Environmental Pollution 206 (2015) 17e23

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Fullerenes(nC60) affect the growth and development of the sediment-dwelling invertebrate Chironomus riparius larvae Greta C. Waissi-Leinonen a, *, Inna Nybom a, Kukka Pakarinen a, Jarkko Akkanen a, €nen b, Jussi V.K. Kukkonen c Matti T. Leppa a b c

Department of Biology, University of Eastern Finland, Joensuu, Finland €skyla €, Finland Finnish Environment Institute, Jyva €skyla €, Finland Department of Biological and Environmental Science, University of Jyva

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 April 2015 Received in revised form 29 May 2015 Accepted 1 June 2015 Available online xxx

The possible toxicity of nanoparticles (NPs) to aquatic organisms needs to be investigated for chronic effects at low concentrations. Chronic effects of carbon NPs, fullerenesC60, on the midges of Chironomus riparius at different life stages on larvae and adult midges were investigated. Sediment associated fullerenesC60 were studied by 10-day growth and 42-day emergence tests with artificial sediment at nominal concentration ranges 0.0004e80 mg/kg dry weight. The body length decreased in the lower tested concentrations (0.0025e20 mg/kg), but the effect vanished with higher concentrations. Delayed emergence rate observed at 0.5 mg/kg. The observed effects correlated with analyzed sediment particle sizes indicating that small agglomerates of fullerene have more significant effects on C. riparius than larger agglomerates observed with higher C60 doses. The results reveal that fullerene may pose risks to benthic organisms, emerging as changes in the ecotoxic parameters studied here which inflects by the survival of the population. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Carbon nanoparticle Nanoecotoxicology Nanotoxicity

1. Introduction The rapidly expanding usage of nanoparticles (NP) in consumer applications has led researchers to focus increasingly on the ecological risks of NPs (Klaine et al., 2008; Grieger et al., 2009; Benn et al., 2011). Recent studies have indicated that fullerenesC60 may have negative effects to organisms and other biological systems (Petersen and Henry, 2012; Waissi-Leinonen et al., 2012). For aquatic organisms this concern was raised by studies where negative influences were observed to have emerged as an outcome of ecotoxicological assays (Tervonen et al., 2010; Waissi-Leinonen et al., 2012; Pakarinen et al., 2011). Results in these studies have raised doubts whether current standardized methods designed for chemicals are suitable for assessing the ecological risks of nanoparticles (NPs) (Handy et al., 2012; Petersen, 2014). These stems from ecotoxicology assays, where fullerenes were observed to cause the immobility of the water flea Daphnia magna (Lovern and Klaper, 2006) and packing in the gut thus causing high

* Corresponding author. E-mail address: greta.waissi-leinonen@uef.fi (G.C. Waissi-Leinonen). http://dx.doi.org/10.1016/j.envpol.2015.06.010 0269-7491/© 2015 Elsevier Ltd. All rights reserved.

concentrations in the organisms’ wet mass basis (Tervonen et al., 2010). On the other hand, acute toxicity tests have reported that fullerenes cause only minor or negligible effects to aquatic organisms (Tervonen et al., 2010; Pakarinen et al., 2011; Fraser et al., 2011; Petersen and Henry, 2012). There is a lack of conclusive data on chronic exposures; some evidence on benthic invertebrates has been reported (Waissi-Leinonen et al., 2012; Pakarinen et al., 2011; Oberholster et al., 2011) and even fewer studies of chronic toxicity to water flea D. magna (Tao et al., 2009). The question of the chronic toxicity of fullerenes to benthic invertebrates covering low concentration ranges has not been solved. Sediments are known to be a major sink of contaminants; this most likely applies to nanomaterials (NMs) as well (Pakarinen et al., 2013; Gottschalk et al., 2009). Benthic invertebrates are an important link between the sediment and other trophic levels. Additionally, small concentrations of fullerenes have been already measured from wastewater effluents (Farre et al., 2010) and sedimentation has also been shown to have an impact on NPs (Pakarinen et al., 2013; Quik et al., 2014). There are several factors which influences on the fate of NPs in the aquatic ecosystems like dissolved organic matter (DOM) or other natural particles (Wang et al., 2011; Quik et al., 2012; Pakarinen et al., 2013). On the other

