Embryonic Exposure to the Benzotriazole Ultraviolet Stabilizer 2‐(2H‐benzotriazol‐2‐yl)‐4‐methylphenol Decreases Fertility of Adult Zebrafish (Danio rerio)

Benzotriazole ultraviolet stabilizers (BUVSs) are emerging contaminants of concern. They are added to a variety of products, including building materials, personal care products, paints, and plastics, to prevent degradation caused by ultraviolet (UV) light. Despite widespread occurrence in aquatic environments, little is known regarding the effects of BUVSs on aquatic organisms. The aim of the present study was to characterize the effects of exposure to 2‐(2H‐benzotriazol‐2‐yl)‐4‐methylphenol (UV‐P) on the reproductive success of zebrafish (Danio rerio) following embryonic exposure. Embryos were exposed, by use of microinjection, to UV‐P at <1.5 (control), 2.77, and 24.25 ng/g egg, and reared until sexual maturity, when reproductive performance was assessed, following which molecular and biochemical endpoints were analyzed. Exposure to UV‐P did not have a significant effect on fecundity. However, there was a significant effect on fertilization success. Using UV‐P‐exposed males and females, fertility was decreased by 8.75% in the low treatment group and by 15.02% in the high treatment group relative to control. In a reproduction assay with UV‐P‐exposed males and control females, fertility was decreased by 11.47% in the high treatment group relative to the control. Embryonic exposure to UV‐P might have perturbed male sex steroid synthesis as indicated by small changes in blood plasma concentrations of 17β‐estradiol and 11‐ketotestosterone, and small statistically nonsignificant decreases in mRNA abundances of cyp19a1a, cyp11c1, and hsd17b3. In addition, decreased transcript abundances of genes involved in spermatogenesis, such as nanos2 and dazl, were observed. Decreases in later stages of sperm development were observed, suggesting that embryonic exposure to UV‐P impaired spematogenesis, resulting in decreased sperm quantity. The present study is the first to demonstrate latent effects of BUVSs, specifically on fish reproduction. Environ Toxicol Chem 2024;43:385–397. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Benzotriazole ultraviolet stabilizers have been detected in aquatic environments.They have been detected in lakes, rivers, streams, sediment, sewage sludge, wastewater effluent, and aquatic organisms, globally (Lu et al., 2016(Lu et al., , 2018;;Montesdeoca-Esponda et al., 2019;Nakata et al., 2009Nakata et al., , 2012;;Parajulee et al., 2018;Peng et al., 2017Peng et al., , 2019;;Zhang et al., 2011).Although concentrations of BUVSs in the environment are frequently below detection limits, when detected, concentrations of certain BUVSs can be as great as 720 ng/g (dry wt) in sediment, 4780 ng/L in heavily polluted surface waters, and 255 ng/g (lipid wt) in muscle tissue of fish, globally (Castilloux et al., 2022;Kameda et al., 2011;Kim et al., 2011;Nakata et al., 2009).One BUVS that has been detected in aquatic environments is 2-(2Hbenzotriazol-2yl)-4-methylphenol (UV-P).Annual production of UV-P has ranged from 227 to 454 tons in the United States, and 1000 to 10 000 tons in Europe (Castilloux et al., 2022).Concentrations of UV-P up to 23 ng/L have been detected in surface waters, and up to 15 ng/g (dry wt) in sediment from various locations globally (Kameda et al., 2011;Vimalkumar et al., 2018).In addition, UV-P has been detected in muscle tissues from 26 species of freshwater and marine fish, with maximum detected concentrations of 222 ng/g (lipid wt; Kim et al., 2011;Vimalkumar et al., 2018).Although UV-P does accumulate in lipid, it can be biotransformed in fish (Zhang et al., 2021).Because BUVSs are additives in plastic, it is plausible that increases in both plastic use and waste (Borrelle et al., 2020;MacLeod et al., 2021) could increase concentrations of BUVSs in the environment, including in biota, therefore it is critical to understand any adverse effects these chemicals might have on aquatic environments.
Little is known about adverse effects of BUVS, but there is evidence that some BUVS, including UV-P, can act as endocrine-disrupting chemicals (EDCs).Using reporter gene assays, UV-P was classified as an antagonist of the of human androgen receptor (AR; Sakuragi et al., 2021).In another study of transactivation of the human AR, UV-P was determined to be a potent antiandrogen, and metabolites from CYP3A4mediated biotransformation of UV-P also acted as antiandrogens (Zhaung et al., 2017).Significant antiandrogenic activity of UV-P was also observed in the yeast androgen screening assay (Fent et al., 2014).These findings are consistent with observations in UV-P-exposed Japanese medaka (Oryzias latipes), where an antiandrogenic mode of action was proposed based on changes in expression of steroidogenic genes and plasma concentrations of 17β-estradiol (E2) and testosterone (T; Fujita et al., 2022).Because most research about the endocrine disrupting effects of BUVSs, including UV-P, has been conducted using mammalian models, further research is needed to understand the endocrine disrupting potential of BUVSs on aquatic biota, including fishes.
Exposure to chemical stressors during early life stages (ELS) can have greater adverse effects on biological processes relative to impacts of exposures during adult life stages, specifically due to heightened sensitivity to contaminants during critical developmental periods (Russell et al., 1999).Embryonic exposure of fish to EDCs can result in disruption of physiological processes later in life, including abnormal gamete development, reduced fecundity, and infertility.For instance, embryonic exposure of Japanese medaka to the EDC, letrozole, impaired reproductive capacity, gonad maturation, and expression of reproductive genes at sexual maturity (Liao et al., 2014).Embryonic exposure of zebrafish (Danio rerio) to benzophenone-3 impaired the development and maturation of ovaries and reduced egg production, and decreased chasing behaviour (Tao et al., 2023).Exposure of Japanese medaka embryos to the brominated flame retardant 1,2,5,6-tetrabromocyclooctane, via maternal transfer, resulted in decreased fecundity and impairment of oocyte maturation (Devoy, Raza, Jones, et al., 2023;Devoy, Raza, Kleiner, et al., 2023).To date, no study has investigated whether embryonic exposure to BUVSs impacts the physiological performance of fishes at sexual maturity, including effects on reproduction.
Studies of effects of ELS exposure to anthropogenic contaminants on later life physiological performance are needed to understand the risk that contaminants pose to organisms.Therefore, the present study aimed to characterize the effects of embryonic exposure to UV-P, a BUVS with anti-androgen activity, on the reproductive performance of zebrafish.Zebrafish embryos were microinjected with UV-P, reared to sexual maturity in clean water, and effects on reproductive performance were assessed, including molecular mechanism(s) of effect.The results provide novel insight into the long-term effects of ELS exposure to UV-P on fish reproduction that fills critical knowledge gaps about the potential risks of this class of chemicals to the aquatic environment.

