Are the TiO2 NPs a "Trojan horse" for personal care products (PCPs) in the clam Ruditapes philippinarum?

In recent years, increasing quantities of personal care products (PCPs) are being released into the environment. However, data about bioaccumulation and toxicity are scarce; and extraction and analytical approaches are not well developed. In this work, the marine clam Ruditapes philippinarum, selected as model organism, has been employed to investigate bioaccumulation, antioxidant enzyme activities and DNA damage due to exposure to TiO2 nanoparticles and bulk TiO2 (inorganic compounds that are frequent components of PCPs, plastics, paints and coatings, foods and disinfectant water treatments). We have also studied the joint effect of both forms of inorganic TiO2 combined with four organic compounds (mixture exposures) commonly used in PCPs: an antimicrobial (triclosan), a fragrance (OTNE) and two UV filters (benzophenone-3 and octocrylene). Bioaccumulation of the inorganic compound, TiO2, was almost immediate and constant over exposure time. With respect to the organic compounds in mixtures, they were mediated by TiO2 and bioaccumulation is driven by reduced size of the particles. In fact, nanoparticles can be considered as a vector to organic compounds, such as triclosan and benzophenone-3. After a week of depuration, TiO2 NPs and TiO2 bulk in clams showed similar levels of concentration. Some organic compounds with bioactivity (Log Kow >3), like OTNE, showed low depuration after one week. The joint action of the organic compound mixture and either of the two forms of TiO2 provoked changes in enzyme activity responses. However, for the mixtures, DNA damage was found only after the depuration period.

Toxicity of TiO2, in nanoparticle or bulk form to freshwater and marine microalgae under visible light and UV-A radiation.

Use of titanium dioxide nanoparticles (TiO2 NPs) has become a part of our daily life and the high environmental concentrations predicted to accumulate in aquatic ecosystems are cause for concern. Although TiO2 has only limited reactivity, at the nanoscale level its physico-chemical properties and toxicity are different compared with bulk material. Phytoplankton is a key trophic level in fresh and marine ecosystems, and the toxicity provoked by these nanoparticles can affect the structure and functioning of ecosystems. Two microalgae species, one freshwater (Chlamydomonas reinhardtii) and the other marine (Phaeodactylum tricornutum), have been selected for testing the toxicity of TiO2 in NP and conventional bulk form and, given its photo-catalytic properties, the effect of UV-A was also checked. Growth inhibition, quantum yield reduction, increase of intracellular ROS production, membrane cell damage and production of exo-polymeric substances (EPS) were selected as variables to measure. TiO2 NPs and bulk TiO2 show a relationship between the size of agglomerates and time in freshwater and saltwater, but not in ultrapure water. Under two treatments, UV-A (6 h per day) and no UV-A exposure, NPs triggered stronger cytotoxic responses than bulk material. TiO2 NPs were also associated with greater production of reactive oxygen species and damage to membrane. However, microalgae exposed to TiO2 NPs and bulk TiO2 under UV-A were found to be more sensitive than in the visible light condition. The marine species (P. tricornutum) was more sensitive than the freshwater species, and higher Ti internalization was measured. Exopolymeric substances (EPS) were released from microalgae in the culture media, in the presence of TiO2 in both forms. This may be a possible defense mechanism by these cells, which would enhance processes of homoagglomeration and settling, and thus reduce bioavailability.

Direct and indirect effects of silver nanoparticles on freshwater and marine microalgae (Chlamydomonas reinhardtii and Phaeodactylum tricornutum).

The last decade has seen a considerable increase in the use of silver nanoparticles (AgNPs), which are found in many every-day consumer products including textiles, plastics, cosmetics, household sprays and paints. The release of those AgNPs into aquatic environments could be causing ecological damage. In this study we assess the toxicity of AgNPs of different sizes to two species of microalgae, from freshwater and marine environment (Chlamydomonas reinhardtii and Phaeodactylum tricornutum respectively). Dissolution processes affect the form and concentration of AgNPs in both environments. Dissolution of Ag from AgNPs was around 25 times higher in marine water. Nevertheless, dissolution of AgNPs in both culture media seems to be related to the small size and higher surface area of NPs. In marine water, the main chemical species were AgCl2- (53.7%) and AgCl3-2 (45.2%). In contrast, for freshwater, the main chemical species were Ag+ (26.7%) and AgCl- (4.3%). The assessment of toxicological responses, specifically growth, cell size, cell complexity, chlorophyll a, reactive oxygen species, cell membrane damage and effective quantum yield of PSII, corroborated the existence of different toxicity mechanisms for microalgae. Indirect effects, notably dissolved Ag ions, seem to control toxicity to freshwater microalgae, whereas direct effects, notably attachment onto the cell surface and the internalization of AgNPs inside cells, seem to determine toxicity to the marine species studied. This research contributes to knowledge on the role of intrinsic and extrinsic factors in determining the behavior of NPs in different aquatic environments and the interaction with microalgae.