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hand DOM stabilizes NPs in water column but aggregate formation with colloids and larger natural particles will increase sedimentation. This indicates that NPs may end up in the sediments and, thus, benthic organisms are exposed. There are a few models of environmentally realistic concentrations, which we could treat as a prognosis about the possible exposure to fullerenes (Gottschalk et al., 2009; Sun et al., 2014; Gottschalk et al., 2013). Based on certain model estimations (Sun et al., 2014), the yearly increase of fullerene concentrations in the sediments in Europe is 0.0004 mg/ kg. These models however do not include factors like environmental modification and degradation which have an influence on accumulation potential of fullerenes. Chironomus riparius is commonly used in sediment ecotoxicology studies and is suitable for chronic toxicity tests (OECD, 2004). The species have numerous characteristics which make them valuable for toxicity tests: their short life cycle consists of the egg, larval (instars one to four) and pupal stage, as well as the adult stage. The larval stage is of special interest due to its complete ongoing metamorphosis during the instar phases, which plays an important role in the life cycle. Likewise, larvae are in contact with the sediment during the entire larval stage (Armittage et al., 1995; Goodyear and McNeill, 1999; Rasmussen, 1984; Pery et al., 2003; Taenzler et al., 2007) and thus growth and development parameters are suitable links to study possible effects of environmental stress. In our previous study, a thin fullerene layer was shown to hinder growth and cause damage to the microvilli (WaissiLeinonen et al., 2012). In addition, sediment spiked with fullerenes affected another benthic species Lumbiculus variegatus causing damage to the skin layer (Pakarinen et al., 2011). On the other hand, exposure concentrations used in these studies were relatively high and represented more hot spots than supposed environmental concentrations. Studies with lower exposure doses, the results of which are closer on modelled environmental concentrations, are highly necessary. In this study the ecotoxicity of the carbon NP, fullerenesC60, on the aquatic organism C. riparius was investigated. The aim was to study whether the fullerenesC60 have chronic effects on C. riparius's growth and development within a wide concentration range. Effects of fullerene in artificial matrix are needed to characterize first when assessing the potential effects to benthic invertebrates. Later on the influences of natural sediments can be analysed and discussed the influence of all other characteristics (e.g., pH, organic matter, ionic strength, etc.) which varies in the natural environment. The most used parameters for this organism were studied; mortality, body length, head capsule length and width, instar stage as well as dry mass which inflects to population survival potential. Ten concentrations ranging from 0.0004 to 80 mg/kg of sediment (dw) were chosen emphasis being at low environmentally relevant concentrations. In addition, the time for emergence was explored; the whole life cycle from the first instars of the larva stage up to the midge adults' stage was covered with four different concentrations determined with growth experiments. The goals in this experiment were to follow ecotoxicological parameters such as mortality, emergence rate and female:male proportions. The effects of fullerenes to the gut epithelia were studied with electron microscopy, and microvilli length was analysed after 10 days of exposure. 2. Materials and methods 2.1. Preparation and characterization of aqueous nC60 suspensions Artificial freshwater (AFW) was prepared by adding inorganic salts (Ca þ Mg hardness 0.5 mmol/L, pH 6.5e7 corresponding soft fresh waters) to MQ water (>18.2 mU) (detailed information in Tervonen et al., 2010). The suspensions of nC60 were made by

