The abundance and persistence of plastic nanoparticles in aquatic habitats are considered a threat to marine and freshwater biota. However, the risk assessment of plastic particles is complicated due to various factors that need to be considered, including composition, size and environmental abundance.

This study investigated the behavioural response of a key river species, Gammarus roeseli, to dietary exposure of plain biodegradable and non-biodegradable plastic as well as to natural small micro- and nanoparticles. Mortality, feeding, swimming velocity and energy assimilation  endpoints were examined by considering four particles sizes ranging from 30 to 1000 nm in two concentrations.

Contrary to our expectations, neither decreasing size nor increasing abundance of each tested particle impacted any of the examined endpoints. Likewise, dietary exposition with biodegradable plain polylactide did not induce other or stronger effects than non-biodegradable plain polystyrene or natural silica micro- and nanoparticles, as all three particle types did not lead to adverse effects on G. roeseli. These findings also suggest that the functional role of Gammarus roeseli as a shredder is not impaired due to particle occurrence within the exposure range of this study.

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MORTALITY

Mortality was monitored daily, and dead gammarids were removed from the test. Gammarids were counted as dead when no movement of antennae or pleopoda was observed.

FEEDING

To determine the mass of food consumed, the loaded phyll-tabs were dried for three days at 45°C in a drying cabinet (Memmert GmbH), and initial dry weight (dwI ) was determined with a fine scale (Sartorius, 0.01 ± 0.02 mg). Afterwards, tabs were pre-wetted in distilled water for a day for easier feeding and transferred into the allocated treatment. After one week of exposure, the tabs were removed, exchanged with new tabs (pre-weighed and pre-wetted) and dried for two days at 45°C in the drying cabinet. The final dry weight (dwF ) was also determined. Simultaneously, tabs of each treatment were handled the same but without gammarids in the beaker to determine weight loss during the week independent of gammarids feeding on the tabs (dwL ). The mass eaten per week of all living gammarids in the beaker was calculated by subtracting the percentual proportion of the calculated dwL from the dwI and then subtracting the dwF. Subsequently, the mass eaten per gammarid and day was calculated by dividing the mass eaten per week by the sum of the number of feeding gammarids on each day.

SWIMMING BEHAVIOUR

The swimming behaviour of the individual gammarids was monitored at the beginning of the experiment and subsequently every seventh day (Bartonitz et al., 2020). First, individual gammarids were carefully removed with a spoon from the beaker and transferred into a Petri dish with a diameter of 5.5 cm filled with 10 mL of artificial water. The dishes were placed on light boards and at a 30 cm distance under a camera. The gammarids were tracked with Ethovision XT 11 (Noldus, Netherlands) for 10 min and with a sample rate of 25 frames per second. Thus, for each gammarid, the velocity in cm/s was measured and summarised in an Excel sheet. Afterwards, gammarids were transferred using a spoon into a new beaker according to the treatment.

Reference: 

- Bartonitz A, Anyanwu IN, Geist J, Imhof HK, Reichel J, Graßmann J, Drewes JE, Beggel S (2020) Modulation of PAH toxicity on the freshwater organism G. roeseli by microparticles. Environmental Pollution 260:113999. https://doi.org/10.1016/j.envpol.2020.113999

ENERGY ASSIMILATION

The determinations of lipid, glucose and glycogen were based on the assays from Charron et al. (2014) with some modifications. The determination of protein content was based on the Bradford assay (Bradford, 1976) as described by Walker (2002).

The dried gammarid tissue (without head and gut) were homogenised in a 1.5-mL tube with a mortar fixated in a dremel (Micromod 50/e, Proxxon). Then, 200 µL methanol was added, and the remains were homogenised the second time. Another 700 µL methanol was rinsed over the mortar into the tube to wash any sample residues. After homogenisation using a vortexer, the sample was divided into three aliquots: 300 µL was transferred into a 1.5-mL tube for lipid measurement, 300 µL was transferred into the second 1.5-mL tube for glucose and glycogen measurement. Next, 2 x 50 µL was transferred to two glass test tubes for protein measurement.

Lipid and glucose/glycogen measurement assays were further conducted as described in Götz et al. (2021) with some adjustments. The lipid aliquot was divided into two samples of 400 µL after cooling for 20 min for measurements in duplicate. The used amount of reagents in each assay was 2.5 mL instead of 5 mL.

For protein measurement, 50 µL distilled water was added to 50 µL of the sample in each glass test tube. Also, 50 µL of distilled water and 50 µL of methanol were pipetted into an extra glass test tube as blank. Finally, 1-mL Bradford reagent was added to each glass test tube. After 2 min, the colour change from brown to blue allows the photometric measurement of the absorption at 595 nm against the blank. The amount of proteins was calculated using a calibration curve.

Reference:

- Charron L, Geffard O, Chaumot A, Coulaud R, Jaffal A, Gaillet V, Dedourge-Geffard O, Geffard A (2014) Influence of molting and starvation on digestive enzyme activities and energy storage in Gammarus fossarum. PLoS ONE 9:e96393. https://doi.org/10.1371/journal.pone.0096393

- Bradford MM (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilising the Principle of Protein-Dye Binding. Analytical Biochemistry:248–254

- Götz A, Imhof HK, Geist J, Beggel S (2021) Moving Toward Standardised Toxicity Testing Procedures with Particulates by Dietary Exposure of Gammarids. Environ Toxicol Chem 40:1463–1476. https://doi.org/10.1002/etc.4990

- Walker JM (ed) (2002) The protein protocols handbook. Humana Press, Totowa, NJ