Bioaccumulation Behaviors of Copper Phthalocyanine Nano-pigments in Adult Zebrafish ( Danio rerio )

. Copper phthalocyanine (CuPc) has the largest application market of blue nano-pigments (NPs), which can release from relative products to aquatic environment during daily use. However, bioaccumulation of CuPc NPs in high trophic-level aquatic organisms is unclear. Therefore, bioaccumulation behaviors of CuPc in zebrafish as model aquatic organism were investigated. In this work, CuPc NPs were irregular in shape with an average size of 76.8 nm, whose crystal phase was a common beta phase according to the XRD spectrum. After 10 d of exposure to CuPc NPs, the weight of zebrafish at both low and high concentrations showed no obvious difference with con-trol, indicating little influence of CuPc on the survival of zebrafish. It was noted that amounts of Cu accumulated in the liver (982.84 ng/g) and skin (609.08 ng/g), and a small accumulation of Cu was observed in the gut, heart, and gill. CuPc NPs could not lead to Cu accumulation in the brain and muscle. This work helps to better understand the bioaccumulation behaviors and potential risks of CuPc NPs.


Introduction
Nano-pigments (NPs) are materials with high performance to give objects bright color, which are widely utilized in coatings, plastics, paints, inks, and construction materials. Copper phthalocyanine (CuPc, C 32 H 16 N 8 Cu) NPs have the largest application in blue pigments, whose market accounting for a quarter in whole artificial pigments [1] . It is noted that CuPc NPs can release from commodities during daily use, resulting in the exposure of humans and environments [2] . CuPc NPs were detected in human tissues and rivers, indicating occurrence in natural environments [3,4] . In addition, CuPc NPs contain heavy metal copper with possible threats to ecosystems. However, the environmental risks of CuPc NPs remain limited. Bioaccumulation reflects both ecotoxicity and risk assessment of nanomaterials [5] . Pang et al. (2021) found the low bioaccumulation potential of CuPc NPs in lower trophic-level aquatic organisms, due to low bioavailability [6] . Bioaccumulation capacities of CuPc NPs in other organisms at higher trophic levels (e.g. fish) are unknown. Fish is an important ecological and economic species, which is tightly related to human health [7] . Zebrafish (Danio rerio) is commonly regarded as a model organism for freshwater fish. Therefore, in this work, the adult zebrafish is selected to explore the bioaccumulation behaviors of CuPc NPs. Results are helpful to understand the biological process of environmental pigments.

Materials and characterizations
CuPc NPs were purchased from Sigma-Aldrich (USA) with irregular particle shapes (< 100 nm). The shape and size were observed by the transmission electron microscope (TEM, JEOL, Japan), which was equipped with an energy dispersive spectroscopy (EDS). The diameters of CuPc particles were counted and calculated by the Image J software according to TEM images. The crystal phase of CuPc was characterized by the X-ray diffraction (XRD, Rigaku Smartlab SE, Japan).

Fish maintenance and exposure
Adult zebrafish (4 months, AB wild-type) were purchased from the China Zebrafish Resource Center (Wuhan, China), and cultured in a flow-through aquatic system with a light/dark cycle of 14:10. Charcoal-filtered fully aerated tap water was used as aquaculture water. Before exposure, zebrafish was acclimated for 14 d in 2 L glass beakers (with 1.5 L of aquaculture water). Then, CuPc NPs were suspended fully in aquaculture water at two concentrations (0.1 µM, 0.5 µM), and exposed to zebrafish for 10 d. Specifically, a total of 15 glass beakers were used, and each beaker cultured 10 zebrafish. There were three exposure settings (non-exposure for the control, 0.1 µM CuPc for a low concentration level, and 0.5 µM CuPc for a high concentration level), and each setting had five replicates. During the exposure, zebrafish were fed with the brine shrimp (Artemia salina) at twice a day, and the exposure solutions were replaced every day. At the same time, the weight of zebrafish was recorded every day using an electronic balance. The weight at each exposure day was minus the weight before exposure to obtain the weight variations.