Animals
Use of animals was approved by the University of Lethbridge Animal Care Committee (Protocol #2105).Adult zebrafish were held in an active breeding group, in a 1:1 ratio of male to female fish, maintained in a ZebTEC Active Blue System (Tecniplast) at the Aquatic Research Facility within the Alberta Water and Environmental Science Building at the University of Lethbridge (Lethbridge, AB, Canada).Fish were under a 14:10-h light:dark photoperiod and were supplied with processed City of Lethbridge municipal tap water at 28 °C.Diets consisted of adult zebrafish feed (Ziegler Bros.), brine shrimp (Artemia salina; Brine Shrimp Direct), and Gemma Micro 300 feed (Skretting,) at a feeding rate of approximately 2% body weight per day.Water quality parameters, including dissolved O 2 , pH, ionized and unionized ammonia, NO 3 − , and NO 2

Microinjections
Zebrafish embryos were microinjected with UV-P based on a previously described protocol (Lane et al., 2019) with minor modification (Dubiel et al., 2022).This method of exposure allows for precise doses of UV-P to be delivered into each embryo, therefore reducing any differences in exposure dosage between embryos, and allowing for full absorption of the chemical by the fish (Lane et al., 2019).To obtain embryos, two male and two female zebrafish were placed in 1.7 L sloped breeding tanks (Tecniplast) the evening prior to microinjections.Embryos were collected 1 h following a breeding event, and dead and unfertilized embryos were discarded.A batch of embryos was used only if more than 80% of eggs were fertilized.Using an IM-400 Electric Microinjector (Narishige Group), zebrafish embryos were injected with 1.5 nL of either a solvent control (100% DMSO), or UV-P at nominal doses of 100 and 1000 ng/g-egg.All microinjections were completed prior to gastrulation, at approximately 6 h postfertilization (hpf).The highest nominal exposure dose was approximately the dose causing 20% lethality based on a previously determined dose-response to zebrafish embryos (Johnson, H. M. 2023.Unpublished raw data on acute lethality of UV-P to zebrafish embryos.University of Lethbridge).Exposures were replicated three times, each with eggs from a discrete breeding event.Approximately 200 embryos per replicate were injected per dose.After injections, embryos were placed in a glass Petri dish containing dechlorinated water for 24 h, after which any dead embryos were discarded.Mortalities were assumed to be caused by the microinjection procedure and therefore were not included in the final mortality determination.Remaining embryos were used for the embryotoxicity assay and reproduction assays, as described in the next sections.For chemical quantification of dose, 1 g of embryos was injected per treatment and immediately frozen at −80 °C (see section Analysis of UV-P in eggs).

