Effect of Copper On Bioconcentration of Benzotriazole Ultraviolet Stabilizers (BUVSs) in Common Carp (Cyprinus Carpio)

Benzotriazole ultraviolet stabilizers (BUVSs) have received increasing attention due to their widespread usage, ubiquitous detection and their adverse ecological effect. However, information about the bioaccumulation potential of BUVSs and their joint exposure with heavy metals remains scarce. In this study, we investigated the bioaccumulation kinetics of 6 frequently reported BUVSs in common carp under different Cu concentration for 48 d, and their tissue-specic distribution patterns (liver, kidney, gill, and muscle tissues) were also evaluated. The bioconcentration factors (BCFs) and half-lives (t 1/2 ) in the tissues ranged from 5.73 (UV-PS) to 1076 (UV-327), and 2.19 (UV-PS) to 31.5 (UV-320) days, respectively. The tissue-specic concentration and BCF values followed the order of liver > kidney > gill > muscle with or without Cu exposure. An increase in BCF with rising Cu concentration was observed, which is caused by the decreased depuration rate (k 2 ) in more than half of treatment groups. These results indicated that BUVSs accumulated in sh and provides important insight into the risk assessment of this group of chemicals. on the bioaccumulation of BUVSs was investigated in this study for the rst time, and the effect varied from the compounds and sh tissues. Cu exposure can apparently affect the uptake and eliminate rates of BUVSs. And an increase of BCF value caused by increasing Cu concentration was observed in half of the treatment groups (11 groups out of 21). However, Cu will not signicantly alter the tissue distribution pattern of 6 BUVSs. Overall, these results provide basic data for the risk assessment of BUVSs and their co-presence with copper. Nonetheless, the mechanism of the interaction between BUVSs and heavy metals remains unclear, which requires further elucidation.


Introduction
Ultraviolet (UV) radiation can cause aging of organic materials as well as human skin, posing a threat to both environment and human body (Correa et al., 2021;Zeng et al., 2018). As a result, UV stabilizers have been widely applied into industrial products and personal care products (PCPs). Among all UV stabilizers, benzotriazole ultraviolet stabilizers (BUVSs) have the largest output and most variety (Li and Li, 2007). BUVSs can absorb full spectrum of UV light from 280 to 400 nm (UV-A and UV-B), and they are widely used as additive in building materials, paint, plastics or sunscreens, creams and shampoos Considering the widespread presence of BUVSs, their adverse effects on organisms have been investigated. Hirata-Koizumi et al. (2009) found a genderrelated hepatic peroxisome proliferative activity of HDBB in rats. And some BUVSs are proved to have partial estrogenic activity or to disturb thyroid hormone pathway, development, and locomotory activity of early-stage zebra sh (Feng et al., 2020;Liang et al., 2017). An activation of aryl hydrocarbon receptor pathway in zebra sh eleuthero-embryos was observed after their being exposed to BUVSs. Besides, these compounds are also reported to cause oxidative stress damages in Daphnia magna and zebra sh (Giraudo et al., 2017;Hemalatha et al., 2020). More recently, Li et al. (2019) and Li et al. (2020) revealed their in ammatory effects in sh, which was potentially caused through the AHR-IL17/IL22 pathways.
BUVSs have been considered to be persistent in environment (Nakata et al., 2010) and their lipophilicity/hydrophilicity (log Kow:4.31-8.28) property is the main factor governing their accumulation potential in aquatic organisms (Hemalatha et al., 2020;Xing et al., 2018). For instance, UV-327 showed a signi cant bioaccumulation property in marine mammals from the western North Paci c Ocean with a bioconcentration factor (BCF) value as high as that of persistent organochlorine pesticide, hexachlorocyclohexane (37 000) (Hemalatha et al., 2020). In addition, high bioaccumulation factor (BAF) of BUVSs were also observed in fresh water aquatic organisms from the Pearl River basin in China, and some of the BUVSs congeners showed trophic magni cation behavior with trophic magni cation factor (TMF) > 1 (Xiong, 2017). More recently,  investigated the accumulation and biotransformation of 6 BUVSs in zebra sh under controlled laboratory conditions and reported the BCF values at the range of 1.04-10400 in different sh tissues.
On the other hand, heavy metals such as Cd, Cr, Pb, Cu, Zn, etc., have been widely detected in environmental media including sediments, water, and soils However, studies on the joint effect of copper and BUVSs are still lacking.
In present studies, combined effects of benzotriazole and copper on organisms have been evaluated, considering benzotriazole is widely used as corrosion inhibitor for copper and its alloy (Grillo et al., 2014;Xing et al., 2017;Xing et al., 2018). As a subgroup of benzotriazole with a phenolic group attaching to the benzotriazole structure, BUVSs are also widely used as additives in automobile components and some sports equipment etc (Nakata et al., 2009). Therefore, in this study, we investigated the bioconcentration and distribution pattern of BUVSs in different tissues (liver, kidney, muscle, and gill) of common carp (Cyprinus carpio) and evaluated the effect of copper on their bioaccumulation, which provided a rst glimpse into the cocontamination by heavy metals and BUVSs.
Individual stock solutions of each BUVSs (1g·L −1 ) were made in isooctane. Further dilutions and mixtures of target species were prepared in n-hexane. All the working solutions were stored in brown glass bottles at −20°C.

