Estimating the Bioaccumulation Potential of Hydrophobic Ultraviolet Stabilizers Using Experimental Partitioning Properties.

Although hydrophobic ultraviolet (UV) stabilizers are an emerging environmental concern because of their widespread occurrence, persistence, and bioaccumulation potential, experimental values of their partitioning properties required for risk assessment are scarce. In this study, n-octanol-water partition (Kow) and lipid-water partition constants (Klipw), which are key parameters for environmental risk assessment, were experimentally determined for five selected hydrophobic UV stabilizers (UV326, UV327, UV328, UV329, and UV531) based on third-phase partitioning among polydimethylsiloxane (PDMS), water, and n-octanol/lipid. The partition constants between PDMS and water (KPDMSw), obtained using the dynamic permeation method were used to derive Kow and Klipw. The obtained log Kow and log Klipw values were in the ranges of 7.08-7.94 and 7.50-8.34, respectively, indicating that the UV stabilizers exhibited a high bioaccumulation potential in aquatic environments. The experimental Kow and Klipw values obtained in this study provide valuable information for the evaluation of the fate, distribution, bioavailability, and toxicity of the UV stabilizers in aquatic environments.

1. Introduction

Ultraviolet (UV) stabilizers have been widely used in personal care products, as additives in polymeric food-contacting materials, and as surface coatings [1,2]. Benzotriazoles and benzophenones are among the most commonly used UV stabilizers [3]. These UV stabilizers are aromatic and highly hydrophobic molecules that are of emerging environmental concern owing to their widespread occurrence, persistence, bioaccumulation potential, and toxicity [4,5]. With the increasing production and consumption of UV stabilizers, their residues have been found in humans (e.g., breast milk and urine) as well as various environmental media (e.g., coastal environments or house dust) [5,6,7,8,9,10,11,12,13,14]. Recently, 2.4-di-tert-butyl-6-(5-chloro-2H-benzotriazol-2-yl)phenol (UV327) and 2-(2H-benzotriazol-2-yl)-4.6-bis(2-methyl-2-butanyl)phenol (UV328) have been listed as substances of very high concern (SVHC) under REACH (Registration, Evaluation, and Authorization of Chemicals) owing to their high potential for bioaccumulation, persistence, and toxicity [15], and (2-hydroxy-4-octoxyphenyl) phenylmethanone (UV531) has been included in the European Community Rolling Action Plan (CoRAP) for evaluation in the upcoming years [16]. Although these benzotriazoles and benzophenones have received considerable attention from researchers and policymakers, it is challenging to experimentally obtain fundamental parameters for risk assessment, such as n-octanol-water partition (Kow) and lipid–water partition constants (Klipw), owing to their strong hydrophobicity.

Kow is the most widely used parameter for predicting the environmental distribution and potential bioaccumulation of persistent organic pollutants (POPs) [17]. However, previous studies have shown thermodynamic differences between the n-octanol–water partitioning system and the actual biological uptake because the interactions of pollutants with highly organized lipid membranes are different from those with bulky octanol solvents [17,18]. To evaluate the fate and behavior of hydrophobic organic chemicals (HOCs) in aquatic environments more precisely, intermediate lipid–water systems have recently been used in pharmacology and environmental toxicology to assess the bioconcentration of organic compounds. Only a few experimental partition constants have been reported for storage lipids, which are the major class of lipids in living organisms [19]. Therefore, to assess the bioaccumulation potential of UV stabilizers in aquatic environments, it is essential to obtain both Kow and Klipw values.