Embryotoxicity assays
Twenty-four of the surviving embryos were placed into 24-well plates (one embryo per well) with 2 mL of dechlorinated municipal tap water in each well.These embryos were reared until 15 days postfertilization (dpf) to ensure complete yolk sac absorption and exposure to the entire dose of UV-P.A 50% water renewal was performed daily.To assess embryos, a dissecting microscope was used.Heart rate as the number of beats per minute was assessed at 48 hpf, where the number of beats in 30 s was recorded, then doubled, for eight randomly selected embryos.Mortality (based on lack of heartbeat) was scored daily, and malformations-including spinal curvature, yolk sac edema, and pericardial edema-were recorded as cumulative totals at 15 dpf.

Reproduction assays
Approximately 150 embryos per replicate were reared at 28 °C until sexual maturity (approximately 6 months; Singleman & Holtzman, 2014) without further exposure to UV-P.During rearing, fish were initially fed a diet consisting of brine shrimp and Gemma Micro 300 feed beginning at 7 dpf.Once fish were of sufficient size (approximately 1 month) their diet was supplemented with adult zebrafish feed (Ziegler Bros.).Delaying initiation of feeding until 8 dpf does not impact the growth or survival of zebrafish (Hernandez et al., 2018).Two assays were performed to assess the effects of embryonic exposure to UV-P on the reproductive performance of adults.The assays were based on Organisation for Economic Co-operation and Development (OECD) Test number 229 (Fish Short Term Reproduction Assay; OECD, 2012) except one male and one female were used per tank so that the fecundity and fertilization success of individual fish could be determined.Due to aggression between male and female fish across all treatments, including the controls, assays were terminated at 14 days or 12 days, rather than 21 days.The first reproduction assay (terminated at 14 days) was conducted using UV-P-exposed male and female fish.The second reproduction assay (terminated at 12 days) was conducted using UV-P-exposed male fish and DMSO-exposed (control) female fish to eliminate the possibility that the decrease in fertilization success was due to decreased egg quality from female fish that had been exposed to UV-P as embryos.To perform the reproduction assays, sexually mature adult zebrafish were placed into 3.5-L tanks in groups of two (one male and one female) in a ZebTEC Active Blue System (Tecniplast), with 10 or eight replicate tanks per treatment for the first and second assay, respectively.Fish were fed adult zebrafish feed at approximately 2% bodyweight daily, as described in Animals.Following a 1-week acclimation period, fecundity was assessed daily as the number of eggs per tank, and fertilization success was assessed daily using a light microscope.Eggs were collected and assessed for successful fertilization within 5 h of being laid.On termination of the reproduction assays, fish were euthanized using MS-222 (250 mg/L, buffered with sodium bicarbonate), and gonads and blood were collected for analysis of molecular and biochemical endpoints.Fulton's condition factor (K), gonadosomatic index (GSI), and hepatosomatic index (HSI) were determined from the mass (g) and length (mm) of the fish, gonad mass (g), and liver mass (g), respectively.Due to the lack of effect on fecundity of females, gene expression, steroid hormone concentrations, and histological analyses were determined only with male fish (Embryotoxicity assays, Reproduction assays, and mRNA abundances of genes involved in reproduction).During the rearing period and reproduction assays, average daily water temperature was 28.0 °C (minimum = 27.9 °C, maximum = 28 °C) and average daily dissolved oxygen was 6.3 mg/L (90% saturation).Total ammonia, nitrite, and nitrates were recorded weekly and average values were 0.01, 0.007, and 3.0 mg/L, respectively.

