Facilitating microplastic ingestion in aquatic models: A verified protocol for daphnia magna as a trojan horse vector

Abstract

Microplastic pollution poses a significant environmental threat due to its persistence, widespread distribution, and inherent toxic potential. Despite the increasing number of publications in this field, a standardized protocol for the laboratory intake of microplastics by Daphnia magna has yet to be established. In this study, we introduce a verified protocol designed to facilitate the ingestion of microplastic particles (MPs) by D. magna, ranging in size from 5-55 µm. This protocol can be further applied to evaluate the toxicity of MPs on D. magna, a crucial organism model in ecotoxicology. Furthermore, this protocol can be used to assess toxicity of MPs in other aquatic species, such as fish, by using daphnids as a vehicle for ensuring the ingestion of these particles. Consequently, this protocol can be applied to study also one of the most pressing concerns regarding exposure to MPs, the transfer of MPs through different trophic levels, which has a great potential for ecotoxicological studies.•The influence of MPs concentration, duration and exposure dynamics and D. magna age/size in MPs intake were tested.•We have determined the optimal conditions for promoting microplastic ingestion by D. magna.

Keywords: Aquatic Microplastic Ingestion and Transfer Protocol (AMITP); Daphnia magna; Food consumption; Microplastics; Pollution assessment; “Trojan Horse” vector.

Graphical abstract

Fig. 1

D. magna images comparing daphnid visualization using A) optical image using the Leica EZ4W microscope (Leica Microsystems, Germany) working at 10X magnification, B) transmission light (monochrome camera) combined with RFP and CFP/YFP fluorescence, C) CFP/YFP fluorescence and D) RFP fluorescence using the EVOS m7000 Imaging system working at 10X magnification. The bright (31%), gamma (0.55) and saturation (140) were adjusted for the optical image acquired using the Leica microscope system without applying any further processing adjustment. The bright, coarse and fine parameters were adjusted in each image acquired by the EVOS system to achieve the best visualization of the MPs in the digestive system of daphnids.

Fig. 2

Clone F D. magna images comparing their size from 0 to 30 days. Images were achieved using a Leica EZ4W microscope working at 30X magnification for D.magna at 0 days, 25X for D.magna at 4 days, 15 X at 7 days and 10X at 12 and 30 days. The bright (31%), gamma (0.55) and saturation (140) were adjusted for the optical images acquired using the Leica microscope system without applying any further processing adjustment.

Fig. 3

D. magna adults exposed to A) 1–10 mg/L of the green particles for 2 h and B) 10 mg/L for 2 h and 24 h. All images were acquired using the EVOS m7000 Imaging system working at 10X magnification combining transmission light (monochrome camera) CFP/YFP fluorescence (green). The bright, coarse and fine parameters were adjusted in each image to achieve the best visualization of the MPs in the digestive system of daphnids.

Fig. 4

Clone F D. magna at 8–10 days old (A) and 12–18 days old (B) exposed to green MPs alone and in combination of 1:2 MPs-20:MPs-50 microplastic mixture visualizing fluorescence signals individually and merged (C). All images were acquired working on an EVOS m7000 Imaging system at 10X magnification using fluorescence RFP (orange) and CFP/YFP (green) light cubes in combination with visible light (monochrome camera), and both fluorescence light cubes individually. The bright, coarse and fine parameters were adjusted in each image to achieve the best visualization of the MPs in the digestive system of daphnids.

Fig. 5

Clone F D. magna at 12–18 days exposed to 1:2 MPs-20:MPs-50 microplastic by static and rotation (2 rpm) conditions. All images were acquired working on an EVOS m7000 Imaging system at 10X magnification using fluorescence RFP (orange) and CFP/YFP (green) light cubes in combination with visible light (monochrome camera). The bright, coarse and fine parameters were adjusted in each image to achieve the best visualization of the MPs in the digestive system of daphnids.

Fig. 6

Clone F D. magna juveniles at 4–6 days old (A) and 7–10 days old (B) exposed to violet MPs, green MPs alone and in combination of 1:2 violet: green (1V:2 G) microplastic mixture under rotation conditions. All images were acquired working on an EVOS m7000 Imaging system at 10X magnification using fluorescence RFP (orange) and CFP/YFP (green) light cubes in combination with visible light (4–6 Days old: color camera; 7–10 days: monochrome camera). The bright, coarse and fine parameters were adjusted in each image to achieve the best visualization of the MPs in the digestive system of daphnids.

Fig. 7

Clone F D. magna at 12–18 days old (A) and 28–30 days old (B) exposed to green MPs alone and in combination of 1:2 MPs-20:MPs-50 microplastic mixture. All images were acquired working on an EVOS m7000 Imaging system at 10X magnification using fluorescence RFP (orange) and CFP/YFP (green) light cubes in combination with visible light (monochrome camera), and both fluorescence light cubes individually. The bright, coarse and fine parameters were adjusted in each image to achieve the best visualization of the MPs in the digestive system of daphnids.

Fig. 8

Clone F D. magna at 12–18 days old (A) and 28–30 days old (B) exposed to 1:2 MPs-20:MPs-50 microplastic mixture 1) before and 2) after the peroxide digestion. All images were acquired working on an EVOS m7000 Imaging system at 10X magnification using fluorescence RFP (orange) light cube individually and in combination with visible light (monochrome camera). The bright, coarse and fine parameters were adjusted in each image to achieve the best visualization of the MPs in the digestive system of daphnids and the MPs on the peroxide digested tissue.

Fig. 9

Graphical illustration of the main steps of the experimental set-up: A) Growing daphnids until they are 12–18 days old; B) starving step in clean ASTM water; C) 10 daphnids are placed in 20 mL vials for the MPs exposure (10 mg/L); D) daphnids are exposed to MPs for 24 h under dynamic conditions (rotary shaker, 2 rpm); E) washing external MPS from daphnids previous to imaging acquisition; F) sample preparation for imaging visualization, embedding daphnids on 3% methylcellulose on regular glass slides.

Fig. 10

D. magna individuals at 12–18 days old exposed to 3 different MP types (PE, PS and PMMA). All these optical images were acquired using a Leica EZ4W microscope system working at 10X magnification. The bright (31%), gamma (0.55) and saturation (140) were adjusted for the optical images acquired using the Leica microscope system without applying any further processing adjustment.