensions and in ISO dilution water (EN ISO 6341: 2014). NOM refers to natural organic matter.
body surface. Immediately after the exposure animals were transferred to 70% ethanol and fullness of the gut and the surface of animals were observed with light microscope (Axioimager.Z1 fluorescent microscope, Zeiss). For scanning electron microscopy (SEM) animals were incubated in modified Karnovski fixative (2.5% glutaraldehyde and 4% paraformaldehyde in the phosphate buffer) for 24 h on 4 °C. The samples were then dehydrated in series of alcohol (30, 50, 70, 80, 90% and absolute ethanol) and chemically dried with hexamethyldisilazane. Daphnids were then transferred to aluminium SEM holders on the carbon tape, sputtered with 7 nm gold-palladium (Sputter coater SCD 050, BAL-TEC) and investigated by SEM/ Energy dispersive X-ray analysis (EDX) (Jeol JSM-6500F equipped with EDX Oxford Instruments INCA X-SIGHT LH2 type detector and INCA ENERGY 450 software). The micrographs were taken on several body surface areas to assess how large surface is covered with the NPs or NOM. EDX on unsputtered samples was used to analyse the chemical composition of selected parts of the animal body surface. 15 kV accelerating voltage of primary electron beam for imaging and EDX analysis was used.
Fig. 2. Immobility of daphnids in acute toxicity experiments with TiO2 NPs after 24 h and 48 h of exposure and after prolonged exposure (24 h post-exposure) in ISO dilution water (DW) (A) or in fresh suspensions of TiO2 NPs (B). Results of three experiments in which 20 animals per group were exposed are combined.
NPs only a few animals were immobilised. The percentage of immobilised animals was not more than 20% after 24 h and 48 h of exposure in any of the experiments. At concentrations as high as 100 mg/ L TiO2 NPs did not show any toxic effects on D. magna, therefore an EC50 was not calculated. However, an additional 24 h in ISO dilution water without NPs and in the freshly prepared NP suspensions caused a significant increase of immobilisation at higher exposure concentrations (Fig. 2). EC50 values were not calculated, because only three concentrations were tested. The NOEC in the case of post-exposure in fresh NPs suspensions and in ISO dilution water was 1 mg/L and the LOEC was in both cases 10 mg/L. When daphnids were exposed to NOM, there was no immobilisation observed after the 48 h acute test or after the prolonged period (24 h) in the ISO dilution water or in freshly prepared NOM up to 100 mg/L. Also the survival of controls was not affected after 24 h prolonged exposure in ISO dilution water. The EC50 value for reference chemical K2Cr2O7 was 0.9 mg/L, which is in accordance with EN ISO 6341:2014 (24 h EC50 is 0.6 −1.7 mg/L). Daphnids used in the experiments were therefore of an appropriate sensitivity and the tests were valid. Similar to our work, low biological reactivity of TiO2 NPs after 48 h of exposure has been reported by several authors (Cupi et al., 2015; Griffitt et al., 2008; Rosenkranz, 2010; Strigul et al., 2009; Van Hoecke et al., 2009; Warheit et al., 2007) while after prolonged exposure (72 h or 96 h) the pronounced effect of TiO2 NPs was evidenced (Zhu et al., 2010; Dabrunz et al., 2011). In our study, the same percentage of immobilized animals was recorded in the case of transfer of animals to ISO dilution water and when the exposure period in TiO2 NPs was
2.5. Data analyses The EC50 value for reference chemical K2Cr2O7 was calculated from the concentration-effect curves by the application of the lognormal model using REGTOX software for Microtox Excel™ (MS Excel macro REGTOX EV7.0.5.xls) which is available online at: http://eric. vindimian.9online.fr/. For calculation of EC50 values the Hill-model (non-linear fit) was used. 3. Results and discussion 3.1. Nanoparticle characteristic Transmission electron microscopy (TEM) showed the anatase crystallite form of TiO2 NPs (Fig. 1, Supplementary material). The DLS measurements were performed on the suspension with the highest concentration used in the tests, 100 mg of NPs/L of ISO dilution water. Results showed that the hydrodynamic size of TiO2 NPs was 375 nm. The ζ-potentials was −17.0 ± 2.4 mV. 3.2. Daphnia magna immobilisation After 24 h and 48 h of acute exposure to 1, 10 and 100 mg/L TiO2 63
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surface was already covered with TiO2 NP aggregates and the same was observed after both the 48 h and the prolonged exposure experiments. The strongly adsorbed aggregates were also seen on animals that have been moved into ISO dilution water without NPs in 24 h post-exposure period (Fig. 5). In the case of NOM exposure there were no particles attached on the surface of the animals up to 100 mg/L (Fig. 2, Supplementary material). Some authors have suggested that the adsorption of NPs to the daphnia body surface is responsible for the post exposure effects mainly due to molt disruption (Baumann et al., 2014; Dabrunz et al., 2011; Gaiser et al., 2011). This has been shown for exposure to TiO2 NPs (Dabrunz et al., 2011), iron oxide NPs (Baumann et al., 2014), CeO2 NPs (Gaiser et al., 2011), and spherical and rod shaped gold NPs (Nasser et al., 2016). However, in our work, a