mRNA abundances of genes involved in reproduction
Abundances of transcripts of genes involved in steroidogenesis and spermatogenesis were quantified in testes from six to eight male fish per treatment, from the first reproduction assay.Total RNA was isolated using TRIzol™ reagent (Ther-moFisher Scientific) according to the manufacturer's protocol.RNA concentrations were quantified by use of a Nanodrop™ One © spectrophotometer (ThermoFisher Scientific), following which complementary DNA (cDNA) was synthesized from 2.5 μg of RNA by use of Superscript™ IV First-Strand Synthesis System (Thermofisher Scientific).The cDNA synthesis protocol included a DNase step.The protocol for quantitative polymerase chain reaction (qPCR) was identical to what was described previously (Fujita et al., 2022).No-template controls were run for each primer set to ensure there was no contamination in the reactions.In addition, melt curves were generated to ensure a single product was amplified.The transcript abundance of each target gene was normalized to 18s rRNA and using the efficiency corrected method, changes in mRNA abundance were calculated relative to the control (Pfaffl, 2001).Efficiencies of qPCR reactions using different primer sets were calculated using fivefold serial dilutions of cDNA templates (Supporting Information, Table S1).

Quantification of steroid hormone concentrations
Enzyme-linked immunosorbent assays were used to quantify concentrations of E2 (Item No: 501890) and 11-ketotestosterone (11-KT; Item No: 582751) in blood plasma from five to eight male zebrafish per treatment from the first reproduction assay, following the manufacturer's protocols (Cayman Chemical).

Histological analysis
Following the second reproduction assay, testes from six males per treatment were extracted and fixed in 10% formalin for 24 h for histological analysis.Following fixation, tissues were dehydrated in a series of ethanol dilutions, cleared in xylene, and embedded in paraffin blocks.Six sections per sample were sectioned at 5 μm and stained with hematoxylin and eosin at Prairie Diagnostic Services (University of Saskatchewan).Histological changes were observed using a brightfield Olympus CX43 microscope (Olympus Corporation).High-resolution images were analyzed using ImageJ Version 1.53 (National Institutes of Health).Proportions of area of the following stages of sperm development were determined: Type A undifferentiated spermatogonia (A und ), Type A differentiated spermatogonia (A diff ), Type B spermatogonia (B), primary spermatocytes (PS), secondary spermatocytes (SS), and spermatids (S).Five to six sections per sample were analyzed.

Analysis of UV-P in eggs
Fish egg samples (1 g wet mass per dose) were homogenized and transferred to a glass test-tube and spiked with 20 ng of UV328-d 4 .A commercially available isotope-labeled UV-P standard was not available at the time of the present study.A previous study showed similar recovery of UV-P and UV328-d4 from fish tissues (Castilloux et al., 2022).The sample was extracted using 5 mL of n-hexane in an ultrasonic water bath for 10 min, followed by 1 min of vortexing and 5 min of centrifugation at 1167g.The top n-hexane extract was transferred to a new glass tube.The extraction was repeated three times, and the solvent extracts were combined.The extract was concentrated to dryness using N 2 .The sample was reconstituted in 1 mL of n-hexane for gas chromatography-mass spectrometry (GC-MS) analysis.Details of GC-MS analysis parameters were previously published (Fujita et al., 2022).Concentration of UV-P was quantified using m/z 225.The m/z 168 was used as qualification ions.The recovery of UV328-d 4 was 113% ± 6% (mean ± standard error, five samples).It was not detected in procedure blanks (n = 3).The limit of detection (LOD), which was estimated as being three times the signal to noise ratio in the control egg extract, was 1.5 ng/g (wet mass).

Statistical analysis
Initial statistical analyses were performed in R, Ver.4.2.1 (R Core Team, 2022).Multivariate analysis of variance (MANOVA) was completed for all endpoints utilizing the same individuals, for example all transcript abundance data were analyzed together and all histopathological endpoints compared with each other.The MANOVA was constructed to eliminate experimental design aspects such as different microplates or different fish housing units as being a source of differentiation in the data.Following the MANOVA analysis, data were transferred to GraphPad Prism 9.3.1 for Mac for subsequent analysis.Data were analyzed for normality and homogeneity of variance using the Shapiro-Wilk and Bartlett's test, respectively.Data that conformed to parametric assumptions were further tested by using a single-factor analysis of variance (ANOVA), followed by a Dunnett's post hoc test to determine significance between the treatments and control.Data that did not conform to parametric assumptions were log 10 transformed.Data that failed to meet the parametric assumption following log 10 transformation were analyzed using Kruskal-Wallis test and post hoc Dunn's test.All data were represented as mean ± standard error of the mean (SEM).Differences were considered significant when p ≤ 0.05.