adding approximately 400 mg of crystalline fullerene (C60) powder (99.5%) (SigmaeAldrich, USA) to 2 L of artificial freshwater and stirring with a magnetic stir plate (at 1000 rpm) for 2 weeks in amber jars covered with aluminium foil to prevent interactions between light and fullerenes (Hwang and Li, 2010). Concentrations of the suspension were determined based on UVeVIS absorption at 335 nm with the standard curve (Cary 50BIO Mulgrave, Australia) (SI Fig. S1). The standard curve was linear at concentrations 0.8e52 mg/L (seven points, r2 > 0.999). Fullerene (C60) powder was additionally analysed for metal traces with the aid of inductively coupled plasma optical emission spectrometry (ICP-OES, IRIS Intrepid ll XSP) measurements, and only a few metal elements were observed (SI Table S1). The analyses were done to reveal potential impurities that could disturb the toxicity results (Petersen et al., 2012; Petersen, 2014). For instance, in some studies metal impurities, for instance nickel and yttrium, have been shown to cause toxic effects rather than the studied carbon nanotubes (Liu et al., 2007; Jakubek et al., 2009). The fullerene agglomerates were characterised with transmission electron microscopy (TEM, Zeiss 900, West Germany, 50 kV incident beam energy) by measuring the diameters (the largest dimension) of the agglomerates, and the results were confirmed with dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments). Additionally, the surface charge of the C60 suspension (Zeta potential) was analysed. For TEM analyses, the stock suspension were diluted (1:20) with artificial freshwater to help decrease particle aggregation during the drying of the grids, and 8 mL of the dilution was added to a Formvar (SPI supplies US) polyvinyl resin-coated 150-mesh copper grid (Leica Wetzlar Germany). Before the TEM analysis (magnification range 3e85 kx), the grids were air dried for one hour and left in a desiccator overnight; three grids with the fullerene suspension used in the experiments were analysed. Particle size distribution measurements were conducted using the imaging program MegaVision by AnalySIS (n ¼ 700) (S1 Fig. S2). 2.2. Preparation of the test sediment The artificial sediment used in this study was made according to the OECD method 218 (OECD, 2004) by mixing 20% of kaolinite clay, 75% of quartz sand (50% of the particles in the range of 50e200 mm), and 5% of peat powder (Sphagnum sp.) on a dry mass basis and the pH of the sediment was adjusted to 7.0 with CaCO3. The food source used in this study (0.5% Urtica sp. dw, particle size 60% and temperature change did not vary by more than ±1  C). The ammonium (NH4þ) content was measured from a composite sample at the end of the experiment. The ammonium content did not rise to a level (>18 mg/L) which could be harmful to a larva and its development (Egeler et al., 2010). The sediment pH was checked at the beginning and end of the experiments. The fullerene concentration in the overlying water was

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determined from the extraction of a 1 ml water sample collected at the end of the experiment day and analysed with a UVeVIS spectrophotometer. The fullerene concentration in the overlying water at the end of the 10th day of exposure without larva present was checked via a similar extraction in the concentrations of 10, 40 and 80 mg/kg (n ¼ 5). Results were compared to the measurements based on the growth experiments’ water concentration analyses, where larvae were present and caused some mixing between the sediment and water phase. The fullerene concentration remained stable in the overlying water without the larvae present (SI Table S3). 2.6. Growth experiments Altogether, ten concentrations (ranges 0.0004e80 mg/kg) and controls were included (S1 Table S2). Due to the large number of replicates and concentrations, the experiments were started separately in sets of three or four concentrations at the same time. Each experimental set had its own control. Two concentrations (1 and 40 mg/kg) were duplicated separately to ensure result repeatability. At day 10 larvae were sieved out from the sediment (ø 200-mm sieve), counted and preserved in ethanol (94%). Afterwards, they were analysed using a stereomicroscope (Nikon SMS 800) to measure body length, head capsule length and width. Larvae dry mass (it was heated at 100  C until no further mass changes occurred) was measured using a microbalance (Sartorius 4503 micro). The exact measurement data are shown in SI Table S4. For TEM analysis, five larvae from each concentration were collected, while the middle part of the larvae was used and examined (concentrations > 0.5 mg/kg). The preparation was made as described elsewhere (Waissi-Leinonen et al., 2012). 2.7. Emergence experiment In the emergence experiment, the concentration range was selected based on the results of the growth data. Thus, four concentrations and controls were included, and experimental setups were performed as previously (S1 Table S2). Moreover the lowest used concentration (0.5 mg/kg) and control were repeated. Because experiments were over 10 days long, larvae were fed with TetraMin

Fig. 1. Mean sediment particle size distribution in control and fullerenes spiked sediments. Whiskers indicate standard deviation, asterisks point out significant differences from untreated sediments (Dunn's Multiple Comparison test, **p < 0.01, ***p < 0.001) (n ¼ 400).

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0.12 mg/larva/day (Ristola et al., 1999) for midge bioassays. Larvae were fed three times a week by stopping aerations and adding the TetraMin-suspension gently to the sediment surface. A net was placed above each container to avoid the adult midges from escaping. Containers were checked daily and emerged adults were collected, counted and their dry weight was measured using a microbalance (the adults were heated at 40  C until no further mass changes occurred). Their sex was determined based on the form of their antennae. 2.8. Calculation and statistics Data was analysed with GraphPad Prism 5 (Graphpad software) and SPSS 19 for Windows. Larval growth was expressed relative to the control group. The growth was analysed with the one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison post-hoc test. The normality of the data was tested with the ShapiroeWilk test for normality, if data was not normally distributed; and the KruskaleWallis test followed by Dunn's multiple comparison post-hoc test was performed. Results were deemed significant if p < 0.05 and are marked as * p < 0.05, **p < 0.01, ***p < 0.001. The linear regression was made between the average sediment particle size distribution and the average body length (0.0004e0.1 mg/kg and 0.05e80 mg/kg) of larvae. The emergence data was tested by comparing survival curves using the Log-rank (ManteleCox) test and the sex ratio of emerging adults with Fisher's exact test.