UV stabilizers (i.e., UV328, UV329, and UV531) are classified as high-production-volume chemicals (>1000 metric tons per producer/importer annually) [20], and their physicochemical properties required for registration have been measured and submitted [21]. However, experimental Kow values have been reported for a few hydrophobic UV stabilizers using the HPLC method (database is available in OECD QSAR Toolbox 4.4.1—https://qsartoolbox.org, accessed on 11 February 2022) [22], which might be limited for highly hydrophobic chemicals (log Kow > 6) owing to the limited number of highly hydrophobic substances used for the calibration of the method. Furthermore, no Klipw values have been reported for UV stabilizers. Experimental methods have been developed for “difficult-to-test” compounds, such as highly hydrophobic UV stabilizers [23,24]. The partition constants for highly hydrophobic organic chemicals can be estimated using high-performance liquid chromatography (HPLC) [25]; kinetic methods, such as the dynamic permeation method [24]; or the third-phase partitioning method [26]. Thus, precisely measured values of Kow and Klipw should greatly help to improve evaluations of the distribution, bioavailability, exposure, and toxicity of UV stabilizers in aquatic environments [27].

The aim of this study was to experimentally determine Kow and Klipw values of five widely used UV stabilizers: 2-(5-chloro-2H-benzotriazol-2-yl)-4-methyl-6-(2-methyl-2-propanyl) phenol (UV 326), UV 327, UV 328, 2-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol (UV329), and UV531. The distribution constants of the selected UV stabilizers between polydimethylsiloxane (PDMS) and water (KPDMSw) were obtained using the dynamic permeation method. Using KPDMSw values, Kow and Klipw values were determined using the third-phase partitioning method. The experimentally determined log Kow and Klipw values were then compared with available estimated values in the literature and used to critically discuss the fate of UV stabilizers and their accumulation in aquatic environments.

2. Materials and Methods

2.1. Materials and Chemicals

The five UV stabilizers, namely UV326 (>98%), UV327 (>98%), UV328 (>98%), UV329 (>98%), and UV531 (>98%), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Detailed information regarding the five target chemicals is presented in Table 1. Fish oil from menhaden was purchased from Sigma-Aldrich. All solvents (methanol, acetonitrile, and water) used in this study were of analytical grade. Medical-grade polydimethylsiloxane (PDMS) sheets (thickness = 1 mm, density = 1170 kg m−3) were purchased from Specialty Silicone Products, Inc. (Ballston Spa, NY, USA). The PDMS sheets were cut into disks (diameter = 6 mm) to determine the partition constants. The custom-cut PDMS disks and sheets were cleaned using n-hexane, followed by methanol for 2 h each and stored in methanol until use.

Table 1

Names, molecular structures, molecular weights, empirical formulas, and CAS numbers of UV326, UV327, UV328, UV329, and UV531.

Compounds	Molecular Structure	Molecular Weight(g mol−1)	EmpiricalFormula	CAS No.
UV326		315.80	C17H18ClN3O	3896-11-5
UV327		357.88	C20H24ClN3O	3864-99-1
UV328		351.49	C22H29N3O	25973-55-1
UV329		323.43	C20H25N3O	3147-75-9
UV531		326.436	C21H26O3	1843-05-6

2.2. Partition Constants between PDMS and Water (KPDMSw)

KPDMSw values were measured using the aqueous boundary layer (ABL) permeation method. The theory and experimental procedure for this method have been described in detail in existing literature [24,26]. Briefly, by neglecting the mass loss in the aqueous solution between the two PDMS disks because of the high holding capacity of PDMS for HOCs, the concentration in the acceptor disk (CacceptorPDMS) is given by Equation (1), where C0 (mM) is the initial concentration of chemicals in the donor PDMS disk and k (s−1) is the mass transfer-rate constant through the ABL.