Concentration of UV-P in eggs
Measured doses of UV-P in embryos were <1.5 (the LOD), 2.8, and 24.3 ng/g-egg in the 0, 100, and 1000 ng/g-egg treatments, respectively.Each treatment is hereafter referred to as control, low, and high.Measured doses of UV-P were approximately 40-fold less than the intended nominal dose.

Effect of UV-P on embryotoxicity of zebrafish
Embryonic exposure of zebrafish to UV-P did not cause any significant changes in cumulative survival, which was greater than 80% across all treatments (Figure 1A) or heart rate (data not shown).The mean prevalence of both spinal curvature and uninflated swim bladder was significantly increased from 1.7% in controls to 12.1% in the low treatment group (Figure 1B,C).No significant difference in the prevalence of pericardial or yolk sac edema was observed among treatments (data not shown).

Effect of embryonic exposure to UV-P on reproductive performance of adult zebrafish
The reproductive capacity of zebrafish was altered following embryonic exposure to UV-P.In the first reproduction assay with UV-P-exposed male and female fish, fecundity in the controls (35.7 ± 7.4) was not statistically different to that in the low (38.4 ± 3.8) and high (38.2 ± 5.1) treatment groups.However, fertilization success was significantly decreased by 8.8% and 15.0% relative to controls in the low and high treatment groups, respectively (Figure 2A).In the second reproduction assay with UV-P-exposed males and control females, fecundity in the controls (55.1 ± 9.7) was not statistically different to that in the low (55.7 ± 9.7) and high (34.5 ± 8.8) treatment groups, but fertilization success was significantly decreased by 11.5% in the high treatment group relative to the control (Figure 2B).There were no significant differences in GSI, HSI, or K of male fish among treatment groups for either reproduction assay (Supporting Information, Table S2).

Effect of UV-P on blood plasma steroid hormone concentrations of male zebrafish
Concentrations of E2 and 11-KT in blood plasma of sexually mature male fish exposed as embryos to UV-P were not significantly different from those in control male fish (Figure 3).

Effect of UV-P on transcript abundance of steroidogenic genes in male zebrafish
Abundances of transcripts of ar, cyp11a1, cyp19a1a, cyp11c1, and hsd11β2 were not significantly different in testes of male fish in either treatment group, relative to the control (Figure 4A-D, F).Transcript abundance of hsd17β3 was significantly decreased by 2.5-fold in testes of male fish exposed to the high treatment, relative to the control (Figure 4E).Although not statistically significant, transcript abundances of cyp11a1, cyp19a1a, and cyp11c1 in testes from male fish were decreased by FIGURE 2: Effect of embryonic exposure to 2-(2H-benzotriazol-2-yl)-4-methylphenol (UV-P) on the fertilization success of adult male zebrafish.Embryos were exposed via microinjection to doses of <1.5 (control), 2.77, and 24.3 ng/g egg and reared to sexual maturity in clean water.Once at sexual maturity, one male and one female zebrafish were assigned to eight to 10 replicate tanks per treatment.Fertilization success of (A) UV-Pexposed male and female zebrafish, following 14-day reproduction assay, and (B) UV-P-exposed male and unexposed female zebrafish, following 12-day reproduction assay, was measured.Data are represented as mean (±SEM) of six to nine replicate tanks.Differences from the control were measured using a one-way analysis of variance, followed by a Dunnett's post hoc test.*Significant differences (p ≤ 0.05).

