Table 3.

Overview of in vivo studies of the immunotoxic effects of nano- and microplastics in invertebrates

Reference	Nano-micro plastics	Dosage	Duration of exposure	Route of exposure	Species	Observed immunotoxic effects
Nanoplastics
Sadler et al., Environ. Pollut. 2019 [102]	Carboxylate- modified polystyrene beads 500 nm	1.25 ± 0.205 particles/L, or 85.6 ± 14.0 mg/L	1 year	Tank water	Cladoceran (Daphnia magna)	↑ Hemocyte counts
Brandts et al., Sci.Total Environ. 2018 [77]	Polystyrene ~ 110 nm	0.005–0.05-0.5-5-50 mg/L	96 h	Tank water	Mussel (Mytiulus galloprovincialis)	Hemolymph ↓total antioxidant capacity (5 mg/L) ↑ DNA damage (all dosages)
Auguste et al., Front.Immunol 2020 [103]	Amino-modified nanopolystyrene 50 nm	10 μg/L	24 h	Tank water	Mussel (Mytilus. galloprovincialis)	Hemocytes One exposure: ↓ mitochondrial membrane potential (MMP), ↑ lysosomal acidification ↓ lysosomal membrane stability ↑ lysozyme release No changes in total hemocyte count, subpopulations, phagocytic activity and ROS production ↓ transcription of PCNA and p53 No change in hemolymph bactericidal activity Two exposures with 72 h resting period between: normal hemocyte lysosomal stability, MMP, and lysozyme activity ↓ lysosomal membrane destabilization ↓ fully mature phagocytes ↑ bactericidal activity ↑ transcription of immune-related genes
Auguste et al. Marine Environmental Research 2020 [104]	Amino-modified nanopolystyrene 50 nm	10 μg/L	96 h	Tank water	Mussel (Mytilus galloprovincialis)	Hemolymph ↓ phagocytosis, ↑ ROS and lysozyme activity ↓ NO production. Hemolymph microbiota composition shift
Microplastics
Paul-Pont et al., Environmental Pollution 2016 [76]	Polystyrene microbeads (2 and 6 μm)	32 μg/L	7 days	Supplied with Chaetoceros mueller algae as a food source	Mussel (Mytilus spp)	↑ hemocytes mortality and ROS production
Liu et al., Sci. Total Environ.,2019 [99]	Polystyrene 5 μm	0.04–0.4-4-40 mg/L	7, 14, and 21 days	Tank water	Crab (Eriocheir Sinensis)	Immune parameters in the hemolymph Hemocyanin content After 7 days: ↑ at 0.04 mg/L After 14 days: ↑ at 0.04 and 0.4 mg/L After 21 days: ↓at all dosages Acid phosphatase activity After 7 days: ↑ at 4 and 40 mg/L After 14 days: ↑ at 0.04 and 0.4 mg/L, ↓ 4 and 40 mg/L After 21 days: ↑ at 0.04 mg/L, ↓ 4 and 40 mg/L Alkaline phosphatase activity After 7 days: ↑ at 0.04 mg/L, ↓ 4 and 40 mg/L After 14 days: ↑ at 0.04 mg/L After 21 days: ↓ at all dosages Lysozyme activity After 7 days: ↓ at 40 mg/L After 14 days: ↓ at 0.4 and 4 mg/L After 21 days: ↓ at 4 et 40 mg/L Phenoloxidase activity After 7 days: ↑ at 0.04, 0.4, 4 mg/L, ↓ 40 mg/L After 14 days: ↓ at 0.4, 4, and 40 mg/L After 21 days: ↓ at all dosages Expression of immune-related genes in the hemocytes Hemocyanin and lysozyme: dose dependent ↓ Caspase: ↑ 0.04 and 4 mg/L, ↓ at 40 mg/L MyD88: ↑ at all dosages
Murano et al. Environmental Pollution 2020 [105]	Polystyrene microbeads (10 and 45 μm)	10 particles /mL	24 h 48 h 72 h	Tank water	Mediterranean sea urchin (Paracentrotus lividus)	↑ total number of immune cells ↑ ratio between red and white amoebocyte (at 3 times for 10 μm beads and only at 48 and 72 h for 45 μm beads) ↑ intracellular levels of reactive oxygen and nitrogen species (at 24 h only for both 10 and 45 μm beads) ↑ total antioxidant capacity (at 72 h for 10 μm beads)
Revel et al., Environ Sci Pollut Res Int. 2020 [106]	Polyethylene and Polypropylene 0.4–400 μm	10–100 μg/L	10 days	Soil	Ragworm (Hediste diversicolor)	Coelomocytes No variation of phagocytosis activity, phenoloxydase, and acid phosphatase
Revel et al., Mar. Pollut. Bull. 2020 [107]	Polyethylene and Polypropylene fragments < 400 μm	0.008–10-100 μg of particles/L	10 days	Tank water	Pacific oyster (Crassostrea gigas)	Hemolymph No variation of ROS production, acid phosphatase activity, and DNA damage
Green et al., Environ. Pollut. 2019 [108]	High density Polyethylene (HDPE) 0.48–316 μm Polylactic acid (PLA) 0.6–363 μm	HDPE 845 particles/L PLA 1296 particles/L	52 days 2 h/day	MP-dosed microalgae Isochrysis galbana	Blue mussel (Mytilus edulis)	Hemolymph proteome HDPE group Dysregulation of 6 protein involved in immune response ↑ three complement C1q domain-containing (C1qDC) proteins (FR715598.1; FR715581; HE609753.1), and fibrinogen-related protein (OPL33687.1) ↓ macrophage migration inhibitory factor (HE609105.1), Microfibril-Associated Glyco 4 (OPL32613.1) PLA group dysregulation of 3 protein involved in immune response ↑ C1Q Domain Containing 1Q19 (FR715598.1) and Fibrinogen-Related (OPL33687.1) ↓ Microfibril-Associated Glyco 4 (OPL32613.1)
Revel et al. Frontiers in Environmental Science 2019 [78]	Commercial polyethylene and polystyrene mixture (< 400 μm)	0.008, 10, 100 μg/L	10 days	Tank water	Mussel (Mytilus spp.)	Hemolymph No variation hemocyte count ↑ acide phosphatase activity (0.008 and 10 μg/L) ↑ DNA damage (10 and 100 μg/L)
Both nanoplastics and microplastics
Shi et al., J. Hazard. Mater. 2020 [109]	Polystyrene beads 500 nm (NP) and 30 μm (MP)	0.29 mg/L	14 days	Tank water	Bivalve mollusk (Tegillarca granosa)	Hemocytes ↓ total hemocytes count ↓ phagocytosis ↓ viability (NP only) ↑ ROS content ↑ Caspase 3 activity ↑ malondialdehyde content ↓ ATP content (NP only) ↓ pyruvate kinase activity ↑ GABA content
Tang et al., Environ. Pollut. 2020 [110]	Polystyrene 500 nm (NP) and 30 μm (MP) and	1 mg/L	4 days	Tank water	Bivalve mollusk (Tegillarca granosa)	Hemocytes ↓ hemocytes count, basophils count, phagocytosis ↓ lysozyme (NP only) ↓ TLR4 (NP only), TRAF6, IKKα, NFκB gene expression ↑ Bcl2 (NP only), Caspase 3, Calmodulin gene expression