Equation (2) can be obtained by rearranging Equation (1), and the mass transfer-rate constant (k) can be determined using a linear regression between and t. Mass transfer resistance in aqueous solution is dominant for highly hydrophobic chemicals because PDMS is an amorphous polymeric material. Thus, KPDMSw (Lw LPDMS−1) can be derived using Equation (3), and the aqueous diffusion coefficient (D in m2 s−1) can be estimated from the relationship with the molar mass of the five UV stabilizers using Equation (4) [24]. where δ is the thickness of the aqueous boundary layer (m), A is the surface area of PDMS (m2), V is the volume of PDMS (m3), and MW is the molecular weight of the chemical (g mol−1). A donor PDMS disk (diameter = 6 mm, thickness = 1 mm) was loaded with each UV stabilizer using methanol as the solvent for 24 h (Figure 1a). Donor and acceptor PDMS disks were placed at opposite ends of a custom-made glass well filled with water. The aqueous solution between the two disks was agitated with a stainless steel disk (diameter = 5.08 mm, thickness = 0.635 mm) at 300 rpm using a VP710F tumble stirrer (V&P Scientific Inc., San Diego, CA, USA) (Figure 1b). The thickness of the ABL was set to 12.5 μm because the experimental system used in this study was the same as that used in previous studies [24,26]. After a designated time, the disks were removed and rinsed with 1 mL methanol:water (6:4, v/v) and 1 mL water. The UV stabilizers in the disks were extracted using 1 mL acetonitrile (ACN) for 24 h. The mass transfer rate was calculated by measuring the changes in the concentrations of both donor and acceptor disks (Equation (2)). KPDMSw was derived using the mass transfer-rate equation (Equation (3)). All experiments were conducted at 25 °C. A schematic diagram for determining KPDMSw is shown in Figure 1a,b.

Figure 1

Schematic diagram of the measurement of the partition constants of the five UV stabilizers. (a) Loading selected UV stabilizers from MeOH solution to donor PDMS; (b) partition constants between PDMS and water (KPDMSw) using the aqueous boundary layer-permeation method [24]; (c) determination of n-octanol-PDMS and fish oil-PDMS partition constants (Kn-octanol-PDMS and Kfishoil-PDMS).

2.3. n-Octanol-Water and Fish Oil–Water Partition Constants

The partition constants between n-octanol or fish oil and PDMS (Koctanol-PDMS or Kfish oil-PDMS) were obtained by measuring the equilibrium concentrations of n-octanol or fish oil and PDMS in a batch system. Then, the Koctanol-PDMS or Kfish oil-PDMS values were used to calculate Kow or Klipw, assuming that the activity coefficients in PDMS were the same in water and in n-octanol or fish oil. A PDMS disk (diameter = 10 mm, thickness = 1 mm) was loaded with each UV stabilizer, dissolved in a 500 or 1000 mg L−1 methanol, for 24 h (Figure 1a). The loaded PDMS disks were rinsed with 1 mL methanol:water (6:4, v/v) and 1 mL water and placed in vials containing 125, 166, and 250 μL of n-octanol or fish oil (Figure 1c). Each vial was agitated at 25 °C and 150 rpm in the dark in a shaking incubator. Preliminary experiments showed that 24 h and 48 h were sufficient to attain equilibrium in the octanol–PDMS and fish oil–PDMS systems, respectively. After equilibrium was established, the PDMS disk was collected from the vial, rinsed using 1 mL methanol:water (6:4, v/v) and 1 mL water, and extracted using 1 mL ACN. Fish oil and n-octanol were diluted in ACN. The values of Koctanol-PDMS or Kfish oil-PDMS were obtained using a linear regression between the concentration in n-octanol or fish oil and that in PDMS. Finally, the values of Kow and Klipw were calculated using the third-phase equilibrium method using Equations (5) and (6):

2.4. Instrumental Analyses

The concentrations of the five tested chemicals were quantified using a Waters ACQUITY ultra performance liquid chromatograph (UPLC) with a photodiode array (PDA) detector. The absorbance was measured at 221 nm (UV326), 204 nm (UV327 and UV328), 218 nm (UV329), and 287 nm (UV531). The UV stabilizers were separated using a C18 column (2.1 mm × 50 mm, 1.7 μm, Waters) at 35 °C. The mobile phase comprised 95% acetonitrile and 5% water in isocratic mode with a flow rate of 0.2 mL min−1.