CeO2 NPs, toxic or protective to phytoplankton? Charge of nanoparticles and cell wall as factors which cause changes in cell complexity.

CeO2 nanoparticles (CeO2 NPs) are well-known for their catalytic properties and antioxidant potential. Recent uses in therapy are based on the Ce+3 ions released by CeO2 NPs. Reactions involving redox cycles between Ce+3 and Ce+4 oxidation stage seem to promote scavenging of reactive oxygen species (ROS), thus protecting cells from oxygen damage. However, the internalization of CeO2 NPs and release of Ce+3 could be responsible for a toxic effect on cells. The literature reports controversial results on the toxicity of CeO2 NPs to phytoplankton. Therefore, we have tested the potential toxic effect of two CeO2 NPs (with positive and negative zeta potential) and bulk CeO2 (at 0.1, 1, 10, 100 and 200mg·L-1) on three species of microalgae from different environments: marine diatom (Phaeodactylum tricornutum), marine chlorophyte (Nannochloris atomus) and freshwater chlorophyte (Chlamydomonas reinhardtii) over 72h in batch cultures. Responses measured in the microalgae population are: growth, chlorophyll a, cell size, cell complexity, percentage of ROS, and percentage of cell membrane damage. Positive zeta potential CeO2 NPs provoked greater cell complexity (up to 78, 172 and 23 times more cell complexity than in controls found for C. reinhardtii, P. tricornutum and N. atomus respectively) than negative zeta potential CeO2 NPs. The SSC signal detected by flow cytometry measured increases of particles entering cells, and this is related to cell viability and levels of intracellular ROS (correlation between SSC and percentage of ROS of 0.72 and 0.97 found for C. reinhardtii and P. tricornutum). When increased cellular complexity over controls is between 2 and 6 times greater, CeO2 (in bulk or nanoparticulate form) seems to protect against ROS. When increased cellular complexity is from 7 to 23 times greater, CeO2 does not provoke toxic responses; however, when increased cellular complexity over controls is very high, from 61 to 172 times, increased ROS production and toxic responses are found. Results show that two factors, the charge of CeO2 NPs and cell wall structure, constitute the primary barrier to the possible accumulation of CeO2 NPs within phytoplankton cytosol.

Homoagglomeration and heteroagglomeration of TiO2, in nanoparticle and bulk form, onto freshwater and marine microalgae.

TiO2 nanoparticles (TiO2 NPs) are employed in many products (paints, personal care products, especially sunscreens, plastics, paper, water potabilization and food products) and are then released into the environment from these products. These nanoparticles present potential risk to freshwater and marine microalgae. The primary toxicity mechanism is adsorption between NPs and microalgae (heteroagglomeration); however, studies of interactions of this kind are scarce. We investigated the heteroagglomeration process that occurs between two forms of TiO2 material, nanoparticles and bulk, and three different microalgae species, and under different environmental conditions (freshwater and marine water), in order to assess the influence of pH and ionic strength (IS). The heteroagglomeration process was examined by means of co-settling experiments and the Derjaguin-Landau-Verwey-Overbeek (DLVO) approach. The homoagglomeration process (only NPs to NPs) did not show differences between culture media (freshwater and marine water). However, in the heteroagglomeration process between NPs and cells, IS played an important role. Ions can compress the electro-double layer between NPs and microalgae, allowing a heteroagglomeration process to take place, as shown by settling experiments. TiO2 NPs presented a settling rate higher than bulk TiO2. The DLVO theory could only partially explain heteroagglomeration because, in this model, it is not considered that NP-NP and Cell-Cell homoagglomeration co-occur. In this study neither the role of exopolymeric substances in the interaction between NPs and cells nor detoxification are considered. The authors suggest that the interaction between NPs and microalgae could be considered as the first stage in the process by which nanoparticles affect microalgae.