Effect of UV-P on morphometry of zebrafish testicular cells
In the testes of male zebrafish exposed as embryos to either dose of UV-P, there was no change in proportion of area of Type A und spermatogonia, Type A diff spermatogonia, Type B spermatogonia, or primary spermatocytes relative to the control (Figure 5B-E).The proportion of area of secondary spermatocytes was nonsignificantly increased by 1.3-fold in the low treatment group and significantly increased by 1.6-fold in the high treatment group, relative to the control (Figure 5F).The proportion of area of spermatids was decreased by 1.5-fold (p = 0.07) in the high treatment group, relative to the control, but was not statistically significant (Figure 5G).There were no incidences of ova-testis in males from either treatment group, FIGURE 3: Effect of embryonic exposure to 2-(2H-benzotriazol-2-yl)-4-methylphenol (UV-P) on concentrations (pg/mL) of (A) 17β-estradiol (E2) and (B) 11-ketotestosterone (11-KT), in blood plasma from adult male zebrafish.Embryos were exposed via microinjection to doses of <1.5 (control), 2.77, and 24.3 ng/g egg UV-P/g-egg and reared to sexual maturity in clean water.Blood was collected and analyzed from five to eight adult male fish per treatment.Data are represented as mean (±SEM).Differences from the control were measured using a one-way analysis of variance, followed by a Dunnett's post hoc test.
FIGURE 4: Effects of embryonic exposure to 2-(2H-benzotriazol-2-yl)-4-methylphenol on abundances of transcripts of steroidogenic genes in testes from male adult zebrafish.Embryos were exposed via microinjection to doses of <1.5 (control), 2.77, and 24.3 ng/g egg and reared to maturity in freshwater.(A-F) Abundances of ar, cyp11a1, cyp19a1a, cyp11c1, hsd17β3, and hsd11β2 transcripts in testes of male zebrafish.Parametric data were analyzed using a one-way analysis of variance, followed by a Dunnett's post hoc test.Nonparametric data were log 10 transformed, and if data remained nonparametric, were analyzed using a Kruskal-Wallis test, followed by a Dunn's post hoc test.Data are represented as mean (±SEM) of six to eight replicates.*Significant differences from control (p ≤ 0.05).
is consistent with gross observations of testis during dissections.

Effect of UV-P on transcript abundance of spermatogenic genes
Transcript abundance of nanos2 was significantly decreased by 2-fold in the low treatment group, relative to the control, whereas the 1.88-fold decrease in the high treatment group was near statistical significance (p = 0.06; Figure 6A).Statistically significant 3-and 3.1-fold decreases in transcript abundance of dazl were observed in the low and high treatment groups, respectively, relative to the control (Figure 6C).Abundances of transcripts of piwil1, insl3, igf3, amh, and wnt5a were not statistically different in UV-P-exposed male fish compared with the controls.