strong adsorption of TiO2 NPs on a large portion of daphnids body surface at exposure concentration 10 and 100 mg/L was revealed, but molting was not altered. Namely, the majority of the animals molted between 3 and 24 h, with no significant differences among the exposure groups but second molting was not observed until 72 h even in controls. This latter finding is in line with literature data (Gaiser et al., 2011) as the second molting under normal feeding conditions can take up to 96 h. A similar pattern of adsorption was observed in all exposure scenarios including postexposure in clean medium, i.e. ISO dilution water. The adsorbed NPs were present until animals underwent the first molting, however new material was adsorbed immediately after the cuticle was renewed. Additionally, even after the 24 h post-exposure period in the clean medium NPs remained adsorbed on the daphnids. To conclude, the adsorption of TiO2 onto the body surface of daphnids occurs instantly after NP exposure and no direct link to molt disruption could be established in our study, so this could not be the only reason for the observed immobility. Another cause of observed post-exposure effects could be gut retention of the NPs in the post exposure cleaning period. It was reported that in the 24 h after the acute exposure period to NPs, daphnids were not able to fully purge NPs from their guts (Petersen et al., 2015; Skjolding et al., 2014; Tervonen et al., 2010). We have also observed that daphnids did not clear theigut content in the 24 h post exposure in the clean media presumably causing delayed effects of TiO2 NPs. The same effect was observed in the work of Jemec et al. (2016), where daphnids were fed on microplastics (MP) following the same
prolonged. This means that daphnids were not able to recover in clean medium after. 48 h of exposure to TiO2 NPs. Other authors have also observed simmilar effect (Blinova et al., 2015; Volker et al., 2013). Similar to our study, many authors have discussed that the effects of different chemicals and NPs on daphnids could not be observed before the transfer from test medium to clean medium (Andersen et al., 2006; Jemec et al., 2016; McWilliam and Baird, 2002; Taylor et al., 1998; Villarroel et al., 1999). Taylor et al. (1998) found that D. magna previously exposed to sublethal concentrations of cadmium exhibited a persistent feeding depression after transfer to uncontaminated medium. The same experimental set up as our study was used by Jemec et al. (2016), where daphnids were fed microplastics (MP). Daphnids were not able to recover from 48 h MP exposure after an additional 24 h incubation period in a MP free medium with algae. According to the results of the present study with low-soluble NPs, we propose prolonging the standard acute test by an additional 24 h during which daphnids are moved to ISO dilution water to observe the possible delayed effects of NPs post exposure.
3.3. Absorption of TiO2 NPs on D.magna body surface, molting of daphnids and retention of NPs in the gut The adsorption of NPs on daphnids exposed to 1, 10 and 100 mg/L TiO2 NPs was inspected under light and electron microscope after 24 h, 48 h and after 24 h prolonged period of exposure in freshly prepared NPs suspensions and in the ISO dilution water without NPs. The control animals and animals exposed to 10 and 100 mg/L of NOM were also inspected. In the case of NP exposure the aggregates of adsorbed material can be seen with light microscope (Fig. 3); however, the percentage of absorbed material on body surface cannot be assessed. Together with adsorption of material on the surface, the gut content was observed with light microscopy in the post-exposure and prolonged exposure period, in all animals at least some fragments of food was still seen in the gut. Using SEM, we observed that on the surface of the animals exposed with up to 1 mg/L TiO2 NPs only a few aggregates were adsorbed, mainly on the head. However, almost the whole body surface of the daphnids exposed to 10 and 100 mg/L was covered with the Ti aggregates (Fig. 4). The EDX analyses confirmed that the observed material is Ti (Fig. 5). After 24 h of exposure to NPs, the larger part of body
Fig. 3. Light microscopy images of control D. magna (A), D.magna exposed to NOM (100 mg/L) (B), D. magna exposed to 10 mg/L of TiO2 NPs (C) and 100 mg/L of TiO2 NPs (D). Please note attached material on the surface of TiO2 NPs exposed animals (red arrow). After three individual experiments at least 5 animals per exposure concentration were investigated. G – gut. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. SEM images of D. magna to TiO2 NPs with concentrations 1, 10 and 100 mg/L for 48 h and D. magna after post-exposed in ISO dilution water for 24 h (previously exposed to 100 mg TiO2 NPs/L). On each exposed animal, aggregates of NPs can be seen. The largest amounts of absorbed material were observed when animals were exposed to the highest concentration (100 mg/L). After post-exposure in water (in the right square) the aggregates were still attached onto the body surface and can be seen attached to the cuticle. After three individual experiments at least 2 animals per exposure concentration were investigated.