3. Results and discussion 3.1. Characterization of aqueous nC60 suspensions The particle size of the suspension was analysed with two methods (TEM, DLS). The hydrodynamic diameter was tested with DLS. Based on the measured images from TEM the fullerene suspension particle size distribution was in the range of 41e12 533 nm and the average particle size was 515 ± 909.2 nm (n ¼ 700; S1 Fig. S2). Size distribution and shape were similar to that of our previous observations from fullerene suspensions (WaissiLeinonen et al., 2012). The majority was in the size range of 100e200 nm (28%). In analysing fullerene suspension with DLS, the average particle size was determined to be 366.7 ± 32.7 nm, indicating a slightly smaller size than that measured with TEM. This is due to the larger agglomerates that are out of the DLS measurement range (>300 nm). According to TEM measurements the largest agglomerates (>1000 nm) constituted 11% of the total amount of particles. The Zeta potential of the fullerene suspension was 24.5 ± 0.76 (n ¼ 3), likewise another similar study, where aqueous nC60 suspensions were made with two weeks of stirring time (Gai et al., 2011). The used fullerene suspension was characterized quite heterogeneous where small aggregates dominated (1000 nm) were present. This lead to situation that analysing the behaviour of the suspension may be affected strongly by these small aggregate sizes. Smaller fullerene aggregates are hydroxylated for the most part compared to larger

Fig. 2. Relative growth rate of larva in a 10-d chronic test. Fullerenes associated sediments are compared to untreated sediment set to 100%. A) body length B) head capsule length C) head capsule width D) dry weight. Graphs indicates the dashed line as control, circles mean values and whiskers standard deviation. Asterisks point out significant differences from untreated sediments (Dunn's and Dunnett's Multiple Comparison test, *p < 0.05, **p < 0.01, ***p < 0.001) (For all parts in this figure n ¼ 30, 30, 30, 30, 27, 29, 28, 28, 27, 23, respectively).

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ones resulting higher toxicity of small fullerene aggregates for instance in bacteria and viruses (Chae et al., 2010) while interactions with sediment particles is not known. 3.2. Fullerene quantification in sediments Fullerene concentrations 1 mg/kg were below the detection limit of the method. For the treatments of 10, 20, 40, 60 and 80 mg/ kg the recovery was good 100 ± 15% (n ¼ 2e4). Sediment particle size characterization was made using TEM images. TEM measurements of the sediment particles (n ¼ 400) showed a clear difference between the control sediment and fullerene spiked sediments (Fig. 1). In the control sediments altogether 800 particles were included in the particle size analyses. Particle sizes in the control sediment (1182 ± 994 nm) significantly differed from the fullerene treatments of 0.0004, 0.025. 0.1, 0.5, 1, 10, 60 and 80 mg/kg (Dunn's multiple comparison test ***p < 0.001), where smaller particles were observed (Fig. 1). Large particles (>1500 nm) dominated in the control, constituting 23% of the particles. On the contrary, in the fullerene spiked sediments, for instance in the concentration of 1 mg/kg, the distribution was focused on the smallest particles (34% < 500 nm). Particle size distributions were similar in controls and spiked sediments ranging from 500 to 1500 nm. 3.3. Larval growth rate The growth clearly decreased at the exposure range of 0.0025e20 mg/kg and in the highest exposure (80 mg/kg) (Fig. 2). Fullerenes did not have significant effects on larval survival. Mortality was generally low (less than 23%), being highest in the exposure of 80 mg/kg (S1 Table S5). Body length was significantly lower at the exposures of 0.0025e20 and 80 mg/kg (Fig. 2A), which also reflected in dry weight. Instead exposures of 0.0004, 40 and 60 mg/kg did not differ significantly from the controls. Head capsule length and width (Fig. 2BeC), which indicate the developmental stage (instar phases one to four) of the larvae, decreased significantly at the exposure of 0.5, 1 and 10 mg/kg, expressing the delayed development of the larvae. In those exposures, the proportions of the larvae that did not reach the fourth and thus the final instar phase, were 80%, 56% and 93% respectively (S1 Table S5). Notably, in the three lowest exposures, the head capsule size did not differ from that of the controls. The mean head capsule length of the control organisms was 0.60 ± 0.11 mm, and for the fullerene treatments of 0.5, 1 and 10 mg/kg the values were 0.41 ± 0.10, 0.49 ± 0.12 and 0.39 ± 0.08 mm respectively (S1 Fig. S3). The dry

