Article pubs.acs.org/est
Nanoplastic Affects Growth of S. obliquus and Reproduction of D. magna Ellen Besseling,*,†,‡ Bo Wang,† Miquel Lürling,† and Albert A. Koelmans†,‡ †
Aquatic Ecology and Water Quality Management Group, Wageningen University, P.O. Box 47, 6700 AA Wageningen, The Netherlands ‡ IMARES − Institute for Marine Resources & Ecosystem Studies, Wageningen UR, P.O. Box 68, 1970 AB IJmuiden, The Netherlands S Supporting Information *
ABSTRACT: The amount of nano- and microplastic in the aquatic environment rises due to the industrial production of plastic and the degradation of plastic into smaller particles. Concerns have been raised about their incorporation into food webs. Little is known about the fate and effects of nanoplastic, especially for the freshwater environment. In this study, effects of nano-polystyrene (nano-PS) on the growth and photosynthesis of the green alga Scenedesmus obliquus and the growth, mortality, neonate production, and malformations of the zooplankter Daphnia magna were assessed. Nano-PS reduced population growth and reduced chlorophyll concentrations in the algae. Exposed Daphnia showed a reduced body size and severe alterations in reproduction. Numbers and body size of neonates were lower, while the number of neonate malformations among neonates rose to 68% of the individuals. These effects of nano-PS were observed between 0.22 and 103 mg nano-PS/L. Malformations occurred from 30 mg of nano-PS/L onward. Such plastic concentrations are much higher than presently reported for marine waters as well as freshwater, but may eventually occur in sediment pore waters. As far as we know, these results are the first to show that direct life history shifts in algae and Daphnia populations may occur as a result of exposure to nanoplastic.
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pseudofeces production and reduced filtering activity have been reported.20 For algae, nanoplastic has been shown to reduce CO2 uptake and enhance the production of reactive oxygen species (ROS).21 As the interaction of organisms with pollutants in particulate form is completely different from that with conventional dissolved chemicals, there is a potential high risk associated with particles.7,22 Given the limited data, there is an urgent need to quantify the effects of nanoplastic on freshwater organisms. Effects of nanoplastic may be related to particle toxicity, toxicity of plastic-associated chemicals, or both and will depend on the characteristics of the nanoplastic, such as particle size, polymer type, and age. However, previous research on nanoparticle behavior and effects was often conducted using pristine particles,23 whereas aged and naturally altered particles are of higher importance considering environmental relevance, which will therefore be addressed in the present study.
INTRODUCTION Pollution with plastic is a growing concern in the marine environment.1 However, emissions from land-based sources reach rivers first, and freshwaters provide an important source of marine plastic pollution through riverine transport.2 Therefore, the occurrence of plastic in the freshwater environment receives increasing attention.3−6 Special concerns exist with respect to nanoplastics because of their large surface area and hypothesized ability to penetrate cells.7−11 Both primary particles from personal care and cosmetic products and secondary particles from degradation of larger plastic items are expected to contribute to pollution of the environment with nanoplastic.12 Recent reports showed the importance of physical abrasion as a source of secondary micro- and nanoplastic.13,14 Yet there are hardly any proven life history effects of micro- and nanoplastic on marine organisms, and effect data for freshwater organisms are lacking. For microplastic, the first reported data on effects on invertebrates relate to survival, feeding, oxidative status, and PCB uptake in lugworms (Arenicola marina).15−17 In marine zooplankton, decreased feeding18 and reduced survival and fecundity have been observed.19 Even less is known about the effects of nanoplastic. For mussels (Mytilus edulis), an increased © XXXX American Chemical Society
Received: June 20, 2014 Revised: September 22, 2014 Accepted: September 30, 2014
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dx.doi.org/10.1021/es503001d | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology
Article
Plastic interacts with man-made organic compounds,24 as studied for several kinds of pollutants and additives.16,24−26 Recently, an exceptionally strong sorption of PCBs to nanoplastic was observed, which might imply a strong transport capacity including increased exposure upon penetration of cells or tissues.27Effects of nanoplastic might also be caused by direct particle toxicity, attachment to algae, reduction of light penetration, reduced food quality, release of additives, or interference with chemical communication. Here we hypothesize that nanoplastic might also interact with natural organic molecules such as kairomones, which may yield unforeseen effects on the interactions among species. Daphnia are known to express life history traits such as altered adult and/or neonate body size and altered neonate quantity in response to the presence of predator kairomones.28,29 Sorption of kairomones to nanoplastic might disturb these life history traits.30 The aim of the present study was to investigate effects of nanoplastic at the first two trophic levels of the freshwater aquatic food chain; algae, represented by Scenedesmus obliquus, and zooplankton, represented by Daphnia magna. Both species are widely used for ecotoxicity tests. Nanosized polystyrene (nano-PS) spheres were used as the test substance, as polystyrene is one of the most widely used commercial plastics in the world and was used in earlier toxicity tests.15,20,21 We investigated direct and indirect effects of a broad range of expected environmentally relevant and elevated concentrations of nano-PS in fresh water bioassays. The bioassay we present here is the first interaction bioassay of nano-PS combined with an interspecific organic molecule: fish kairomone. We took the interaction time between plastic particles and algae into account by using both pristine and aged dispersions of nano-PS, thereby providing novel information about the potential role of particle aging.