specific adsorption properties which cause the observed effects on daphnids. It seems that the extent of adsorption that was observed in the case of TiO2 NPs is not similar to you would expect in nature by NOM. To conclude, the standard acute D. magna imobilisation test (ISO 6341:2014) for assesing the effects of NPs with low dissolution potential can be more reliable with adding some aditional steps in the existing protocol. This modification includes new steps detailed as follows: a) post-exposure observation of daphnids with transfer of animals to clean medium for an additional 24 h and recording of immobility, b) observing the surface adsorption of NPs and c) observing presence of particles in the gut. These test modifications are based on NP specific
experimental set up as in study presented here. Daphnids were not able to recover from 48 h MP exposure after an additional 24 h incubation period in a MP free medium with algae. Jemec et al. (2016) concluded that the plastic attachment on the surface together with ingested MP affect the survival of daphnids during the post-exposure period. This leads to the conclusion that the increased immobility of daphnids seen upon TiO2 NP exposure was most probably a result of a joint action of NP adsorption and NP gut retention. The observed effect of NPs were not seen in the case of NOM. In the same exposure scenarios (acute test and post-exposure in fresh suspensions and in the clean medium), we did not observe any increase of immobility or any absorption of the NOM on the body surface of animals. This means, that TiO2 NPs possess
Fig. 5. SEM image of a part of the D. magna cuticle with adsorbed material after 24 h exposure to 100 mg TiO2 NPs/L. Corresponding EDX elemental maps are showing composition and phase distribution of O, Na, Ca, P and Ti elements. We can see that TiO2 NPs are evenly distributed across the whole observed surface. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article
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characteristic, such as adsorption, therefore the relevance of the modified D. magna immobilisation test is improved. This is of significant importance for nanomaterial environmental risk assessment.
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Acknowledgements We thank Alenka Malovrh for her contribution. We thank Craig Mayall for English language editing. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2018.01.007. References Adam, V., Loyaux-Lawniczak, S., Quaranta, G., 2015. Characterization of engineered TiO2 nanomaterials in a life cycle and risk assessments perspective. Environ. Sci. Poll. Res. 22, 11175–11192. Amiano, I., Olabarrieta, J., Vitorica, J., Zorita, S., 2012. Acute toxicity of nanosized TiO2 to Daphnia magna under UVA irradiation. Environ. Toxicol. Chem. 31, 2564–2566. Andersen, T.H., Tjornhoj, R., Wollenberger, L., Slothuus, T., Baun, A., 2006. Acute and chronic effects of pulse exposure of Daphnia magna to dimethoate and pirimicarb. Environ. Toxicol. Chem. 25, 1187–1195. Artells, E., Issartel, J., Auffan, M., Borschneck, D., Thill, A., Tella, M., Brousset, L., Rose, J., Bottero, J.Y., Thiery, A., 2013. Exposure to cerium dioxide nanoparticles differently affect swimming performance and survival in two daphnid species. Plos One 8. Asghari, S., Johari, S.A., Lee, J.H., Kim, Y.S., Jeon, Y.B., Choi, H.J., Moon, M.C., Yu, I.J., 2012. Toxicity of various silver nanoparticles compared to silver ions in Daphnia magna. J. Nanobiotechnol. 10. Baumann, J., Koser, J., Arndt, D., Filser, J., 2014. The coating makes the difference: acute effects of iron oxide nanoparticles on Daphnia magna. Sci. Total Environ. 484, 176–184. Baun, A., Hartmann, N.B., Grieger, K., Kusk, K.O., 2008. Ecotoxicity of engineered nanoparticles to aquatic invertebrates: a brief review and recommendations for future toxicity testing. Ecotoxicology 17, 387–395. Blinova, I., Kanarbik, L., Irha, N., Kahru, A., 2015. Ecotoxicity of nanosized magnetite to crustacean Daphnia magna and duckweed Lemna minor. Hydrobiologia 1–9. Bondarenko, O.M., Heinlaan, M., Sihtmae, M., Ivask, A., Kurvet, I., Joonas, E., Jemec, A., Mannerstrom, M., Heinonen, T., Rekulapelly, R., Singh, S., Zou, J., Pyykko, I., Drobne, D., Kahru, A., 2016. Multilaboratory evaluation of 15 bioassays for (eco) toxicity screening and hazard ranking of engineered nanomaterials: FP7 project NANOVALID. Nanotoxicology 10, 1229–1242. Coll, C., Notter, D., Gottschalk, F., Sun, T., Som, C., Nowack, B., 2016. Probabilistic environmental risk assessment of five nanomaterials (nano-TiO2, nano-Ag, nano-ZnO, CNT, and fullerenes). Nanotoxicology 10, 436 (1205-1205). Cupi, D., Hartmann, N.B., Baun, A., 2015. The influence of natural organic matter and aging on suspension stability in guideline toxicity testing of silver, zinc oxide, and titanium dioxide nanoparticles with Daphnia magna. Environ. Toxicol. Chem. 34, 497–506. Dabrunz, A., Duester, L., Prasse, C., Seitz, F., Rosenfeldt, R., Schilde, C., Schaumann, G.E., Schulz, R., 2011. Biological surface coating and molting inhibition as mechanisms of TiO2 nanoparticle toxicity in Daphnia magna. Plos One 6. Das, P., Xenopoulos, M.A., Metcalfe, C.D., 2013. Toxicity of silver and titanium dioxide nanoparticle suspensions to the aquatic invertebrate, Daphnia magna. Bull. Environ. Contam. Toxicol. 