3. Results

Figure 2 shows the mass transfer kinetics of the selected UV stabilizers from the donor PDMS to the acceptor PDMS for the determination of KPDMSw. The desorption rate constants (k) for the five test chemicals were obtained using Equation (2). Except for data for UV531 (R2 = 0.81) and UV328 (R2 = 0.76), the experimental data fit well (R2 > 0.9). The KPDMSw values were then calculated using Equation (3), and log KPDMSw ranged from 6.14 to 7.22, and the uncertainties of the KPDMSw were calculated as the 95% upper and lower limits of the error propagation from the standard error of regression and the estimated uncertainties of the thickness of the ABL (Table 2). These values were used to estimate the Kow and Klipw values.

Figure 2

Mass transfer kinetics of (a) UV326, (b) UV327, (c) UV328, (d) UV329, and (e) UV531 for the determination of KPDMSw using the aqueous boundary layer (ABL) permeation method. The solid lines denote regression calculated using Equation (2).

Table 2

Values of Dw, log KPDMSw, log Klipw, and log Kow (experimentally determined and model predicted values) of the five UV stabilizers.

Chemicals	Dw a(m2·s−1)	Experimental Data				Predicted Data
		log KPDMSw(KPDMSw in Lw·LPDMS−1)	log Klipw(Klipw in Lw·Llip−1)	log Kow(Kow in Lw·Lo−1)	log Kow(ECHA) b	log Kow(EPI Suite) c	log Kow(ALOGPS) d
UV326	4.54 × 10−10	6.57(6.53, 6.62)	7.86(7.81, 7.91)	7.38(7.34, 7.43)	>6.5(23 °C, pH 6.4)	5.55	5.70
UV327	4.15 × 10−10	6.56(6.53, 6.60)	7.69(7.75, 7.64)	7.31(7.27, 7.35)	—	6.91	6.27
UV328	4.21 × 10−10	7.22(7.13, 7.33)	8.34(8.24, 8.45)	7.94(7.85, 8.05)	>6.5(23 °C, pH 6.4)	7.25	6.54
UV329	4.46 × 10−10	6.14(6.06, 6.20)	7.40(7.34, 7.46)	6.91(6.86, 6.97)	>6.5(23 °C, pH 6.4)	6.21	5.83
UV531	4.43 × 10−10	6.19(6.07, 6.38)	7.50(7.37, 7.68)	7.08(6.96, 7.27)	—	6.96	6.12

Values in parentheses are the lower and upper 95% confidence intervals. The confidence limits of Klipw and Kow were calculated using error propagation. a Dw values are calculated using Equation (4) [28]. b the partition coefficient was measured using HPLC. OECD TG 117, Registration Dossier, ECHA [29]. c Estimated values from EPI Suite version 4.11 [30]. d Estimated values from the ALOGPS 2.1 program [31].

The values of Kow and Klipw were obtained from the slopes of the linear regression between the concentration in n-octanol or fish oil and that in PDMS (Figure 3 and Figure 4). As shown in Figure 3 and Figure 4, good linear relationships were observed for the five UV stabilizers within the concentration range investigated (R2 > 0.97). The resulting values of log Kow and log Klipw, calculated using Equations (5) and (6), were in the ranges of 7.08–7.94 and 7.50–8.34, respectively (Table 2). As shown in Table 2, log Klipw values were consistently greater than the log Kow values by 0.38–0.49 log units. Table 2 presents the literature values of log Kow submitted to the European Chemical Agency (ECHA) using the HPLC retention time method [29] and those estimated using EPI Suite version 4.11 [30] and the ALOGPS 2.1 program [31]. It should be noted that the Kow and Klipw values in this study were measured at 25 °C, the standard thermodynamic temperature for providing reference values. Further studies on the effects of environmental factors (e.g., temperature and electrolytes) on Kow and Klipw values would extend the applicability of those partition constants under various environmental conditions [19,32].

Figure 3

Regression between n-octanol and PDMS of (a) UV326, (b) UV327, (c) UV328, (d) UV329, and (e) UV531. The solid lines denote linear regression lines.