Fig. 3. The linear regression in fullerenesC60 treatments between the average sediment particle size distribution and the average body length of larvae (concentrations 0.0004e0.1 mg/kg and 0.5e80 mg/kg). Experimental sets were separated. There was no significant difference between slopes.

Fig. 4. Cumulative emergence (%) of the midges exposed to fullerenesC60 in the sediment. Asterisks indicate significant differences from controls (Log-rank ManteleCox test, *p < 0.05, **p < 0.01).

weight of the larvae differed significantly from the control within the exposures of 0.0025e10 mg/kg (Fig. 2D). In this study, decreased growth was observed already at the exposures of 0.0025 mg/kg up to 20 mg/kg and in the highest exposure of 80 mg/kg, causing a bell-shaped “doseeresponse curve”. Decreased growth rate in case of the same food concentration (0.5% dw Urtica) was shown our previous observations when larvae were exposed to high concentrations of fullerenes, using a fullerene layer on top of the sediment to imitate freshly settled fullerenes (Waissi-Leinonen et al., 2012). The larvae did not reach the fourth and final instar phase which indicates delayed development in the exposures ranged between 0.5 and 10 mg/kg and in the exposure of 80 mg/kg, The head capsule width and length did not differ in the two lowest exposures (0.0025 and 0.1 mg/kg), indicating that the larvae were in their fourth instar phase. Otherwise the body length and dry weight were significantly smaller. The growth patterns studied in this

Fig. 5. Development time of the females and males between the treatments. The symbol “þ” indicates mean values, and whiskers indicate minimum and maximum values.

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research were results from individually exposed larvae aiming that the factor of larval density can be ruled out. This factor, the densitydependence, is expected to have an impact on larval survival and growth due to competition. When studying larval growth it is essential to expose them in an individual chambers (Ristola et al., 1999). Nevertheless, when studying the effect at the population level exposures in groups should be preferred. There was a linear regression between average body length and sediment particle size in the fullerene exposures of 0.0004e0.1 mg/ kg and 0.5e80 mg/kg (y ¼ 0.0011x þ 9.2009 R2 ¼ 0.27 and y ¼ 0.0035x þ 4.6263, R2 ¼ 0.70) (Fig. 3). This may imply that fullerenes interact with the sediment or food particles, forming aggregates that disturb feeding behaviour and cause starvation. If fullerene binds the food particles the nutritional value might be shifted. This concentration dependence does not apply with regard to the highest tested treatment concentration (80 mg/kg), in which the mean particle size diminished again. This may indicate that the sediment became saturated with fullerenes in the highest concentration. The causality is not only based on C60 quantity or concentration, but also to the aggregate state in which the C60 is present. The mechanism behind these measured adverse effects is probably related to the dominance of the small fullerene particles in the food of larvae. 3.4. Emergence of adults Although fullerenes significantly decreased growth at certain concentrations, a reduction in emergence was only seen with the lowest dose of fullerenes (0.5 mg/kg p ¼ 0.0011) (Fig. 4). The test was repeated with the lowest concentration and the same result was gained; the emergence significantly reduced with a p value of 0.0449 (SI Fig. S4). Interestingly, the time to emergence in other treatments was significantly faster compared to the control group (10 mg/kg p ¼ 0.0272, 40 mg/kg p ¼ 0.0441 and 80 mg/kg p ¼ 0.0064) (Fig. 4). The median time to emergence was 22 days for midges in the control and 26.5 for midges exposed to 0.5 mg/kg of fullerenes. For treatments 10, 40 and 80 mg/kg the median time to emergence was 19.5, 22 and 20 respectively. The length of emergence period was in the control 14 days, whereas in the exposures (0.5, 10, 40, 60 ad 80 mg/kg) it was 22, 11, 10, 10 days. Time to first emergence were observed at the day 17 in all other treatments and control besides the treatment 0.5 mg/kg of fullerenes where it was at the day 19. In many ecotoxicological studies the emergence rate of C. riparius has been observed to increase when organisms are subjected to stress by exposing them to chemicals, increasing the effort put in reproduction (Ristola et al., 1999). This occurred in the fullerene concentrations of 10, 40 and 80 mg/kg used in this study. Therefore, as hormonal regulation plays an important role in the development of aquatic invertebrates, studies on potential endocrine disruption are needed (Matthiessen et al., 2012; Taenzler et al., 2007). To our knowledge, there are no such studies on carbon nanoparticles and C. riparius's emergence rates. On the other hand, metal nanoparticles have been observed to cause changes in adult emergence. Silver nanoparticles caused a decrease in adult emergence and pupation, and even alterations in the sex ratio have been documented (Nair et al., 2013). The development time of the females was longer than that of males in all treatments (Fig. 5). Female emergence occurs normally later than males (Ineichen et al., 1979; Liber et al., 1996). In current study median emergence for control males was 20 and females 25 days but 23 and 32 days in the fullerene exposure of 0.5 mg/kg, respectively. This delay between males and females may lead to situation that they do not have possibility to mate taking into account adult's short living time. In other fullerene exposures (10, 40 and 80 mg/kg) median emergences for males were 19, 18 and 19