91, 76–82. Gaiser, B.K., Biswas, A., Rosenkranz, P., Jepson, M.A., Lead, J.R., Stone, V., Tyler, C.R., Fernandes, T.F., 2011. Effects of silver and cerium dioxide micro- and nano-sized particles on Daphnia magna. J. Environ. Monit. 13, 1227–1235. Garcia, A., Espinosa, R., Delgado, L., Casals, E., Gonzalez, E., Puntes, V., Barata, C., Font, X., Sanchez, A., 2011. Acute toxicity of cerium oxide, titanium oxide and iron oxide nanoparticles using standardized tests. Desalination 269, 136–141. Gottschalk, F., Sonderer, T., Scholz, R.W., Nowack, B., 2009. Modeled Environmental Concentrations of Engineered Nanomaterials (TiO2, ZnO, Ag, CNT, Fullerenes) for Different Regions. Environ. Sci. Technol. 43, 9216–9222. Griffitt, R.J., Luo, J., Gao, J., Bonzongo, J.C., Barber, D.S., 2008. Effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms. Environ. Toxicol. Chem. 27, 1972–1978. Heinlaan, M., Ivask, A., Blinova, I., Dubourguier, H.C., Kahru, A., 2008. Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus. Chemosphere 71, 1308–1316.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
The significance of nanomaterial post-exposure responses in Daphnia magna standard acute immobilisation assay: Example with testing TiO2 nanoparticles
T
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Sara Novaka, , Anita Jemec Kokalja, Matej Hočevarb, Matjaž Godecb, Damjana Drobnea a b
Department of Biology, Biotechnical Faculty, University of Ljubljana, 111, Jamnikarjeva 101,1000 Ljubljana, Slovenia Institute of Metals and Technology, Lepi pot 11, 1000 Ljubljana, Slovenia
A R T I C L E I N F O
A B S T R A C T
Keywords: Acute toxicity test TiO2 nanoparticles Adsorption Scanning electron microscopy Delayed effects
One of the most widely used aquatic standarized tests for the toxicity screening of chemicals is the acute toxicity test with the freshwater crustacean Daphnia magna, which has also been applied in the toxicity screening of manufactured nanoparticles (NPs). However, in the case of non-soluble NPs most of the results of this test have showed no effect. The aim of the work presented here was to modify the standardized test by the least possible extent to make it more sensitive for non-soluble particles. The standard acute immobilisation assay with daphnids was modified by prolonging the exposure period and by measuring additional endpoints. Daphnids were exposed to TiO2 NPs in a standard acute test (48 h of exposure), a standard acute test (48 h of exposure) followed by 24 h recovery period in clean medium or a prolonged exposure in the NPs solutions totaling 72 h. Together with immobility, the adsorption of NPs to body surfaces was also observed as an alternative measure of the NPs effects. Our results showed almost no effect of TiO2 NPs on D. magna after the 48 h standard acute test, while immobility was increased when the exposure period to TiO2 NPs was prolonged from 48 h to 72 h. Even when daphnids were transferred to clean medium for additional 24h after 48h of exposure to TiO2 NPs the immobility increased. We conclude that by transferring the daphnids to clean medium at the end of the 48 h exposure to TiO2 NPs, the delayed effects of the tested material can be seen. This methodological step could improve the sensitivity of D. magna test as a model in nanomaterial environmental risk assessment.
1. Introduction The standarized toxicity test with D. magna (ISO 6341:2014) fulfils criteria for the testing of different chemicals. This test is also a part of a regulatory framework in different countries. Recently, the test with D. magna has been applied to the toxicity screening of manufactured nanoparticles (ISO/TR 16197:2014). However, a shortcoming of this assay is its low sensitivity for non-soluble nanoparticles (NPs) (Bondarenko et al., 2016). The most produced and broadly applied nanomaterial is titanium dioxide (TiO2), with up to 7800–38,000 t/year produced in the United States alone (Piccinno et al., 2012; Keller and Lazareva, 2014). It is used in a variety of products including sunscreens, clothing, paints, coatings, electronics, cosmetics, medicine, as well as in the environmental decontamination of air, soil, and water (Adam et al., 2015). Due to the large quantities of TiO2 NPs being produced, it can be expected in the environment (Gottschalk et al., 2009). In freshwater, TiO2 NPs have
been detected in concentrations in the range of a few mg/L following runoff from painted-house facades (Kaegi et al., 2008). In Europe, the highest levels of Ti are expected in landfill and soil (Keller and Lazareva, 2014; Coll et al., 2016). Bondarenko et al. (2016) published an extensive comparative study on 15 bioassays using a variety of NPs and found that only one test was ranking TiO2 NPs as potentially adverse due to the ‘‘entrapment’’ of algal cells into TiO2 NP agglomerates. These authors have reported a suitability of the D. magna test for NPs prone to dissolution (Ag, CuO and ZnO). No effect of TiO2 NPs on D. magna up to 100 mg/L of exposure concentration was found by several authors (Heinlaan et al., 2008; Cupi et al., 2015; Lovern and Klaper, 2006; Strigul et al., 2009; Zhu et al., 2009; Wiench et al., 2009), however, some authors are reporting variable 24 h EC50 values, ranging from 7.6 to 143.4 mg/L (Li et al., 2017). The high variability of the toxicological results is dependent on different TiO2 NP properties as well as different exposure protocols used in the studies (Li et al., 2017). In the work of Garcia et al.