Figure 4

Regression between fish oil and PDMS of (a) UV326, (b) UV327, (c) dUV328, (d) UV329, and (e) UV531. The solid lines denote linear regression lines.

4. Discussion

The experimental values of Kow and Klipw can be obtain by using direct-measurement methods (e.g., slow-stirring, shake-flask, and generator column method) or indirect methods (e.g., reverse-phase high-performance liquid chromatography). However, shake-flask and generator column method and reverse-phase high-performance liquid chromatography might be not appropriate for determining of Kow and Klipw for highly hydrophobic compounds (log Kow > 6) [17,33]. The slow-stirring method has been reported in literature for measuring the Kow and Klipw for highly hydrophobic chemicals; however, the time required to reach to equilibrium generally takes from a few days up to a few weeks [17,34,35]. To overcome the difficulties in measuring the Kow and Klipw for highly hydrophobic chemicals (i.e., time-consuming, extremely low water solubility, degradation of parent compound during experiment period), the third-phase method, in which a polymer serve as a reference partitioning phase, was proposed to determine the Kow and Klipw for hydrophobic chemicals with log Kow > 6 [36,37]. As shown in Table 2, the log Kow values of the five tested UV stabilizers varied substantially depending on the experimental and estimation methods. The experimental log Kow data for the UV stabilizers have been as yet reported in literature using a HPLC-based method [29]. The experimental log Kow values in this study were consistently higher and could be more accurate than those reported in the registration dossier to ECHA [29], which reported only lower bounds estimated using the HPLC retention time (Table 2). Because the HPLC retention times of chemicals with experimental log Kow values between 0 and 6 were used in the calibration of the method (OECD Guideline 117), greater uncertainties resulted when the log Kow values were above 6. However, for highly hydrophobic substances, log Kow values are often estimated using quantitative structure–property relationship (QSPR) methods (e.g., ALOGPS, KOWWIN) (Table 2). Although the predicted log Kow values by KOWWIN or ALOGPS are lower than the experimental values in this study, the prediction for Kow by the QSPR methods agree quite well with experimental data in this case, especially by KOWWIN (the difference within 1 log unit, except for UV326). The predicted log Kow might not be accurate because the predicted Kow values from the commercial software packages are derived from the experimental Kow values of their nearest analogs [38,39]. An important additional reason here is that at least UV-328 can make an intramolecular hydrogen bond that is not captured in EPISuite [40]. Therefore, the lack of measured data for highly hydrophobic chemicals leads to the inevitable accuracy of the QSPR methods [39]. Although there are certain limitations, such as the long experimental time, high cost, and expensive instruments to detect trace concentrations of analyte to obtain reliable Kow values, these experimental methods are highly recommended for evaluation of UV stabilizers. Ab initio methods are also used as an alternative to predict partition constants for HOCs because they do not rely on the quality of experimental data in the training set. A commonly used program from this is the COSMOtherm program, which is based on quantum chemical descriptors [41,42]. Interestingly, the log Kow of UV327 and UV328 in this study agrees well with estimations made using COSMOtherm (log Kow of UV327 and UV328 are 7.91 and 8.5, respectively [40,43]. The difference between the predicted values by COSMOtherm and the experimental data for log Kow is within 0.6 log unit, supporting the usefulness of these ab initio methods for “difficult-to-test” substances.