Effects of TiO2 nanoparticles and sunscreens on coastal marine microalgae: Ultraviolet radiation is key variable for toxicity assessment.

Given the large numbers of sunbathers on beaches, sunscreen compounds are being released into the coastal aquatic environment in significant amounts. Until now the effect of these potential pollutants on microbiota has been not well-known. Phytoplankton is a key component of the microbiota community. It forms the basis of the aquatic trophic networks, and any change in the natural population of phytoplankton can affect the structure of aquatic biota. This paper describes an experiment performed outdoors (in natural sunlight conditions including ultraviolet radiation (UVR) and with UVR blocked) on mixed microalgae populations (four species from different key marine taxonomic groups, Nannochloropsis gaditana, Chaetoceros gracilis, Pleurochrysis roscoffensis and Amphidinium carterae), for three days, exposed to a range of concentrations of three commercial sunscreens (with variable TiO2 concentrations: highest concentration for sunscreen C, followed by sunscreen A; and sunscreen B did not contain TiO2 in its composition). With regard to UVR effect, in the absence of sunscreens, the most sensitive species is the centric diatom, Chaetoceros gracilis, and the least is Nannochloropsis gaditana; this last species presented the same behavior in the absence of UVR and with high sunscreen concentrations. The toxicity gradient obtained for sunscreens and nanoparticles under UVR is: TiO2 NPs>Sunscreen C>Sunscreen A>Sunscreen B. The differential sensitivity of microalgae to sunscreens and TiO2 NPs can produce a change in the dynamics of phytoplankton populations and provoke undesirable ecological effects (such as giving dinoflagellates more prominence). The effects of UVR, commonly neglected in bioassays, could alter the results in important ways and should be considered when performing environmentally-relevant bioassays. The toxicity mediated by hydrogen peroxide production associated with the concentration of TiO2 NPs cannot be considered the only factor responsible for the toxicity: the organic compounds in the sunscreens must also be taken into account.

European bee-eater (Merops apiaster) populations under arsenic and metal stress: evaluation of exposure at a mining site.

Two populations of the European bee-eater were studied, one living at a reference site and the other at a metal mining site. The concentration of arsenic and 11 metals (Al, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, and Zn) was measured in feathers and regurgitated pellets collected at both sites. Cadmium, chromium, mercury, nickel, and lead were at least twofold higher in feathers of birds from the contaminated site than in the reference site, suggesting that this population was exposed to higher metal levels. Similarly, levels of aluminum, arsenic, cadmium, cobalt, chromium, iron, and lead were also at least twofold higher in pellets from the contaminated area. The obtained results suggested that the impacted population of Merops apiaster is at risk due to the exposure to some metals.

The use of marine benthic diatoms in a growth inhibition test with spiked whole-sediment.

The benthic diatom Cylindrotheca closterium was exposed to whole-sediment spiked with the synthetic surfactant Linear Alkylbenzene Sulphonate (LAS), as commercial mixture or individual homologues (C10-LAS, C11-LAS or C12-LAS). Separately, the diatoms were exposed to C12-LAS in a water-only system. The algal growth was determined after 72 h. The 72-h ErC50 values ranged from

Suitability of the marine prosobranch snail Hydrobia ulvae for sediment toxicity assessment: A case study with the anionic surfactant linear alkylbenzene sulphonate (LAS).

Individuals of the mudsnail Hydrobia ulvae (Pennant) (Mollusca: Prosobranchia) were exposed to sediments spiked with increasing concentrations (1.59-123.13mgkg(-1) dry weight) of the anionic surfactant linear alkylbenzene sulphonate (LAS) which is employed in the formulation of laundry powders and liquids, as well as hand dishwashing products. The suitability of the selected organism, H. ulvae for routine sediment toxicity testing was evaluated by measuring acute toxicity recording survival. Sublethal toxicity was evaluated as total number of produced veliger larvae per treatment throughout the test (9d). Mortality has shown to be a reliable and reproducible indicator of acute toxicity. LC(50) values were comprised between 203.4 (48h) and 94.3mgkg(-1) (9d) dry weight. As sublethal endpoint, the total number of produced larvae showed to be a useful indicator of toxicity for this organism. The number of produced larvae increased at lower exposure concentrations, whereas at the highest LAS concentration, the number of produced larvae decreased. This is the first report of acute and sublethal toxicity of sediment associated LAS for this species.

