Embryonic Exposure to Benzotriazole Ultraviolet Stabilizer 327 Alters Behavior of Rainbow Trout Alevin

Benzotriazole ultraviolet (UV) stabilizers (BUVSs) are used in great quantities during industrial production of a variety of consumer and industrial goods. As a result of leaching and spill, BUVSs are detectable ubiquitously in the environment. As of May 2023, citing concerns related to bioaccumulation, biomagnification, and environmental persistence, (B)UV(S)‐328 was recommended to be listed under Annex A of the Stockholm Convention on Persistent Organic Pollutants. However, a phaseout of UV‐328 could result in a regrettable substitution because the replacement chemical(s) could cause similar or unpredicted toxicity in vivo, relative to UV‐328. Therefore, the influence of UV‐327, a potential replacement of UV‐328, was investigated with respect to early life development of newly fertilized rainbow trout embryos (Oncorhynchus mykiss), microinjected with environmentally relevant concentrations of UV‐327. Developmental parameters (standard length), energy consumption (yolk area), heart function, blue sac disease, mortality, and behavior were investigated. Alevins at 14 days posthatching, exposed to 107 ng UV‐327 g−1 egg, presented significant signs of hyperactivity; they moved on average 1.8‐fold the distance and at 1.5‐fold the velocity of controls. Although a substantial reduction in body burden of UV‐327 was observed at hatching, it is postulated that UV‐327, due to its lipophilic properties, interfered with neurological development and signaling from the onset of neurogenesis. If these results hold true across multiple taxa and species, a potential contributor to neurodevelopmental disorders might have been identified. These findings suggest that UV‐327 poses an unknown hazard to rainbow trout embryos and alevins, rendering UV‐327 a potential regrettable substitution to UV‐328. However, a qualified statement on a regrettable substitution requires a comparative investigation on the teratogenic effects between the two BUVSs. Environ Toxicol Chem 2024;43:762–771. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.