DISCUSSION
Benzotriazole ultraviolet stabilizers are environmental contaminants that have been detected in a variety of environmental matrices, including aquatic biota.Some BUVSs have endocrine-disrupting potential, but there is a lack of knowledge regarding the effects of BUVSs on reproduction.The present study investigated the effects of embryonic exposure to environmentally relevant concentrations of UV-P on the reproductive performance of adult zebrafish.In the environment, UV-P has been found at concentrations as great as 81.5 ng/g lipid weight in muscle tissues of fish, but concentrations of UV-P in fish eggs have not been reported (Kim et al., 2011;Vimalkumar et al., 2018).Thus, the doses measured in embryos in the present study-2.77and 24.3 ng/g-egg-fall into the range of reported environmental concentrations of UV-P in biota (Kim et al., 2011;Vimalkumar et al., 2018).It is not clear why measured doses are approximately 40-fold less than FIGURE 5: Effects of embryonic exposure to 2-(2H-benzotriazol-2-yl)-4-methylphenol on spermatogenesis of adult male zebrafish.Embryos were exposed via microinjection to doses of <1.5 (control), 2.77, and 24.3 ng/g egg and reared to maturity in freshwater.(A) Representative histological sections of male zebrafish testes and (B) proportion of testicular components.Type A undifferentiated spermatogonia (A und ), Type A differentiated spermatogonia (A diff ), Type B spermatogonia (B), primary spermatocytes (PS), secondary spermatocytes (SS), and spermatids (S) are indicated.Parametric data was analyzed using a one-way analysis of variance, followed by a Dunnett's post hoc test.Nonparametric data were log 10 transformed, and if data remained nonparametric, were analyzed using a Kruskal-Wallis test, followed by a Dunn's post hoc test.Data are represented as mean (±SEM) of six replicates.*Significant differences from control (p ≤ 0.05).
nominal.Eggs were frozen at −80 immediately after injections, meaning biotransformation UV-P would not have occurred.In addition, there was no evidence that UV-P had precipitated out of solution.Exposure of zebrafish embryos to UV-P impaired the fertilization success of adult male zebrafish, but the fecundity of female zebrafish was unaffected.Although a clear mechanism of this effect was not identified, the evidence suggests that altered expression of genes involved in sex steroid synthesis and spermatogenesis, as well as decreased abundance of later stages of sperm development, might be playing a role.
Microinjection of embryos with UV-P had negligible effect on embryos.There were no significant effects on cumulative survival or heart rate.There was a significant, albeit small (6.7%), increase in the prevalence of spinal curvature and uninflated swim bladder in UV-P-exposed embryos in comparison with the controls.However, these increases occurred only in embryos exposed to the low treatment of UV-P.In previous studies, UV-P has been reported to be an agonist of the aryl hydrocarbon receptor (AhR) in zebrafish embryos based on greater transcript abundance of cytochrome P450 1A (cyp1a), a commonly used biomarker for AhR activation, as well as activation of the zebrafish AhR in a luciferase reporter gene assay (Fent et al., 2014;Johnson et al., unpublished).Activation of the AhR by some xenobiotics can cause developmental malformations, such as spinal curvature, yolk sac edema, and pericardial edema (Dubiel et al., 2022;Elonen et al., 2009;Larigot et al., 2018).In a previous study where zebrafish embryos were injected with UV-P at nominal doses of 4167, 12 500, and 37 500 ng/g-egg, there were 13%, 14%, and 18% incidences of malformed larvae, respectively (Johnson et al., unpublished).The lower incidences of malformations in the present study are likely due to lower doses injected into the embryos.
Following embryonic exposure of zebrafish to UV-P, fish were reared to sexual maturity in clean water and reproductive performance was assessed.First, reproductive performance was FIGURE 6: Effect of embryonic exposure to 2-(2H-benzotriazol-2-yl)-4-methylphenol on transcript abundances of genes involved in spermatogenesis in adult male zebrafish.Embryos were exposed via microinjection at doses of <1.5 (control), 2.77, and 24.3 ng/g egg and reared to sexual maturity in freshwater.(A-G) Abundances of nanos2, piwil1, dazl, insl3, igf3, amh, and wnt5a transcripts in testes of male zebrafish.Parametric data were analyzed using a one-way analysis of variance, followed by a Dunnett's post hoc test.Nonparametric data were log 10 transformed, and if data remained nonparametric, were analyzed using a Kruskal-Wallis test, followed by a Dunn's post hoc test.Data are represented as mean (±SEM) of six to eight replicates.*Significant differences from control (p ≤ 0.05).
using male and female fish that had been to UV-P.There were no impacts on fecundity, but fertilization success was significantly decreased by both doses of UV-P.Because fertilization is usually associated with fertility of males, fertilization success was re-assessed in a second reproduction assay that paired UV-P-exposed males with unexposed females to determine whether the males were responsible for this decline in fertility.Similar to the first reproduction assay, the results of this second reproduction assay showed a dose-dependent decrease in fertilization success, although the decrease was statistically significant only in the high treatment group.Ours is the first study that demonstrates impaired reproduction of fish exposed to a BUVS.In a previous study where sexually mature Japanese medaka were exposed to UV-P via their diet, there was no impacts on fecundity or fertilization success despite evidence of an antiandrogenic effect based on changes in gene expression and plasma concentrations of E2 and T (Fujita et al., 2022).Reasons for the differences in effects between these two studies are unknown, but it has been demonstrated that critical stages of embryo development are more sensitive to contaminants relative to adult life stages, thus these differences are likely the result of differences in the exposed life stage (Russell et al., 1999).