Sediment toxicity tests involving immobilized microalgae (Phaeodactylum tricornutum Bohlin).

Populations of calcium-alginate immobilized marine diatom Phaeodactylum tricornutum were exposed to two sediments containing different levels of surfactant (LAS). Toxic responses were compared for free and immobilized algae. Although there is a direct relation between LAS content in sediment and inhibition, immobilized algae suffered less inhibition than free cells, over all when fluorescence is chosen as a biomarker for biomass. When cells are counted from dissolved beads, inhibition of growth is closer to the values found for free cells. Immobilization can be useful for in situ experiments but protection of cells inside the alginate beads against toxic capacity of xenobiotics must be taken into account.

Calcium alginate immobilized marine microalgae: experiments on growth and short-term heavy metal accumulation.

Growth of 11 calcium alginate immobilized marine microalgal species belonging to eight taxonomical groups has been checked in the present work. Cellular densities inside the calcium alginate beads were monitored during 17 days. Good growth and maintenance of the structure of the beads were both found for some of the assayed species. One of those species (Tetraselmis chui, Prasinophyceae) was selected in order to perform a short term (up to 24 h) heavy metal accumulation experiment. Beads of calcium alginate containing (or not) cells of T. chui were exposed to 820 microg L(-1) Cu and 870 microg L(-1) Cd separately during a 24 h period, and accumulation of heavy metals in the beads was measured after this time and compared. Concentration of each metal in the supernatants was monitored at 5, 10, 60 min and 24 h from the beginning of the experiment. After 24 h, practically all Cu was removed by the beads. Beads with immobilized algae removed around 20% of total Cd, while beads without algae removed half of that percentage.

Sublethal effects of linear alkylbenzene sulphonate on larvae of the seabream (Sparus aurata): histological approach.

Neonate (< 24 h) larvae of the seabream, Sparus aurata, were exposed to sublethal concentrations (0.1-1.0 mg x L(-1)) of the anionic surfactant Linear Alkyl-benzene Sulphonate (LAS) for 72 h under laboratory conditions. The first histopathological changes, such as peri-yolk sac edema, were observed at concentrations of 0.2 mg x L(-1). Higher exposure concentrations provoked disorganisation of the nervous system, trunk musculature and trophoblastic sincitium as well as in the digestive epithelium. Immunohystochemical CYP1A analysis, however, was not shown to be an adequate indicator of sublethal effects produced by exposure to this type of anionic surfactant

Sediment toxicity tests using benthic marine microalgae Cylindrotheca closterium (Ehremberg) Lewin and Reimann (Bacillariophyceae).

A new method for sediment toxicity testing using marine benthic pennate noncolonial diatom (Cylindrotheca closterium, formerly Nitzschia closterium) has been developed. This microalgae showed a good growth rate during the experimental period, even when low enriched media were used. Sediment spiked with heavy metals [cadmium (Cd), copper (Cu), and lead (Pb)] was employed to determine the EC(50) values, using microalgal growth inhibition as the endpoint. The obtained results were as follows: Three heavy metals (Cd, Cu, and Pb), previously spiked on experimental sediment, were separately assayed in toxicity tests. The EC(50) values for these heavy metals in microalgal growth inhibition tests resulted to be 79 mg kg(-1) for Cd, 26 mg kg(-1) for Cu, and 29 mg kg(-1) for Pb (in experimental sediment). The influence of sediment granulometry on the growth of microalgal population was also studied, finding that the growth of the microalgal population on media containing sediment with a relation sand-size:silt size of 9:1 was not different from optimal growth (occurring in media containing 100% sand-sized sediment). The diatom C. closterium proved to be a suitable organism for sediment toxicity tests, due to its sensitivity and fast growth even in poorly enriched media.

Can early life-stages of the marine fish Sparus aurata be useful for the evaluation of the toxicity of linear alkylbenzene sulphonates homologues (LAS C10-C14) and commercial LAS?