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Corresponding author. E-mail addresses: [email protected] (S. Novak), [email protected] (A. Jemec Kokalj), [email protected] (M. Hočevar), [email protected] (M. Godec), [email protected] (D. Drobne). https://doi.org/10.1016/j.ecoenv.2018.01.007 Received 23 August 2017; Received in revised form 11 December 2017; Accepted 3 January 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.
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drying the suspension of NPs in dilution water used for experiments at room temperature on a transparent carbon foil supported on a copper grid. Dynamic light scattering (DLS) measurements of the hydrodynamic sizes of particles in the dilution water (100 mg/L) were performed using a Fritsch Analysette 12 DynaSizer (Idar-Oberstein, Germany). The ζ-potentials of the suspended TiO2 NPs in dilution water were measured with a Brookhaven Instruments Corp., ZetaPALS (NY, USA).
(2011) and Lee et al. (2009) where the genotoxic effect of TiO2 NPs on daphnids was studied, the authors concluded that the TiO2 NPs were practically inert for D. magna. Amiano et al. (2012) also reported no effect of TiO2 NPs under darkness, while UVA irradiated TiO2 NPs did have adverse effects for D. magna. Das et al. (2013) provided evidence that the 48 h LC50 value for uncapped TiO2 NPs was 7.75 mg/L and no mortality was observed when daphnids were exposed to carboxyfunctionalized capped TiO2 NPs at concentrations up to 30 mg/L. Many researchers have suggested improvements to the D. magna test to increase its relevance and sensitivity for different NPs. Dabrunz et al. (2011) proposed a prolonged exposure of D. magna from the standard 48 h exposure period to a 72 h and 96 h exposure durations which detected effects of TiO2 not observed at shorter exposures (Dabrunz et al., 2011) (72 h EC50 = 3.8. mg/L and 96 h EC50 = 0.73 mg/L). Zhu et al. (2010) also concluded that TiO2 NPs exerted minimal toxicity to daphnia within the 48 h exposure time (NOEC less than 50 mg/L, 48 h EC50 and LC50 values greater than 100 mg/L), but when the exposure time was extended to 72 h (72 h EC50 = 1.62 mg/L; 72 h LC50 = 2.02 mg/L) adverse effects were evident. The prolonged exposures in test with daphnids were also conducted with CeO2 NPs, which were not acutely toxic at concentrations up to 10 mg/L in the terms of mortality but they caused inhibition of molting and growth of the daphnids (Gaiser et al., 2011). In the standard acute D. magna toxicity test, the assessed endpoint selected for the evaluation of the toxic impact is the “immobility” criterion, i.e. the inability of the test organisms to resume swimming within 15 s after gentle agitation. In the case of NP exposure, it is well reported that nanomaterials adsorb onto the animals body surface and appendages which could be the mode of action that causes the observed immobility (Asghari et al., 2012; Artells et al., 2013; Nowack and Bucheli, 2007; Baumann et al., 2014; Baun et al., 2008; Dabrunz et al., 2011; Gaiser et al., 2011; Li et al., 2017). In addition, adhesion of material to body surfaces may also lead to the disruption of the molting process (Asghari et al., 2012; Artells et al., 2013; Baumann et al., 2014; Baun et al., 2008; Dabrunz et al., 2011; Gaiser et al., 2011; Nowack and Bucheli, 2007). Many studies have provided evidence that ingested material filled the gut which might then interfere with digestive processes (Asghari et al., 2012; Kwon et al., 2015). A detailed ultrastructural analyses of the gut epithelium of D. magna exposed to TiO2 NPs revealed that TiO2 NPs did not pass into the epithelium cells and were present only in the gut lumen (Kwon et al., 2015). Our study tested if immobilisation of daphnids changed in a postexposure period in clean medium and whether this modification could be implemented as a Supplementary endpoint to immobilisation after the standard 48 h test with D. magna. In addition, molt frequency alterations and adsorption of the tested NPs on the animal body surface were observed. The aim of this study was to provide an alternative way to measure the effects of NPs while changing the standard tests as little as possible. Daphnids were exposed under three different scenarios: (i) standard acute test (48 h of exposure), (ii) standard acute test (48 h of exposure) followed by 24 h recovery period in clean medium and (iii) prolonged (72 h) exposure in the NP solution. In each of the exposure scenarios, immobilisation, adsorption of NPs onto the animal's body surface and retention of material in the gut were investigated. Molting of individual daphnids was followed as well.