INTRODUCTION
In May 2023, benzotriazole ultraviolet (UV) stabilizer (BUVS) 328 (2-[2H-benzotriazol-2-yl]-4,6-di-tert-pentylphenol; herein referred to as UV-328) was recommended to be added to Annex A of the Stockholm Convention on Persistent Organic Pollutants (European Chemicals Agency [ECHA], 2022).This decision marked the initiation of a process toward a global ban and a sunset date of UV-328 by January 2026 and 2044, respectively.This recommendation is unique and represents the first time a nonhalogenated compound has been recommended for Annex A listing (POPRC-18/2); however, and as highlighted throughout the second draft of the Risk Management Evaluation of UV-328 by the ECHA (2022), there is a substantial risk in replacing UV-328 with another BUVS in a so-called one-to-one substitution because such a replacement could result in a regrettable substitution (Maertens et al., 2021).Hence, there is an inherent risk in substituting a somewhat empirically investigated chemical, like UV-328 (Fent et al., 2014;Giraudo et al., 2020;Hemalatha et al., 2020;Liang et al., 2023;Sakuragi et al., 2021;Zhao et al., 2022), with a less investigated one throughout global supply chains (ECHA, 2022).The rationale supporting this phaseout and ban is in line with, and cites similar concerns related to, environmental persistence as stated in the 2015 European Union's preamble on several BUVSs, following quantitative structure-activity relationship assessment (Malm, 2015).As a result, several BUVSs are to be completely phased out across the European Union by mid-November 2023 under Annex XIV of the REACH directive (Registration, Evaluation, Authorisation and Restriction of Chemicals;European Commission, 2020).
Toxicological properties, although only vaguely understood, are known to vary between BUVSs (Fent et al., 2014;Kim et al., 2022;Sakuragi et al., 2021).Empirically, a commonality shared by most BUVSs, but not all, is a propensity to bioaccumulate and biomagnify (Wang et al., 2022;Zhang et al., 2021), while partitioning into soil and sediment (Cantwell et al., 2015) and dispersing heterogeneously in vivo (Zhang et al., 2021), properties which are linked to high log K OW (Apel et al., 2018;Cantwell et al., 2015;Wick et al., 2016).In addition, Zhang et al. (2021) established the bioconcentration factors (BCFs) for six BUVSs, all of which varied by compound, organ, and tissue specifically, with a greater BCF for BUVSs with a higher log K OW and in lipid-rich organs.In fish, exposure to certain BUVSs induces hepatotoxicity (Hemalatha et al., 2020), which has been linked to increased levels of oxidative stress and disruption of cellular metabolism (Giraudo et al., 2020).Antiandrogenic effects as well as disruption of fatty acid and thyroid metabolism have been reported in zebrafish (Danio rerio) exposed to UV-P (2-[2H-benzotriazol-2-yl]-4-methylphenol) (Fent et al., 2014), while a potential for estrogenic effects has been reported in male and female medaka (Oryzias melastigma) exposed to 1H-benzotriazole (Tangtian et al., 2012).The potential for endocrine disruption by BUVSs is supported by Fujita et al. (2022), who report perturbed steroidogenesis in female Japanese medaka (Oryzias latipes) exposed to UV-P, at environmentally relevant concentrations, perturbations that potentially can impair reproduction in more sensitive teleost species.Phase I metabolism of BUVSs, but not necessarily every BUVS, follows the aryl hydrocarbon receptor (AhR) pathway, as indicated by upregulated gene expression of cytochrome P450a1 (cyp1a; Fent et al., 2014) combined with subsequent Phase II metabolism via the glutathione pathway (Hemalatha et al., 2020).Glutathione-mediated Phase II metabolism of BUVSs indicates oxidation of the substrate and the formation of electrophilic BUVSs, which in turn can form covalent bonds with unintentional molecular targets, thereby contributing to toxicity (Boelsterli, 2007).Activation of AhR, by certain BUVSs, can therefore trigger toxicity, although parallel non-AhR pathways are likely to exist and can therefore influence and contribute to BUVSmediated toxicity in vivo.
With a global phaseout and a fixed sunset date of UV-328 on the horizon among signature nations of the Stockholm Convention on Persistent Organic Pollutants, other BUVSs are likely to be used as a substitute for UV-328.Even with the best of intentions, there is a substantial risk that replacing UV-328 with other BUVSs will result in a regrettable substitution, especially when considering unknown adverse effects following exposure to BUVS(s), including developmental toxicity and behavioral alterations.In the present study, the influence of environmentally relevant concentrations of  phenol) on early lifestages of rainbow trout (Oncorhynchus mykiss) was investigated.As a potential substitution for UV-328, UV-327 has a similar molecular weight, structure, log K OW (6.91 compared to 7.25 for UV-328), and environmental omnipresence (summarized in Supporting Information S1, Table S2; Cantwell et al., 2015).Rainbow trout was selected as a model organism due to the species' economic and scientific importance, while also being an integral part in environmental risk assessments (Bobe et al., 2016;D'Agaro et al., 2022).Newly fertilized embryos were microinjected with UV-327 at environmentally relevant concentrations (nominally ≤429 ng UV-327 g −1 egg; Brorström-Lundén et al., 2009;Nakata et al., 2012;Wick et al., 2016) and sampled throughout the developmental period until compete yolk absorption.Microinjection was selected as the means of delivery to mimic maternal transfer (or uptake from contaminated sediment), a plausible scenario because UV-327 has an ovary-specific BCF of 1.04 × 10 4 L kg −1 in zebrafish exposed to 0.5 µg UV-327 L −1 (Zhang et al., 2021), which reflects the lipophilic environment of eggs (Heiden et al., 2005).Several endpoints related to developmental exposure were investigated (biometric endpoints, cardiotoxicity, body burden, and behavior) to assess if UV-327 could present unknown risks and hazards, which may render it a potential regrettable substitution for UV-328, while increasing the general understanding on how a selected BUVS might influence early life development of fish.

