Testing for suitable particles with Daphnia magna

Polystyrene particles with different surface modifications, charges, sizes (25 nm to 330 nm) and at a range of concentrations (0.005 g/L to 0.150 g/L) were tested for toxicity towards Daphnia magna. On day 1, 100 ml algae or water were added to each bottle together with particles with different concentrations except for the control bottle, which received only water (Table 1). The bottles were then shaken for 2 minutes, and the algae were allowed to ingest the particles for 24 hours. On day 2, 900 ml water was added to the algae/water together with 10 adult Daphnia magna with an approximate size of 3 mm. The number of dead Daphnia was counted every hour for 24 hours and the bottles were then gently stirred to distribute the algae or water evenly. To rule out a potential batch dependent toxicity of 52 nm amino modified polystyrene particles, the same type of particles but in a size of 53 nm, 57 nm as well as 58 nm were also tested.

Nanoparticle preparation and characterization

Positively charged amino-modified polystyrene (PAO 2 N) particles with diameters of 52 nm, 53 nm, 57 nm, 58 nm, 120 nm, 180 nm and 330 nm were purchased from Bang laboratories (Fisher, IN, USA). The particles were dialyzed with fresh tap water for 24 hours. The particle size was measured, to ensure that particles size remained constant during the experiment, with Dynamic Light Scattering (DLS) both before and after dialysis, as well as one week after the dialysis. No change in particle size was recorded during the study. We chose to not use surface labelled particles since this may affect the surface chemistry. Moreover, it is unknown how passage through the digestive systems of Daphnia and especially fish might affect the labelling and vice versa.

Fish experiment

Two sizes of particles were chosen for the fish experiment, one with the size of 53 nm that was shown to affect the Daphnia and one larger, 180 nm, that did not show any toxicity towards Daphnia. The particle size were confirmed with DLS and measured 56 nm (PI: 27%) and 174 nm (PI: 18%) in the water used during the experiment. Twenty-four aquaria with three fish in each were divided into four groups. The first group (the 180 nm group) received 180 nm particles at a concentration of 0.1 g/L. The second group received the same mass concentration (0.1 g/L) of 53 nm particles (the 53 nm mass group). The third group also received 53 nm particles, but at a lower concentration corresponding to the same surface area as the group receiving 180 nm particles (the 53 nm surface area group, concentration 0.029 g/L). The results from this treatment are, for clarity, presented in Supplementary material, (Table S1). The fourth group, the control group, did not receive any nanoparticles. All fish were measured and weight before the experiment started. The study was performed under the permission from the Malmö/Lund Ethical committee (D nr 14 13–12) and was performed according to the current laws in Sweden.

Food chain

Algae (Scenedesmus sp.) with a diameter of approximately 25 μm were cultivated in aquaria. On day 1, 500 mL algae with a concentration of 450 \(\mu g/L\) were mixed with water and particles to a total volume of 1 L in four different test bottles (except for the control bottle, which received only water). After 24 hours Daphnia magna (20 Daphnia/fish) were added to the algae medium. After 2 hours, the Daphnia were collected on a net with a mesh size of 150 \(\mu m\) and washed two times with 150 mL water. Each fish (Crucian carp, Carassius carassius) was then served 20 Daphnia, i.e. 60 Daphnia per aquarium.

We replicated this natural food chain such that the fish eventually ingested, via algae and Daphnia, the same type of amino-modified polystyrene nanoparticles as used for the Daphnia toxicity with diameters of 53 nm and 180 nm (Fig. 2). To distinguish between size and mass effects, two concentrations of the 53 nm particles were used, one that corresponded to the same surface area and one that corresponded to the same mass as the 180 nm particles. The three groups: 180 nm, 53 nm surface area (Table S1) and 53 nm mass were studied together with the control group, that did not receive any nanoparticles. Sixty Daphnia individuals were introduced as food to each fish aquarium every third day for a period of 67 days.

Video analysis

On day 62, we monitored the hunting behaviour of the fish by video recording each aquarium separately during 2 minutes before the fish received food and 10 minutes after. Since the smaller particles were toxic to the Daphnia and thereby possibly affected their interaction with the fish, all groups of fish were on the 62nd day fed with Daphnia that had not received any nanoparticles. Each fish position was registered each second during the whole tracking period using the software ImageJ. The feeding time – the time it took for the fish to consume 50% of the provided food (Daphnia) – was registered. An ANOVA post hoc was used to test differences between treatments.

Brain analysis

On day 64, all fish were collected and anaesthetized using benzocaine. They were measured and weighed before the neck was cut and the brain was extracted. All samples were stored at −80 °C. The brain was weighed and an image was recorded with Olympus SZX7 microscope with an Infinity 1 camera and then freeze-dried and weighed again before it was homogenized in PBS buffer. The area of two gyri in all brain images was measured in pixels2 using ImageJ and further calculated with Matlab. Finally, three brains from each group were analysed with CytoViva hyperspectral microscope. This microscope was equipped with an enhanced darkfield illuminator and visible-near infrared (400–1000 nm) hyperspectral imaging components. The homogenized brain samples were imaged under 60x magnification. Each image captured one pixel line at a time using an automated stage. These pixel lines were compiled to form hyperspectral images, also known as datacubes, which contain spatial and spectral data for each pixel. For each image of exposed brain that was acquired, a spectral library corresponding to polystyrene was created. This was accomplished by gathering several regions of interest from each exposed brain image and filtering the spectra associated with those regions against 3 negative control images (homogenized brain with no polystyrene). Any spectra that matched spectra in the negative controls were eliminated from the polystyrene spectral libraries. Then, the polystyrene was spectrally mapped and identified in the exposed brain images using the Spectral Angle Mapper algorithm.