Most commercial household cleaning agents and personal care products contain the anionic surfactant linear alkylbenzene sulphonates (LAS) as the active compound. After their use they are discharged, theoretically after adequate wastewater treatment, into receiving waters finally reaching estuaries and coastal waters. Laboratory toxicity tests are useful tools in determining at which concentration a certain wastewater compound becomes hazardous for an existing group of organisms. Early life-stage toxicity tests include exposure during the most sensitive development period of the organism. In fish, this type of assay has shown to predict accurately maximum acceptable toxicant concentration (MATC) values (comprised in the range defined by the NOEC and LOEC) in fish early life-stage tests. For this reason, larvae of the seabream, Sparus aurata, were exposed to increasing concentrations of LAS homologues (C10-C14) and commercial LAS. Obtained LC50 values ranged between 0.1 and 3.0 mg l(-1) and were compared with LC50 values of previous hatching experiments with the same species. Larvae proved to be more sensitive to LAS exposure of individual homologues than eggs, except in the case of commercial LAS. LC50 values can be directly employed to determine their potential risk in a concrete environment with known pollutant concentrations. Dividing the LC50 value with the found homologue concentration and extrapolating with certain security factors proposed by different environmental organisms, potentially hazardous pollutant concentrations may be detected. Average estuarine or coastal LAS concentrations are generally below toxicity limits for this kind of organism, considering that the average alkyl chain length of commercial LAS is 11.6 carbon atoms.

Acute toxicity of LAS homologues in marine microalgae: esterase activity and inhibition growth as endpoints of toxicity.

The toxicity of two linear alkylbenzene sulfonate (LAS) homologues (C(10) and C(13)) was evaluated in four marine microalgae (Nannochloropsis gaditana, Tetraselmis suecica, Rhodomonas salina, and Isocrysis galbana), using growth inhibition rate and esterase activity (measured by flow cytometry) as endpoints. The inhibitor effect was higher for the C(13) LAS homologue than for C(11), in both responses analyzed. When both endpoints were compared, the growth inhibition rate was between 2 and 5 times more sensitive than esterase activity. Among microalgae species, R. salina exhibited the highest sensitivity.

Marine microalgae toxicity test for linear alkylbenzene sulfonate (LAS) and alkylphenol ethoxylate (APEO).

Different microalgal species have been used in growth-inhibition tests to determine the toxic concentrations of anionic and non-ionic surfactants to phytoplankton. The species used were selected from different taxonomic groups, all of considerable ecological relevance to marine environments. The toxicity of the C13 LAS homologue to the microalgal species selected was usually one order of magnitude greater than that of the C11 homologue. The toxicity of a commercial LAS mixture to different microalgal species was also checked. For this material and C. gracilis, cellular counting by means of a Neubauer chamber and by use of a flow cytometer were compared; differences between the two methods were insignificant. The toxicity of decaethoxylated nonylphenol non-ionic surfactant to C. gracilis was also checked; the EC50 value for this compound was 1.0 mg L(-1).

Influence of cellular density on determination of EC(50) in microalgal growth inhibition tests.

Growth inhibition tests for copper were carried out on four marine microalgal species: Chlorella autotrophyca, Nannochloris atomus (Chlorophyceae), Phaeodactylum tricornutum (Bacillariophyceae), and Isochrysis aff. galbana (Primnesiophyceae). The test initial cellular densities were reduced to 50 and 10% from the recommended initial cellular density in most of standardized assays. OECD test protocol (originally described for freshwater) was adapted for seawater. The EC(50) values were reduced when initial cellular density decreased. The green algae used in this study exhibited lower sensitivity than P. tricornutum and quite lower than I. aff. galbana. The latter species was found to be very sensitive to copper. The concept of cellular toxic quote (amount of toxic per cell) is defined in order to improve the results of toxicity tests.

In vitro populations of rotifer Brachionus plicatilis Müller demonstrate inhibition when fed with copper-preaccumulating microalgae.

Four marine microalgal species (Chlorella autotrophyca, Nannochloropsis gaditana, Tetraiselmis chuii, and Isochrysis aff. galbana) were exposed for 24 h to 1 mg L(-1) dissolved copper and then transferred to fresh medium. After that, a group of 10 neonate rotifers were fed with these four microalgal species. The levels of accumulated copper in cellular concentrations of the microalgae were checked, with the result of around 40% of original concentration, with the exception of I. aff. galbana (25% of original concentration). In all cases, cells with preaccumulated metal caused a delay of 1 or 2 days in populational development of rotifers (increase in "lag phase"). The microalgae that were not fed to rotifers (disposed in parallel series) did not significantly transfer metal to the medium after the first day.