Experimental setup, rearing, and sampling
Newly fertilized rainbow trout embryos (PLPL strain), provided by Allison Creek Brood Trout Station (Coleman), were transported to the Aquatic Research Facility at the University of Lethbridge under dark and cool conditions.Fertilized eggs were pooled from two females, while milt was pooled from eight males.On arrival, the embryos were acclimatized for 1 h in a preaerated, static, 10 °C plastic holding tank containing dechlorinated City of Lethbridge municipal tap water (9 L).Visually healthy embryos (nonopaque) were microinjected with UV-327 by use of an IM-400 Electronic Microinjector (Narishige) connected to a Discovery.V12 SteREO microscope (Zeiss), under constant flow of nitrogen gas, to avoid clogging of the needle.In total, 135 embryos per dose, in two sets for a total of 270 embryos, were injected with approximately 25 nL of either pure DMSO (procedural control) or UV-327, at nominal doses of 429, 107, 26.8, 6.7, 1.68, and 0.42 ng UV-327 g −1 embryo; the lowest dose corresponded to concentrations in surface water, while the remaining doses represent those measured in biota, soil, and sediment (Supporting Information S1, Tables S1  and S2; Brorström-Lundén et al., 2009;Nakata et al., 2012;Wick et al., 2016).A set of 270 noninjected embryos was also maintained so that effects of microinjection on experimental endpoints could be evaluated.
Embryos were reared in temperature-controlled flow-through systems (water renewed 10-12 times per hour) and provided constant aeration under a 16: 8-h light: dark photoperiod.The setup was checked twice per day, while temperature (maintained at 10.1 ± 0.2 °C) and oxygen saturation (87.7%-106.7%)were recorded daily; dead embryos were removed from the tanks using a baster with a broaden tip, and the number was recorded (note that after day 14 postinjection, opaque embryos were left, to minimize disturbances, because mortality started to increase at this point).Water quality was maintained within acceptable parameters (Organisation for Economic Co-operation and Development [OECD], 2013): pH (8.33-8.44)and concentrations of nitrogen species measured once per week (Permachem reagents for ammonium, 0-0.54 µg L −1 ; nitrite, 0-8.44 µg L −1 ; and ammonia, 0-2.10 µg L −1 ; DR/850 Colorimeter, program 51, 60, and 64; HACH).

Sampling
Embryos and alevins were sampled at selected developmental stages throughout the experiment (at hatching and at days 11, 14, and 18 posthatching).Alevins sampled at hatching and until complete yolk absorption were always anesthetized in tricaine mesylate (MS-222; 1 g L −1 , buffered to pH 7 using 1 mg L −1 of sodium bicarbonate; CAS no.886-86-2; Syndel) before being analyzed for specific endpoints.Symptoms of blue sac disease (BSD), including pericardial edema, yolk sac edema, hemorrhages, craniofacial deformities, and spinal curvature, were recorded and scored (absent or present; 0 or 1); and BSD indices, per dose, were calculated as per established convention (Scott & Hodson, 2008;Villalobos et al., 2000).

Heart rate
Heart rate, as beats per minute, was quantified from video recordings of 11-day-old alevins.Sedation of alevins, without causing anesthesia, was achieved following a brief immersion in MS-222 (20 mg L −1 , pH 7).Videos were captured using a Discovery.V12 SteREO microscope fixed on the heart.The recording output (720 × 576 resolution, 25 frames s −1 ) was analyzed using OpenShot (Ver.3.0.0)and the heart rate established from the number of frames between each beat and in relation to frame rate.Interbeat variability was established from the average of the standard deviation between each beat (in milliseconds).

Behavior
At day 14 posthatching, 10 individuals in two groups of five from each dose were randomly selected and transferred to a glass Petri dish (product no.C3160100; Pyrex, 95 ⌀ mm diameter; Fisher Scientific) along with sufficient tank water to allow for free swimming but not for vertical movement/surfacing (40 mL).One dish was observed per behavioral trial, and the order of behavioral trials was randomized; the process was repeated twice.Following addition of alevins, the dish was immediately placed into a DanioVision Observation Chamber (Noldus Information Technology) equipped by the manufacturer with a thermal camera, white light stimulus, and temperature control unit.The dish was maintained in a recirculating water bath held at 10 °C.Alevins were acclimated to the unit in darkness for 10 min, following which the behavioral trial commenced by video recording of the entire dish.The behavioral trial consisted of a 10-min period of darkness (white light stimulus off), followed by a 10-min period of brightness (white light stimulus on).This sequence was repeated three times, totaling 60 min of recorded behavior per trial (Scarlett et al., 2022).
Video files were analyzed using EthoVision XT video analysis software (Ver.15).To determine the photokinetic response, the average velocity of alevins in the first 30 s following the white light stimulus being activated was analyzed.The shoal ratio, the amount of time spent apart (>1 cm apart) divided by the time spent in a shoal (<1 cm apart), was calculated during dark (white light stimulus off) periods.The total distance moved over the entire trial, less the already analyzed photokinetic periods, was analyzed to represent general swimming behavior (Scarlett et al., 2022).