2.2. Test organism Adult water fleas (Daphnia magna Straus 1820) were obtained from the National Institute of Chemical Physics and Biophysics, Tallinn, Estonia. Daphnids were held in 4 L aquariums containing 2 L of modified M4 media at a constant room temperature of 21 ± 1 °C and the 16:8 h light/dark photoperiod. Daphnids were fed a diet of the pure algae Spirulina platensis dry powder (PET, Dajana professional, Czech republic) corresponding to 0.15 mg carbon/daphnia per day. 2.3. Toxicity tests with Daphnia magna All acute toxicity tests were carried out according to EN ISO 6341:2014. Each experiment was carried out in three parallels. Neonates less than 24 h old were transferred into the glass test containers with increasing concentrations of nanoparticles. Tested concentrations tested were 1, 10 and 100 mg/L TiO2 NPs. In the standard acute tests also two lower concentrations were tested, 0.1 mg/L and 0.5 mg/L, however as there was no effect on immobility these two concentrations were not tested in prolonged experiments. Controls containing only ISO dilution water (defined in EN ISO 6341: 2014) without NPs were included in all experiments. In individual experiment 20 animals for each test concentration were divided in four groups of 5 animals. For 5 animals 10 mL of test solution was provided. For the observation of adsorption of NPs on the surface of animals with scanning electron microscope (SEM) some additional animals were exposed in each group in one of the experiments. In one additional experiment daphnids (20 animals per group in two parallels) were exposed only to natural organic matter (NOM) with concentrations 10 and 100 mg/L in dilution water in order to observe the differences between body absorption of NOM and NPs. This additional control was included to investigate if the extent of NPs adsorption is similar as you would expect in the natural environment by NOM. The exposure time to NPs was 48 h and after that the animals were transferred to ISO dilution water for 24 h to depurate the gut or transferred to freshly prepared NPs suspensions to extend the exposure for additional 24 h (Fig. 1). In one additional experiment daphnids were exposed individually in the microtiter plates (12 well) in order to observe the molting. In that one well individual animal was exposed in 2 mL of suspension or ISO dilution water. 40 pre-fed neonates per exposure group were exposed individually to TiO2 NPs for 48 h, and then 20 animals per group were transferred to ISO dilution water for 24 h or to freshly prepared NPs suspensions for 24 h. The presence of exuvia in each well was observed in several time points using stereomicroscope. During the experiment all test containers were maintained in a temperature controlled chamber (21 ± 1 °C) on 16:8 h light/dark photoperiod. After exposures daphnids were inspected for immobility and the results were presented as the percentage of immobile animals for each concentration. The sensitivity of the laboratory cultures was determined with exposing the animals for 24 h to potassium dichromate (K2Cr2O7) in dilution water according to ISO 6341:2014.
2. Material and methods 2.1. Nanoparticle suspensions characterization We received the tested TiO2 NPs as a pigment and we are not allowed to disclose the name of the producer. According to the manufacturer the TiO2 NPs were in anatase form, with crystallite size ~5 nm and without coating. Transmission electron microscopy (TEM) of all suspensions in dilution water were done using a JEOL 2100 (Tokyo, Japan), operated at 200 kV. The specimens for TEM were prepared by
2.4. Adsorbtion of NPs on daphnids body surface and gut retention After the TiO2 NPs and NOM exposure daphnids were chemically fixated and prepared for observations of adsorbed material on their 62
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Fig. 1. Scheme of the experimental set-up. Daphnids were exposed in group when immobility, NPs body absorbtion and gut retention were observed. When molting of daphnids was investigated daphnids were exposed individually in 12-well plates. As no effect of TiO2 NPs was observed, the test was prolonged for additional 24 h in fresh TiO2 NPs suspensions and in ISO dilution water (EN ISO 6341: 2014). NOM refers to natural organic matter.
body surface. Immediately after the exposure animals were transferred to 70% ethanol and fullness of the gut and the surface of animals were observed with light microscope (Axioimager.Z1 fluorescent microscope, Zeiss). For scanning electron microscopy (SEM) animals were incubated in modified Karnovski fixative (2.5% glutaraldehyde and 4% paraformaldehyde in the phosphate buffer) for 24 h on 4 °C. The samples were then dehydrated in series of alcohol (30, 50, 70, 80, 90% and absolute ethanol) and chemically dried with hexamethyldisilazane. Daphnids were then transferred to aluminium SEM holders on the carbon tape, sputtered with 7 nm gold-palladium (Sputter coater SCD 050, BAL-TEC) and investigated by SEM/ Energy dispersive X-ray analysis (EDX) (Jeol JSM-6500F equipped with EDX Oxford Instruments INCA X-SIGHT LH2 type detector and INCA ENERGY 450 software). The micrographs were taken on several body surface areas to assess how large surface is covered with the NPs or NOM. EDX on unsputtered samples was used to analyse the chemical composition of selected parts of the animal body surface. 15 kV accelerating voltage of primary electron beam for imaging and EDX analysis was used.
Fig. 2. Immobility of daphnids in acute toxicity experiments with TiO2 NPs after 24 h and 48 h of exposure and after prolonged exposure (24 h post-exposure) in ISO dilution water (DW) (A) or in fresh suspensions of TiO2 NPs (B). Results of three experiments in which 20 animals per group were exposed are combined.