The detection of Copper content in biological tissues
After exposure, zebrafish were euthanatized by ice water mixture. Then, fish tissues (gut, liver, brain, heart, skin, gill and muscle) were collected by dissection, and the weight of tissues were recorded. All the exposure and euthanasia procedures were based on the OECD guideline (2012) for the measurement of Cu contents in fish tissues [8] . Fish tissues were digested by 3 mL HNO 3 (Sinopharm, China) and 0.1 mL HClO 4 (Sinopharm, China) in the microwave digestion system (GEM MARS 6, USA). After dilution, the Cu concentrations in tissues were detected using an inductively coupled plasma mass spectrometer (ICP-MS, PerkinElmer NexION 300D, UK).

Statistical analysis
For Cu detection, five independent samples were respectively treated for ICP-MS analysis. Statistical differences were determined using SPSS 26.0 by one-way analysis of variance (ANOVA) followed by a least significant difference (LSD) test (p < 0.05 for the statistical significance). Data are presented as means ± standard, and blank concentrations were subtracted from actual samples to revise experimental contamination.

Characterization of CuPc NPs
According to TEM image, CuPc NPs were irregular in shape and tended to form particle agglomerates, indicating poor dispersion in the deionized water ( Figure 1A). Size distribution showed that the average size of CuPc NPs was 76.8 ± 33.5 nm ( Figure 1B). The main elements in CuPc NPs were C, N and Cu, suggesting high purity of experimental materials ( Figure 1C). The most important crystal forms of CuPc NPs were alpha and beta forms, which showed different characteristic peaks in XRD spectrum (6.74° and 7.25° for alpha form, 12.60° and 9.20° for beta form) [9] . XRD spectrum showed that the CuPc in this work was beta form, which had a more stable structure and larger application than the alpha form ( Figure 1D).

Weight of zebrafish during exposure
The weight variation of zebrafish was shown in Figure 2. After 10 d of CuPc NPs exposure, both low and high contraction treatments showed no significant difference with the control in the weight. The weight fluctuation range of the high contraction treatment (0.5 µM) was larger than the control (Figure 2A, C). Results indicated that CuPc NPs caused little influence on the survival of the zebrafish. This result was consistent with Kühnel et al., who found that CuPc had no toxicity effect on zebrafish embryos at an extremely high concentration of 100 mg/L [10] .  Figure 3 showed the Cu contents in different zebrafish tissues after exposure to CuPc NPs. Results showed that CuPc NPs could not lead to Cu accumulation in the brain and muscle. Apparently, amounts of Cu accumulated in the liver (982.84 ± 221.93 ng/g) and skin (609.08 ± 166.93 ng/g) at a high concentration of CuPc NPs (0.5 µM), which is probably because the liver was the target organ of pollutants and CuPc NPs can adsorb in the skin, respectively [11] . Also, a small quantity of Cu could accumulate in the gut, heart, and gill. Therefore, exposure to CuPc NPs could cause heavy metal Cu to accumulate in zebrafish. The result was different from low bioaccumulation of CuPc in lower trophic-level aquatic organisms (e.g. algae (Raphidocelis subcapitata) and crustaceans (Daphnia magna)), which might attribute to higher bioavailability of zebrafish [6] . Overall, the present work first found the Cu accumulation by CuPc NPs in the fish, revealing the potential risk to food chain delivery.

Conclusion
In this work, the bioaccumulation behaviors of CuPc NPs in adult zebrafish were investigated. The main results are as follows: (1) The CuPc NPs were irregular in shape with an average size of 76.8 nm. The XRD spectrum showed CuPc NPs were beta crystal phase.
(2) The weight of zebrafish was stable during exposure, indicating little influence of CuPc NPs on the survival of zebrafish.
(3) CuPc NPs could cause heavy metal Cu accumulation in zebrafish tissues, especially for the liver and skin. There was risk of CuPc NPs to environmental species by bioaccumulation and potential food chain delivery.