Chemistry and body burden
Frozen rainbow trout embryos (injected and noninjected) and alevins (at hatching and day 14 posthatching) were prepared for chemical analysis by homogenization in n-hexanes (CAS no.110-54-3; Fisher Scientific): 333 µL per embryo (except for samples containing >100 embryos, which were homogenized in 1 mL g −1 biomass), 1 mL per six to eight newly hatched alevins, and 1 mL per 10 alevins (day 14).Embryos, due to their toughness, were manually crushed using a broad-tipped pincer, while alevins were homogenized using a handheld motorized homogenizer with a replaceable pestle (VWR International).The hexane phase was collected following centrifugation of the homogenate (9600 g for 4 min at 4 °C; AccuSpin 17R; Fisher Scientific).The pellet phase was resuspended in n-hexane and the supernatant pooled.Surrogate standard UV-328-d 4 (20 ng) was added to the sample, and ultrasound-assisted extraction was conducted for each sample for 10 min, followed by 5 min of centrifugation (1170 g).The extract was transferred to a new glass tube.The extraction was repeated three times, and the extracts were combined.When the combined solvent extract was >15 mL, the sample was concentrated to 5 mL using rotary evaporation and then concentrated to dryness under a gentle stream of N 2 .For those combined extracts <15 mL, the sample was directly concentrated to dryness under gentle N 2 .The sample was reconstituted in 200 μL of n-hexane, sonicated for 5 min, and centrifuged for 5 min before instrument analysis.
The sample was analyzed using gas chromatography-mass spectrometry.Details of instrumental conditions can be found in Fujita et al. (2022).The mass-to-charge ratio (m/z) 342 was used as the quantification ion for UV-327, while the qualification ion was m/z 344.The recovery of UV-328-d 4 was 108 ± 5% (mean ± standard error; 36 samples).The limit of detection (LOD) was estimated using three times the signal-to-noise ratio in the fish extract.The corresponding LODs were 0.01 to 0.2, 0.2 to 0.3, and 0.15 to 0.2 ng g −1 (wet wt) in the embryonic stage, newly hatched, and 14-day-old alevins, respectively.

Statistics
Statistical analyses were performed in R-studio (Ver.2022.12.0) utilizing R (Ver.4.2.2).Normality was determined using the Shapiro-Wilks test, and normality was assumed if α >0.05; if ≤0.05 or if n ≤ 4, a nonparametric statistical approach was utilized.Possible significant differences in mortality (day 18 posthatching), relative to control, were established using Fisher's exact test; note that mortality over the first 24 h following microinjection was omitted from the analysis.Developmental endpoints were assessed, depending on normality, with either oneway analysis of variance (ANOVA) with Tukey's post hoc test or Kruskal-Wallis with Dunn's post hoc test; p values were adjusted for multiple comparisons using Bonferroni's method or Tukey contrasts (behavior).Behavioral analyses were performed using a multivariate ANOVA to assess all behavioral endpoints simultaneously (photokinetic response, shoaling, and total distance moved).Preliminary tests were conducted to confirm if there were no differences between replicate tanks of the same treatment or between housing unit systems or temporal differences between trials completed earlier or later in the day.The null hypothesis was rejected if α ≤0.05.

Mortality
Microinjection, irrespective of UV-327 dose, caused elevated levels of mortality within the first 24 h, compared with noninjected embryos, when only three embryos were found dead within the same time period (data not shown).From day 2 until termination of the experiment at complete yolk absorption (day 18 posthatching, which equates to day 39 postinjection), the cumulative number of dead embryos and alevins followed a sigmoidal trajectory and reached 60% to 79% by the end of the experiment (Table 1; Supporting Information S1, Figure S1).Mortality was significantly lower among the noninjected embryos (45% dead) compared to the DMSO-injected controls (73% dead; Table 1), which was within the range observed in a prior microinjection study of rainbow trout embryos (Black et al., 1988).Hence, background mortality was greater than mortality from microinjection.No dose response with regard to mortality was observed, although a near significantly fewer number of dead embryos/alevins was observed between the highest nominal dose of UV-327 and control (Fisher's p = 0.072).By the end of the experiment, there was a significant (28 percentage unit) difference in mortality between control and noninjected embryos (Fisher's p = 0.0001, adjusted for initial mortality).