NPs only a few animals were immobilised. The percentage of immobilised animals was not more than 20% after 24 h and 48 h of exposure in any of the experiments. At concentrations as high as 100 mg/ L TiO2 NPs did not show any toxic effects on D. magna, therefore an EC50 was not calculated. However, an additional 24 h in ISO dilution water without NPs and in the freshly prepared NP suspensions caused a significant increase of immobilisation at higher exposure concentrations (Fig. 2). EC50 values were not calculated, because only three concentrations were tested. The NOEC in the case of post-exposure in fresh NPs suspensions and in ISO dilution water was 1 mg/L and the LOEC was in both cases 10 mg/L. When daphnids were exposed to NOM, there was no immobilisation observed after the 48 h acute test or after the prolonged period (24 h) in the ISO dilution water or in freshly prepared NOM up to 100 mg/L. Also the survival of controls was not affected after 24 h prolonged exposure in ISO dilution water. The EC50 value for reference chemical K2Cr2O7 was 0.9 mg/L, which is in accordance with EN ISO 6341:2014 (24 h EC50 is 0.6 −1.7 mg/L). Daphnids used in the experiments were therefore of an appropriate sensitivity and the tests were valid. Similar to our work, low biological reactivity of TiO2 NPs after 48 h of exposure has been reported by several authors (Cupi et al., 2015; Griffitt et al., 2008; Rosenkranz, 2010; Strigul et al., 2009; Van Hoecke et al., 2009; Warheit et al., 2007) while after prolonged exposure (72 h or 96 h) the pronounced effect of TiO2 NPs was evidenced (Zhu et al., 2010; Dabrunz et al., 2011). In our study, the same percentage of immobilized animals was recorded in the case of transfer of animals to ISO dilution water and when the exposure period in TiO2 NPs was
2.5. Data analyses The EC50 value for reference chemical K2Cr2O7 was calculated from the concentration-effect curves by the application of the lognormal model using REGTOX software for Microtox Excel™ (MS Excel macro REGTOX EV7.0.5.xls) which is available online at: http://eric. vindimian.9online.fr/. For calculation of EC50 values the Hill-model (non-linear fit) was used. 3. Results and discussion 3.1. Nanoparticle characteristic Transmission electron microscopy (TEM) showed the anatase crystallite form of TiO2 NPs (Fig. 1, Supplementary material). The DLS measurements were performed on the suspension with the highest concentration used in the tests, 100 mg of NPs/L of ISO dilution water. Results showed that the hydrodynamic size of TiO2 NPs was 375 nm. The ζ-potentials was −17.0 ± 2.4 mV. 3.2. Daphnia magna immobilisation After 24 h and 48 h of acute exposure to 1, 10 and 100 mg/L TiO2 63
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surface was already covered with TiO2 NP aggregates and the same was observed after both the 48 h and the prolonged exposure experiments. The strongly adsorbed aggregates were also seen on animals that have been moved into ISO dilution water without NPs in 24 h post-exposure period (Fig. 5). In the case of NOM exposure there were no particles attached on the surface of the animals up to 100 mg/L (Fig. 2, Supplementary material). Some authors have suggested that the adsorption of NPs to the daphnia body surface is responsible for the post exposure effects mainly due to molt disruption (Baumann et al., 2014; Dabrunz et al., 2011; Gaiser et al., 2011). This has been shown for exposure to TiO2 NPs (Dabrunz et al., 2011), iron oxide NPs (Baumann et al., 2014), CeO2 NPs (Gaiser et al., 2011), and spherical and rod shaped gold NPs (Nasser et al., 2016). However, in our work, a strong adsorption of TiO2 NPs on a large portion of daphnids body surface at exposure concentration 10 and 100 mg/L was revealed, but molting was not altered. Namely, the majority of the animals molted between 3 and 24 h, with no significant differences among the exposure groups but second molting was not observed until 72 h even in controls. This latter finding is in line with literature data (Gaiser et al., 2011) as the second molting under normal feeding conditions can take up to 96 h. A similar pattern of adsorption was observed in all exposure scenarios including postexposure in clean medium, i.e. ISO dilution water. The adsorbed NPs were present until animals underwent the first molting, however new material was adsorbed immediately after the cuticle was renewed. Additionally, even after the 24 h post-exposure period in the clean medium NPs remained adsorbed on the daphnids. To conclude, the adsorption of TiO2 onto the body surface of daphnids occurs instantly after NP exposure and no direct link to molt disruption could be established in our study, so this could not be the only reason for the observed immobility. Another cause of observed post-exposure effects could be gut retention of the NPs in the post exposure cleaning period. It was reported that in the 24 h after the acute exposure period to NPs, daphnids were not able to fully purge NPs from their guts (Petersen et al., 2015; Skjolding et al., 2014; Tervonen et al., 2010). We have also observed that daphnids did not clear theigut content in the 24 h post exposure in the clean media presumably causing delayed effects of TiO2 NPs. The same effect was observed in the work of Jemec et al. (2016), where daphnids were fed on microplastics (MP) following the same
prolonged. This means that daphnids were not able to recover in clean medium after. 48 h of exposure to TiO2 NPs. Other authors have also observed simmilar effect (Blinova et al., 2015; Volker et al., 2013). Similar to our study, many authors have discussed that the effects of different chemicals and NPs on daphnids could not be observed before the transfer from test medium to clean medium (Andersen et al., 2006; Jemec et al., 2016; McWilliam and Baird, 2002; Taylor et al., 1998; Villarroel et al., 1999). Taylor et al. (1998) found that D. magna previously exposed to sublethal concentrations of cadmium exhibited a persistent feeding depression after transfer to uncontaminated medium. The same experimental set up as our study was used by Jemec et al. (2016), where daphnids were fed microplastics (MP). Daphnids were not able to recover from 48 h MP exposure after an additional 24 h incubation period in a MP free medium with algae. According to the results of the present study with low-soluble NPs, we propose prolonging the standard acute test by an additional 24 h during which daphnids are moved to ISO dilution water to observe the possible delayed effects of NPs post exposure.