In this study, the Klipw values were generally higher than the Kow values, which can be explained by the fact that lipid storage has a highly organized structure compared to the less organized structure of n-octanol, and the driving forces for partitioning into saturated lipid storage can be differentiated from those for n-octanol [18]. There have been studies that experimentally determined lipid–water partitioning constants for a broad range of compounds, including ionic substances, strongly hydrophobic chemicals, and nanomaterials, via different methods, such as potentiometry, the equilibrium dialysis technique, and methods using solid-supported lipid membranes [18,44,45]. To the best of our knowledge, this is the first report of the Klipw values of the selected UV stabilizers. Although this study used a storage lipid (e.g., fish oil) as a representative lipid material, the lipid accumulation properties of chemicals in storage lipids might differ from those in membrane lipids (i.e., phospholipids). Previous studies have demonstrated that the Klipw values of hydrophobic chemicals toward storage lipids were similar to those of hydrophobic chemicals toward a model membrane (i.e., the difference was less than 1 log unit) [19,46]. In addition, storage lipids from different origins (i.e., olive oil, milk fat, fish oil, goose oil, and soybean oil) do not differ in their accumulation properties for various polar and nonpolar organic chemicals [19]. Thus, the Klipw values in this study are good indicators of the biopartitioning of UV stabilizers. For highly hydrophobic organic chemicals, the time required to attain a steady state is often longer than the lifespan of aquatic species, necessitating the consideration of other kinetic parameters, such as growth and metabolic transformation rates, to obtain the bioconcentration factor [47]. Bioaccumulation of UV stabilizers occurs primarily after their ingestion by organisms. There is evidence of the bioaccumulation of UV328 in fish, crustaceans, marine mammals, and algae [48]. Furthermore, biomagnification of UV531 was observed by Peng et al., and the lipid normalized concentration of UV531 in mantis shrimp was higher than that in its prey, sword and Kurumu prawns [49]. In another study, UV326, UV328, and UV329 were found at concentrations ranging from 1.34 to 45.6 ng g−1 (dry weight, dw) on Gran Canaria Island (Spain) [50]. Hasegawa et al. recently showed similar accumulation patterns between direct ingestion from the water column and indirect exposure via the trophic transfer pathway for UV327 in marine fish [51]. In a recent monitoring study, the sum of nine benzotriazole UV stabilizers (including the five target chemicals in this study) were found in river water (17.0–32.5 ng L−1) and sediment (2.0–22.6 ng g−1, dw) in Korea [52]. Furthermore, the benzotriazole (UV326, UV327, UV328, and UV329) bioaccumulation in crucian carp muscle ranged from 0.345 to 5.94 (ng g−1, wet weight), with UV329 being the dominant compound in all crucian carp. The field-derived log bioaccumulation factor (BAF) and biota-sediment accumulation factor (BSAF) for UV329 and UV326 were also calculated during 2018–2020 as 2.89, 2.67, −0.02, and −0.02, respectively [52].

These observations were consistent with the extremely high log Kow and log Klipw values obtained in this study. However, the log BAF and estimated partition coefficients between sediment and water were lower than those estimated from log Kow and log Klipw. As previously mentioned, competition between partitioning processes and other processes may explain these differences. In addition, there are other aspects to be considered for a better understanding of the environmental behavior of UV stabilizers because large amounts of UV stabilizers are used as plastic additives, and they are likely to leach very slowly from these plastics [53]. As microplastics have long environmental residence time [54], they may contribute to the long-range transport of UV stabilizers in the oceans. Because UV stabilizers do not readily biodegrade [43,55], the evaluation of their long-range transport and bioaccumulation potential is important to determine whether they should be listed as POPs. Therefore, future studies on their plastic-mediated, long-range transport as well as bioaccumulation in aquatic species are required. The experimental partitioning properties reported in this study are of considerable value for the evaluation of environmental flux by calculating fugacities in different media [56].

5. Conclusions

In this study, n-octanol-water partition (Kow) and lipid–water partition (Klipw) constants were experimentally measured to estimate the bioaccumulation of five UV stabilizers in aquatic environments. The partition constants between polydimethylsiloxane (PDMS) and water (KPDMSw) of UV stabilizers were obtained using the dynamic permeation method. Consequently, the partition constants between octanol and water (Kow) and lipid and water (Kfishoil-water) were determined using the third-phase equilibrium method. The results showed that the values of log Kow and log Klipw were in the ranges of 7.08–7.94 and 7.50–8.34, respectively, indicating the high potential bioaccumulation of the five UV stabilizers in aquatic environments. The experimental log Kow and log Klipw values also provide valuable information for risks assessment of UV stabilizers.