Uptake and depuration experiments
The experiments were conducted in a semi-static aquarium system with constant aeration. Common carp (5.8 ± 0.6 cm, 3.3 ± 0.7 g) were obtained from a local aquarium in Dalian, Liaoning Province. Fishes were acclimated for two weeks with dechlorinated tap water (dissolved oxygen 7.9 mg·L −1 ± 0.5, pH 7.3-8.0, and at 23 ± 1°C) in advance.
Common carp were exposed to a mixture of six frequently detected BUVSs under constant concentration (10 µg·L −1 for each) in glass tanks containing 100 L of water. According to OECD 305, the chemical concentration in water for bioconcentration tests should not exceed 1% of its 96 h LC 50 . Considering and Cu-high (25.6 µg·L −1 ), respectively. And for each exposure group, there were two parallel groups. Besides, there was a non-spiked tank served as blank group.
Three sh were sampled randomly from each tank on d 2, 4, 8, 12, 16, 20, 24, and 28 during exposure period and on d 30, 32, 36, 40, 44 and 48 during depuration period. Half of the water in tanks were renewed over a period of 24 h to keep the test solution fresh. The length and weight of each sh were measured after being sampled. Fish were anesthetized on ice and then dissected, their four tissues including liver, kidney, gills, and muscle were carefully removed, weighed and stored at −20°C for subsequent analysis.

Sample extraction and analysis
Sample pretreatment was performed using a procedure described by Kim et al. (2011b) and Carpinteiro et al. (2010) with some modi cations. After being homogenized in mortar with the addition of 200 ng internal standard (Allyl-bzt) and anhydrous sodium sulfate, the sh tissue was transferred into 50 mL polypropylene centrifuge tubes. And the samples were extracted with a mixture of dichloromethane and n-hexane in a volume ratio of 8:1. For a better interaction between solvent and sample, the tube was vortexed for 1 min then deposed into ultrasonic bath for 30 min. Hereafter, the tube was centrifuged at 12,000 rpm for 10 min. The extraction step was repeated twice and the supernatant was evaporated to dryness under a gentle stream of nitrogen with heating. The residue was reconstituted in 2 mL n-hexane for a further clean-up by glass column loaded with anhydrous sodium sulfate and silica gel. Dichloromethane was used for eluting and then evaporated to dryness and reconstituted in 1 mL n-hexane then ltered through a 0.22 µm membrane lter for GC-MS analysis. The details of GC-MS analysis and quantitation are listed in the Supporting Information (Text S1 and Table S2). Extractions of BUVSs in water samples followed the steps in the Supporting Information (Text S2).

Quality assurance and quality control (QA/QC)
All glasswares and mortars being used were calcined in mu e furnace at 400°C for 4 h in advance. Procedural blanks going through the same treatment as samples were determined with each batch to monitor potential contamination during analysis. The method detection limit (MDLs) and method quanti cation limit (MQLs) were calculated as three and ten times the standard deviation (SD) of the mean procedural blanks (n = 6), and ranging from 0.05 to 0.81 ng·g −1 wet weight (ww) and 0.15 to 2.70 ng·g −1 ww, respectively. Fish tissues spiked with standards of 3 levels were detected to processed the reproducibility and accuracy of method (n = 3). The recoveries ranged from 85.6-111.2% with relative standard deviations (RSD) ranging from 6.9-23.5% (Table S4).

Data analysis
Bioconcentration kinetic parameters were estimated with a mass balance model (Mackay and Fraser, 2000), where uptake and depuration process can be described by eq. (1): Where C B (ng·g −1 , ww) and C W (ng·L −1 ) are concentrations of chemicals in sh tissue and aquatic environment, respectively. k 1 (L·(kg·d) −1 ) and k 2 (d −1 ) are the rate constants for uptake and depuration process, respectively. t is the exposure time (d). In this study, the elimination of BUVSs caused by metabolism and growth were ignored. Then k 1 can be calculated by tting a nonlinear regression to the eq. (2): And k 2 can be obtained by eq. (3): In this case, the BAF ∞ ifish would correspond to eq. (4): And the half-lives (t 1/2 ) of BUVSs in sh samples can be calculated according to the eq. (5):