Body burden
Temporally, the body burden of UV-327 decreased throughout the developmental period in an exponential trend (Table 2; Supporting Information S1, Figure S2 and Table S3).Note that the measured body burden among embryos was consistently greater than the injected nominal dose.This is likely due to the constant flow of nitrogen gas during the microinjection procedure, which was required to prevent the needle from regularly clogging and requiring recalibration.By the time the alevins hatched, the body burden had decreased by 46% to >97% depending on the initial dose of UV-327; no UV-327 was detected in embryos injected with DMSO or among noninjected embryos.By day 14 posthatching (day 35), UV-327 was still detectable in the carcasses of alevins microinjected with either dose.The rate of metabolism, or clearance, of UV-327 increased with increasing injected dose, ranging from 0.1 to 33.1 ng UV-327 g −1 biomass day −1 between initiation of exposure and hatching (day 21 postinjection) and from 0.1 to 4.9 ng UV-327 g −1 biomass day −1 between hatching and 14 days posthatching (Table 2; Supporting Information S1, Figure S2; one replicate per dose established from 10 to 135 alevins).

Development and cardiotoxicity
No significant effect(s) on development was observed at hatching or by day 11 or 18 posthatching between control and UV-327-exposed alevins.However, newly hatched noninjected alevins were significantly longer than DMSO-injected controls (Table 3; Supporting Information S1, Table S4).However, developmental gains from hatching until days 11 and 18 were lower among UV-327-exposed alevins (Supporting Information S1, Figure S3), especially following exposure to 107 ng g −1 egg (measured 273.4 ng), which resulted in, on average, a 56% smaller planar yolk area while being 163% longer than at hatching.By comparison, DMSO control alevins utilized 64% of the planar yolk area and were 190% longer by day 18 than at day 1 posthatching.No signs of BSD were observed among alevins at any dose of UV-327, DMSO control, or noninjected.
No signs of cardiotoxicity, with regard to heart rate or interbeat variability, were observed by day 11 posthatching (Table 3).Although nonsignificantly impacted, the heart rate among DMSO control alevins was on average lower than that among alevins exposed to UV-327 and noninjected alevins.In addition, interbeat variability was on average nonsignificantly lower among UV-327-exposed alevins, irrespective of microinjected dose, compared to DMSO control and noninjected.