3.3. Absorption of TiO2 NPs on D.magna body surface, molting of daphnids and retention of NPs in the gut The adsorption of NPs on daphnids exposed to 1, 10 and 100 mg/L TiO2 NPs was inspected under light and electron microscope after 24 h, 48 h and after 24 h prolonged period of exposure in freshly prepared NPs suspensions and in the ISO dilution water without NPs. The control animals and animals exposed to 10 and 100 mg/L of NOM were also inspected. In the case of NP exposure the aggregates of adsorbed material can be seen with light microscope (Fig. 3); however, the percentage of absorbed material on body surface cannot be assessed. Together with adsorption of material on the surface, the gut content was observed with light microscopy in the post-exposure and prolonged exposure period, in all animals at least some fragments of food was still seen in the gut. Using SEM, we observed that on the surface of the animals exposed with up to 1 mg/L TiO2 NPs only a few aggregates were adsorbed, mainly on the head. However, almost the whole body surface of the daphnids exposed to 10 and 100 mg/L was covered with the Ti aggregates (Fig. 4). The EDX analyses confirmed that the observed material is Ti (Fig. 5). After 24 h of exposure to NPs, the larger part of body
Fig. 3. Light microscopy images of control D. magna (A), D.magna exposed to NOM (100 mg/L) (B), D. magna exposed to 10 mg/L of TiO2 NPs (C) and 100 mg/L of TiO2 NPs (D). Please note attached material on the surface of TiO2 NPs exposed animals (red arrow). After three individual experiments at least 5 animals per exposure concentration were investigated. G – gut. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. SEM images of D. magna to TiO2 NPs with concentrations 1, 10 and 100 mg/L for 48 h and D. magna after post-exposed in ISO dilution water for 24 h (previously exposed to 100 mg TiO2 NPs/L). On each exposed animal, aggregates of NPs can be seen. The largest amounts of absorbed material were observed when animals were exposed to the highest concentration (100 mg/L). After post-exposure in water (in the right square) the aggregates were still attached onto the body surface and can be seen attached to the cuticle. After three individual experiments at least 2 animals per exposure concentration were investigated.
specific adsorption properties which cause the observed effects on daphnids. It seems that the extent of adsorption that was observed in the case of TiO2 NPs is not similar to you would expect in nature by NOM. To conclude, the standard acute D. magna imobilisation test (ISO 6341:2014) for assesing the effects of NPs with low dissolution potential can be more reliable with adding some aditional steps in the existing protocol. This modification includes new steps detailed as follows: a) post-exposure observation of daphnids with transfer of animals to clean medium for an additional 24 h and recording of immobility, b) observing the surface adsorption of NPs and c) observing presence of particles in the gut. These test modifications are based on NP specific
experimental set up as in study presented here. Daphnids were not able to recover from 48 h MP exposure after an additional 24 h incubation period in a MP free medium with algae. Jemec et al. (2016) concluded that the plastic attachment on the surface together with ingested MP affect the survival of daphnids during the post-exposure period. This leads to the conclusion that the increased immobility of daphnids seen upon TiO2 NP exposure was most probably a result of a joint action of NP adsorption and NP gut retention. The observed effect of NPs were not seen in the case of NOM. In the same exposure scenarios (acute test and post-exposure in fresh suspensions and in the clean medium), we did not observe any increase of immobility or any absorption of the NOM on the body surface of animals. This means, that TiO2 NPs possess
Fig. 5. SEM image of a part of the D. magna cuticle with adsorbed material after 24 h exposure to 100 mg TiO2 NPs/L. Corresponding EDX elemental maps are showing composition and phase distribution of O, Na, Ca, P and Ti elements. We can see that TiO2 NPs are evenly distributed across the whole observed surface. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article
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characteristic, such as adsorption, therefore the relevance of the modified D. magna immobilisation test is improved. This is of significant importance for nanomaterial environmental risk assessment.
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