Uptake and depuration of BUVSs
During the exposure periods, the concentrations of 6 BUVSs in water were maintained relatively stable (10 µg·L −1 ) ( Table S7). The bioconcentration pro les of 6 BUVSs in common carp under different Cu concentrations are presented in Fig.1. For most of the groups, BUVSs showed similar accumulation patterns, except for UV-PS, UV-326, and UV-329 in muscle, where no increasing tendency was observed. In the uptake phase, the concentrations of BUVSs kept increasing, rapid uptake of BUVSs was observed in all treatment groups. As for the depuration phase, the concentrations of BUVSs showed a sharp drop in the rst 2-4 days then decreased slowly. What's more, most BUVSs remained at a comparatively high level in some sh tissues at the end of the depuration phase, suggesting their latent high persistence in sh.
When it comes to the effect of copper, we found signi cant differences between BUVSs concentrations in sh tissues under different Cu exposure (19 groups out of 21, P < 0.05, Fig. S1). For more than half of the treatment groups (12 groups out of 19), the concentrations of BUVSs decreased as Cu concentration increasing (Fig. 2), suggesting an inhibitive effect of Cu on the accumulation of BUVSs. However, in the rest of the treatment groups (mostly in kidney 3/7 and muscle 2/7), the concentrations of BUVSs under low Cu concentration were apparently higher than those in another two groups (Fig. 2), which means low Cu concentration may also stimulate the accumulation of BUVSs in sh.

Bioconcentration factor of BUVSs
The uptake and elimination kinetics of 6 BUVSs in liver, kidney, gill, and muscle of sh for all treatment groups were investigated respectively. The uptake, elimination rate constants (k 1 , k 2 ) were acquired through the mass balance model (Mackay and Fraser, 2000), results are shown in Table 1 Table 1 Kinetic parameters of 6 BUVSs in common carp at concentration of 10 µg·L −1 under different Cu concentrations.
The existence of Cu can signi cantly affect the BCF values (Fig. 3). In general, high-dose Cu exposure led to an increase of BCF values, mainly because of the apparent decrease of k 2 . For the same reason, t 1/2 were also found to be longer in those groups, the longest t 1/2 (31.5 d) was recorded for UV-320 in liver under high-dose Cu exposure. In half of the treatment groups, the BCF values increased as Cu concentration increased. However, low-dose Cu might also cause a decrease of BCF values in some of the groups, which mostly occurred in liver and kidney. Besides, an opposite trend was found in a few groups, the increase of Cu concentration decreased the BCF values. Similarly, in a controlled experimental study of FQs, Zhao et al. (2018a) reported signi cant increase of BCF values of enro oxacin in liver and gill tissues with decreasing Cu exposure concentration, but the opposite trend was observed for enro oxacin and o oxacin in muscle of zebra sh. In another study, the presence of Cu was found to decrease the uptake of PFOA by earthworms, thus causing lower biota-to-soil accumulation factors (BSAF) (Zhao et al., 2018b). It was speculated that the decrease was a result of enhanced sorption of PFOA to soil in the presence of divalent cations (Cu 2 ), which reduced its concentrations in the pore water and desorption from ingested soil particles in earthworm gut. However, this inference cannot be applied into sh, as we conducted our experiment only in aquatic environment. Overall, further study is required to investigate the speci c mechanisms of Cu and organic pollutants co-exposure in sh.

Tissue distribution of BUVSs in common carp
For a better understanding of the bioaccumulation of BUVSs in common carp, tissue distribution was investigated by calculating concentration percentages of each compound in liver, kidney, gill, and muscle at the end of the uptake phase, as shown in Fig. 4.
In general, BUVSs concentrations in sh tissues followed a trend of the order of liver > kidney > gill > muscle. Among 6 BUVSs, the largest relative In the present study, Cu exposure did not signi cantly change the distribution patterns of BUVSs in sh tissue (P > 0.05), the order of BUVSs concentrations in each tissue remains same as that in Cu-blank groups, which is in accordance with the conclusion from the study of FQs in zebra sh (Zhao et al., 2018a) and PFOS and PFOA in Carassius auratus (Feng et al., 2015). However, as the concentration of Cu increased, the relative proportion of UV-PS increased while that of UV-320 and UV-327 decreased in liver. Meanwhile, their percent in kidney showed opposite trends. For UV-329, its percent increased in liver and decreased in gill. As for the proportion of BUVSs in muscle, slight variation was observed after Cu exposure.

Conclusion
In brief, our study indicated that the bioaccumulation of BUVSs differed among sh tissues. Their tissue-speci c BCF and concentration generally followed the order of liver > kidney > gill > muscle. Besides, effect of copper on the bioaccumulation of BUVSs was investigated in this study for the rst time, and the effect varied from the compounds and sh tissues. Cu exposure can apparently affect the uptake and eliminate rates of BUVSs. And an increase of BCF value caused by increasing Cu concentration was observed in half of the treatment groups (11 groups out of 21). However, Cu will not signi cantly alter the tissue distribution pattern of 6 BUVSs. Overall, these results provide basic data for the risk assessment of BUVSs and their copresence with copper. Nonetheless, the mechanism of the interaction between BUVSs and heavy metals remains unclear, which requires further elucidation.    Tissue distribution of 6 BUVSs in common carp after 28 days exposure.

Supplementary Files
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