DISCUSSION
In the present study, we investigated whether exposure to UV-327 during embryonic and early life development of rainbow trout could bring about a plausible regrettable substitution, through unknown risk and hazard(s), vis-à-vis what is empirically understood about UV-328.Exposure to UV-327, relative to DMSO control, did not significantly impact mortality, time to hatching, development, yolk consumption (measured as planar yolk sac area over time), or heart function, nor were Temporal differences in and average metabolic rate per day between each sampling are also shown.Note that n = 1 because embryos and alevins were pooled to reach above the theoretical limit of detection (0.01-0.2, 0.2-0.3, and 0.15-0.2ng g −1 in the embryonic stage, newly hatched, and 14-day-old alevins, respectively).dph = days posthatching; <LOD = below the limit of detection; NA = not applicable; DMSO = dimethyl sulfoxide.
any symptoms related to BSD observed at any dose.These results were anticipated because effects related to the aforementioned endpoints are usually observed at higher concentrations of BUVSs, as exemplified by Japanese medaka embryos chronically exposed (42 days) to 40 mg L −1 of 1,2,3benzotriazole, which caused stunted growth and condition index (Shin et al., 2022).By comparison, adult female zebrafish exposed semistatically to a wide variety of BUVSs, including UV-327 and UV-328 (0.5 and 10 µg L −1 ) for 28 days did not experience adverse effects on development, behavior, mortality, or growth (Zhang et al., 2021).Cardiotoxicity has not been observed in fish larvae exposed to BUVSs previously, nor was cardiotoxicity observed among alevins exposed to UV-327 (as per the present study).Yet, we know from Hirata-Koizumi et al. ( 2007) that male rats exposed to 0.5 mg UV-320 kg −1 body weight for 28 days suffered from both immune cell infiltration of the heart and degradation and hypertrophy of the myocardium, symptoms they observed to persist even after 14 days of depuration.It is unknown if similar effects might occur when teleosts are exposed to UV-320 or BUVSs other than UV-327.Holistically, exposure to UV-327, at environmentally relevant concentrations (nominally ≤429 ng g −1 egg),  can be considered to not affect apical endpoints in rainbow trout alevins following embryonic exposure, while subsequent effects in adults remain unknown.
The significantly lower incidence of mortality among noninjected embryos compared to DMSO-injected controls emphasizes how invasive microinjection is, compared to other routes of exposure, such as waterborne exposure.Although mortality rates were high, ranging from 60% to 79%, these are in agreement with Black et al. (1988), who reported increased mortality rates among microinjected eyed-stage rainbow trout embryos (which corresponds to day 12 postfertilization).It seems though that rainbow trout embryos are sensitive to microinjection because microinjection of zebrafish embryos caused a lower mortality incidence (Dubiel et al., 2022).Nonetheless, the exact impact of microinjection on mortality cannot fully be assessed without comparing with mock injection, which was not performed in the present study.Increased mortality can potentially stem from a weakening of the chorion at the site of injection because several exposed embryos were quantitatively observed to hatch prematurely, irrespective of microinjection treatment, throughout the period preceding hatching.In addition, it seems that DMSO had some effect(s) on development, which could stem from DMSO altering the lipophilic environment of the embryos.It must be noted that the nominal volume injected (25 nL), 0.046% (v/v; range 0.039%-0.054%),was less than the upper limit of 0.1% (v/v) recommended by OECD Guideline 236 (2013)-assuming an average radius of 0.235 ± 0.011 cm per embryo (n = 12), which corresponds to a volume of 54,300 nL (range 46,450-63,310 nL).Yet, microinjection, as a method and compared to water-borne exposure, serves an important function because doses are consistent and standardized across individuals, it mimics maternal transfer, and it circumvents the chorion which protects the developing larvae from xenobiotics and environmental stressors.
Exposure to UV-327, relative to control, had no statistically significant effects on growth or development irrespective of dose, even though the average developmental gains differed greatly when assessed temporally (days 11 and 18, relative to the day of hatching; Supporting Information S1, Figure S3).Exposure to 107 ng g −1 egg (nominal) resulted in the smallest developmental gains compared to control (percentage units).Interestingly, exposure to 107 ng g −1 egg induced what we consider as hyperactive behavior by day 14 posthatching (Figure 1A).Hyperactivity is a fairly uncommon observation in developmental toxicology, and this finding would suggest that embryonic exposure to UV-327, plausibly, has the potential to alter neuroactivity in rainbow trout alevins (Figure 1A; Legradi et al., 2018;Scott & Sloman, 2004).Because newly fertilized rainbow trout embryos are rich in lipids and yolk, it can be proposed that UV-327 was initially homogenously distributed within the newly fertilized embryo, as per the high log K OW .With time, and as yolk is consumed and the embryo develops, UV-327 could plausibly distribute heterogeneously to lipid-rich organs and tissues, including the diminishing yolk sac as well as the brain and the nervous system; these neurological structures form before day 6 postfertilization and become discernible by day 8 (Finch et al., 2010).Even though embryos were proficient at metabolizing UV-327 and able to scale up the rate of metabolism with increasing dose (Table 2), potentially via hydroxylation and glutathione-mediated Phase II metabolism (Hemalatha et al., 2020), it can be postulated that UV-327 was present in the precursor brain before the establishment of the blood-brain barrier.Therefore, UV-327 would be able to interfere with neurological development and neurological signaling already from the onset of neurogenesis.Yet, these findings must be considered cautiously because the significantly altered behavior was established from 10 alevins and observed in one specific dose at one specific time point and developmental stage.Additional studies are required to confirm or disprove our findings.
Because of the experimental design, a specific neural region (s), tissue(s), pathway(s), or mechanism(s) that might have been affected by exposure to UV-327 cannot be identified.It is only possible to correlate embryonic exposure with altered behavior and infer that UV-327 may have interfered with neurological and central nervous system functions and development, based on the log K OW and propensity to accumulate in lipid-rich tissue (Zhang et al., 2021).This notion is supported by Li et al. (2019), who reported shifts in the neurological transcriptome of zebrafish exposed to any of several BUVSs, although at much higher concentrations than utilized in the present study; their results indicate a relationship between neurotoxicity, mitochondrial dysregulation, and inflammation.Sluggish behavior was observed in zebrafish exposed to UV-234 (2-[benzotriazol-2-yl]-4,6-bis[2-phenylpropan-2-yl]phenol) under dark, but not light, conditions (Liang et al., 2019).In addition, exposure to BUVSs is known to cause oxidative stress in fish (Fent et al., 2014), which in turn has been linked to neurotoxicity (Berntssen et al., 2003;Sayre et al., 2008).Because the rate of xenobiotic metabolism in neurological tissue is known to be lower than in liver (Ravindranath et al., 1995), it can be speculated that the parental form of UV-327 and its metabolites is likely to persist in brain and other lipophilic tissues (Zhang et al., 2021), thereby prolonging the exposure and contributing to altered behavior.Holistically, these aforementioned studies on a relationship between exposure to certain BUVSs and neurological toxicity in fish provide support to the observed hyperactive behavior reported within the present study but no finite conclusion.
Because a photokinetic response is a natural behavior of alevins (Dill, 1977;White, 1915), changes in behavior, including hyperactivity, although uncommon, can have repercussions, especially in situ.Previously, hyperactivity has only been reported in a handful of studies investigating the effects of xenobiotics on development and behavior in fish (Legradi et al., 2018).Exposure to Dylox, an organophosphate insecticide, resulted in hyperactive behavior and a shift to positive phototropic and jerky photokinetic response in rainbow trout alevins (Matton & LaHam, 1969).Exposure to cadmium, a neurotoxic heavy metal (B.Wang & Du, 2013), caused hyperactive behavior initially, which with time shifted to lethargy and mortality (Peterson et al., 1983;Rombough & Garside, 1984).Conversely and in the present study, the significant decrease in activity following exposure to the highest dose of UV-327 (429 ng g −1 egg) relative to the second highest dose (107 ng g −1 egg) could be due to saturation of (multiple) neurological and physiological functions and pathways, thereby linking exposure to a behavioral outcome (Huang et al., 2021;Isingrini et al., 2023;Li et al., 2019).In addition, it was quantitatively noted during the daily maintenance and when alevins were randomly selected for the behavioral assay that alevins exposed to the largest dose were sluggish and slow to react, and therefore less active, but still on par with controls when assessed in the behavioral assay (Figure 1A).Therefore, it is plausible that the nonsignificant changes in behavior (highest exposure group compared to controls), and as reported by Zhang et al. (2021), could stem from a saturation of and crossactivation of counteracting neurological and central nervous system feedback mechanisms.
The behavioral changes observed in the present study are indicative of potentially detrimental effects of embryonic and posthatching developmental exposure to UV-327.In situ, and when considered holistically, altered swimming behavior and photokinetic response could potentially alter an individual alevin's ability to escape and evade predation.However, that a widely used chemical such as UV-327, which has been produced and used since the 1950s and is currently detected as an omnipresent pollutant (from nanograms per liter of water to micrograms per gram of dry soil and tissue), can alter the of fish larvae warrants further investigation.Follow-up studies should elucidate potential mechanisms and assess effects of other BUVSs during early life development.This includes the effect(s) of BUVS mixtures, using different (non)model organisms.
Supporting Information-The Supporting Information is available on the Wiley Online Library at https://doi.org/10.1002/etc.5807.

