DOI: 10.5061/dryad.547d7wmbj
title: Raw data on mortality, feeding rates, swimming behavior and energy reserves: Goetz et al 2022

description: 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.
description: &lt;Include links or references to publications or other
 documentation containing experimental design or protocols used in data
 collection&gt; 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