and for females 25, 23 and 21, 5 days respectively. This suggests that the effect of exposure on the population emergence rate was mainly modified by the female development. Observations revealed also potential changes in the female and male proportions although difference was not significant. In the control female:male percentage ratio was 52:48 and in exposures of 0.5, 10, 40, 80 mg/kg ratios were 40:60, 30:70, 53:47, and 48:52, respectively. This phenomenon, a possible chemically induced sex ratio alteration caused by the toxic potential of disturbing hormonal processes, needs to be studied more thoroughly because it may reflect to population level. 3.5. Transmission electron microscope observations of C. riparius On the basis of our previous study, the middle parts of the larvae were used (Waissi-Leinonen et al., 2012), as that part was proved to be the most relevant when using C. riparius as a test organism. The gut structure was detected clearly and possible morphological changes were observed with TEM samples, where the peritrophic membrane and microvilli were visible. Microvilli length did not differ significantly from the control in the fullerene treatment of 1 mg/kg. In all treatments (0.5, 10, 20, 40, 60 and 80 mg/kg) results differed significantly from the control (Man Withney test, *p < 0.05, ***p < 0.0001, n ¼ 400) (S1 Fig. S5). There was no evidence indicating the absorption of the fullerenes from the gut to the surrounding tissues. Surprisingly, the longest microvilli were observed in the highest fullerene concentration (80 mg/kg), where the mean length was 4840 ± 1457 nm. The mean length in the control was 2577 ± 1139 nm. Previous study with fullerene layer on the top of the sediment led to shortened microvilli and damages on microvilli layer (Waissi-Leinonen et al., 2012) but spiking method did not cause damages on the microvilli structures. This reveals the importance of comparing and analysing various exposure scenarios and to observe different mechanisms behind these effects. Current observations corresponds to the previous results of research with another benthic organismdL. variegatus (Pakarinen et al., 2011). When sediments are spiked with fullerenes, mechanical abrasion does not seem to occur, unlike in the study where C. riparius were exposed to a mass of fullerene layers on the top of the sediment, causing damage to the microvilli layer and the length of microvilli (Waissi-Leinonen et al., 2012). 4. Conclusions This study assessed the sublethal chronic effects of fullerenesC60 associated artificial sediment to C. riparius and the relevance of chronic experiments. Highlighting that effects are due to unknown state of C60 in sediments these results showed that the potential effect of fullerenesC60 to the benthic invertebrate C. riparius appeared already at the test concentration of 0.025 mg/kg. The exposure to fullerenes resulted in a bell-shaped doseeresponse relationship in view of the relative growth patterns studied here. This observed divergent doseeresponse curve complicates ecological risk assessment of fullerenes because some effects expressed at low concentrations. In addition, effects on emergence rate may reflect to population level. Studied ecotoxicological parameters suggested that the working mechanisms of adverse effects causing the decreased growth and delayed development of C. riparius are dependent of sediment particle sizes, related to sediment particle fullerene interactions modifying the exposure. There probably is an ecotoxicologically relevant C60 state which varies across treatments and is needed to be more studied. The mechanical damage on microvilli layer caused by the fullerene rupturing was ruled out based on TEM investigations. Development and reproduction are regulated by hormone system and their

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