FIGURE 1 :
FIGURE 1: Boxplot representation of the total distance moved (A), shoal ratio (B), and photokinetic response (C) during the behavior assessment, using DanioVision, and in relation to exposure.Significant differences are denoted using different lowercase letters (simultaneous tests for general linear hypotheses combined with multiple comparisons of means, Tukey contrasts).n = 10.Noninj.= noninjected; DMSO = dimethyl sulfoxide.

TABLE 1 :
Effect of UV-327 on survival of rainbow trout early life stages, as per cumulative mortality at hatching (day 21 postmicroinjection) and days 7 and 18 posthatching, while the numbers within parentheses represent the cumulative percentage of dead embryos *Significant difference in cumulative mortality (percentage) in relation to dimethyl sulfoxide-injected control (day 18; Fisher's exact test).A total of 270 embryos were injected with UV-327.Note that mortality observed during the first 24 h is omitted from any statistical analysis.

TABLE 2 :
Measured body burden of UV-327 in rainbow trout embryos, in newly hatched alevins (day 21 postmicroinjection), and at 14 days posthatching

TABLE 3 :
Summary of developmental parameters (standard length, planar yolk area) and effects on heart rate (beats per minute) and interbeat variability in relation to exposure (nanograms per gram of egg) as well as number of animals measured Significant difference, compared to the respective DMSO-injected control (Kruskal-Wallis and Dunn's post hoc test).Data are presented as average ± standard deviation.NA = not available. *