were kept far below toxicity thresholds of Daphnia35,36 and Scenedesmus. Absence of toxicity to Scenedesmus was confirmed in separate pilot tests with SDS, which are provided as Supporting Information (SI). Similarly, because of its hydrophobicity and high volatility with reported half-lives of 1−3 h in lake water,37 presence of styrene monomers in the aqueous phase can be assumed negligible. The polystyrene beads had a primary nominal size of ∼70 nm (confirmed by transmission electron microscopy) and contained 0.01% on mass basis of the hydrophobic fluorescent dye (Nile Red), which was immobilized by the polymer matrix. Consequently, presence of Nile Red in the aqueous phase can also be assumed negligible, which is consistent with the use of Nile Red as a tracer in numerous studies of biological systems.38−40 Furthermore, even if all Nile Red in the polystyrene would have been bioavailable, the concentration would still have been a factor 1.5 × 104 below the effect concentration reported by Wu et al.41 (Calculation provided as SI). To better represent nano-PS occurring in products and in the environment,42 the spheres were functionalized with carboxylic acid groups. As the glass−liquid transition temperature of polystyrene43 is much higher than the maximum temperature in our bioassay (21 °C), leaching of chemicals from the polymer matrix and therewith their occurrence in the exposure dispersions is negligible. The form of nano-PS in aqueous suspension was extensively characterized before (see SI Figure S1).27 Scenedesmus Bioassay. Scenedesmus obliquus were exposed to 44−1100 mg nano-PS/L in 80 mL of WC medium in a 72-h bioassay. Details about the used concentration range are provided as SI. Algae populations with an initial density of approximately 3 × 106 cell/mL were used. A growth inhibition test was performed three times with controls in 6-fold and nano-PS treatments in triplicate.35,44 Cell densities were determined using a CASY counter (CASY model TT, INNOVATIS) at the start and after every 24 ± 1 h. At the end of two of the bioassays, Chlorophyll-a (Chl-a) was extracted and determined by spectrophotometry (Beckman Coulter, DU 730 Life Science UV/vis) to assess photosynthetic capacity and biomass following a hot ethanol extraction method with phaeopigment correction.45 Daphnia Bioassay. Daphnia magna were exposed individually to 80 mL nano-PS test dispersion in a 21-day bioassay, according to OECD guidelines.46,47 Four types of nano-PS test dispersions were tested, which are referred to as (1) pristine, (2) pristine-kairomone, (3) aged, and (4) aged-filtered (Figure 1). (1) Pristine refers to the treatment where the exposure of the Daphnia started immediately after mixing algae and nanoPS. Nano-PS dispersions were dilutions of nano-PS stock in RT medium to which algae were added just before use in the bioassay. Pristine exposures were applied at ten nanoplastic concentrations in the range of 0.22−150 mg nano-PS/L. Details about the used concentration ranges are provided as SI. (2) For the pristine-kairomone dispersions, the only difference from the pristine dispersions was the presence of fish kairomones in the initial RT medium. Fish kairomones were kindly obtained from a parallel study at our university, where three individuals of Perca fluviatilis (total overall length ±12 cm) were inhabited in 20 L of aerated RT medium for a week. Perca f luviatilis is a predator known to induce life history responses in Daphnia.48,49 Three times a week, the fish were fed with Daphnia. Before use in the Daphnia bioassay, the RT medium with fish kairomones was filtered over a 0.45-μm membrane filter (Whatman cellulose nitrate membrane, grade
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EXPERIMENTAL PROCEDURES Bioassays were performed with algae (S. obliquus), and with D. magna fed with these algae. Organisms. Scenedesmus obliquus SAG 276/3A was obtained from the University of Göttingen, Germany and was maintained in modified algal growth medium (WC-medium).31 Stock cultures and the Scenedesmus bioassay were maintained similar to previous procedures at 20 °C in a climate chamber with 24 h continuous light (∼ 100 μmol quanta m−2 s−1) and 100 rpm rotational shaking.32 Algae inoculum was prepared 3 days ahead of the Scenedesmus bioassay, to obtain exponential growth at the start of the test. Daphnia magna originated from lake Zwemlust, Nieuwesluis,33 The Netherlands and were cultured in artificial growth medium (RT medium33) with a pH of 7.7−8.1. The Daphnia cultures and bioassay were kept at a temperature of 21 ± 1 °C with the natural spring daylight regime (low beam day conditions