Previous studies have linked impaired fertilization success in zebrafish to disruption of steroidogenesis resulting in decreased E2 or T following contaminant exposure (Meng et al., 2023;Qian et al., 2020).The results of the present study suggest that decreased fertility of male zebrafish exposed as embryos to UV-P might be a result of disruption of steroidogenesis.Sex steroids are synthesized from cholesterol through a series of enzyme mediated reactions.First, cholesterol side-chain cleavage enzyme (CYP11A1) converts cholesterol to pregnenolone, which is a precursor for the synthesis of sex steroids (Bacila et al., 2021;Young et al., 2005).Testosterone can be converted to E2 by aromatase (CYP19A) or can be converted to 11-KT in a multistep pathway involving CYP11C1 and HSD11β2 (Bacila et al., 2021;Young et al., 2005).However, the dominant pathway for synthesis of 11-KT in zebrafish involves HSD17β3 (de Waal et al., 2008;Oakes et al., 2020;Tokarz et al., 2015).Binding of 11-KT to ARs initiates spermiation, allowing immature spermatids to differentiate and become mature, flagellated spermatozoa (Golshan & Alavi, 2019;Schulz & Miura, 2002;Schulz et al., 2010).Transcript abundances of cyp11a1, cyp19a1a, and cyp11c1 were decreased by 1.4-1.8-fold in testes from males exposed to UV-P, relative to the control, although these changes were not statistically significant.Transcript abundance of hsd17β3 was decreased by 1.5-and 2.5-fold in testes of male fish exposed as embryos to the low and high dose of UV-P, respectively, with the decrease being statistically significant in the high treatment group.There were no changes in abundances of transcripts of hsd11β2 or ar in testes of fish exposed to UV-P.Decreases in transcript abundances were small (less than threefold) and generally not statistically significant, but there was a maximal 50.0%decrease in E2.However, there was a maximal 32.4% increase in 11-KT, which is the opposite of what would be expected if the decreased transcript abundances of cyp11c1 and hsd17β3 resulted in decreased protein abundances.The absence of any changes in abundances of transcripts of hsd11β2 and ar suggests that embryonic exposure to UV-P likely did not inhibit synthesis of 11-KT or decrease AR signaling (Tokarz et al., 2015).In sexually mature male three-spot wrasse (Halichoeres trimaculatus) exposed to exemestane, an aromatase inhibitor, concentrations of E2 were decreased, concentrations of 11-KT were increased, and although there was an impairment in spermatogonial proliferation of the testis which was able to be rescued by exogenous E2, fertility was not determined (Kobayashi et al., 2010).These patterns in hormone concentrations are similar to those observed in the present study and could result from the decreased transcript abundance of cyp19a1a.However, a study with aromatase-deficient zebrafish found no significant changes in fertilization success (Tang et al., 2017).Other studies involving chemical inhibition of aromatase also found no impairment of fertility, although impairments in the hatching rate of embryos fertilized from exposed males, irreversible damage to sperm, and sperm motility, thus impacting sperm quality, were observed (McAllister & Kime, 2003;Thresher et al., 2011).
Another mechanism by which embryonic exposure to UV-P might have impaired fertilization success is by interfering with the expression of genes regulating sperm development and quantity.Expression of several genes, such as PIWIL1, WNT5A, and NANOS2, is essential for early spermatogenesis.The PIWIL1 gene is predominantly expressed in Type A und and Type A diff spermatogonia and is a marker for early stages of spermatogenesis because it has major functions in germ cell maintenance (Almeida et al., 2022;Houwing et al., 2007;Ye et al., 2023).Abundance of transcripts of piwil1 was decreased by 1.4-fold in the high treatment group, but the change was not statistically significant.In addition, expression of wnt5a, which is involved in spermatogonial self-renewal, was not different in UV-P-exposed males (Safian, Ryane, et al., 2018).The RNA-binding protein, NANOS2, is required for the maintenance and renewal of germline stem cells and is highly expressed in Type A und spermatogonia (Sada et al., 2009;Suzuki & Saga, 2008).Embryonic exposure to UV-P decreased transcript abundance of nanos2 in the low and high treatment groups by 2-and 1.9-fold, respectively, and the change in the low treatment group was statistically significant.However, the abundance of neither Type A und nor Type A diff spermatogonia was altered in testes of UV-P-exposed males.The quantity of Type A und and Type A diff spermatogonia is controlled by balancing increased self-renewal of Type A und spermatogonia, and spermatogonia proliferation forming Type A diff and Type B spermatogonia (Schulz et al., 2010).Given the lack of any change in abundance of Type A spermatogonia, either there was no corresponding change in protein levels or larger decreases in transcript abundance are needed to drive decreases in Type A und spermatogonia.Overall, these results suggest that decreased fertility of male zebrafish exposed as embryos to UV-P is not due to a decrease in spermatogonial self-renewal.
There is evidence that embryonic exposure to UV-P might have impacted the mitotic phase of spermatogenesis.Insulinlike peptide 3 (INSL3) and insulin-like growth factor-3 (IGF3) are both involved in recruiting and signalling spermatogonia into differentiation (Assis et al., 2015;Morais al., 2017;Safian, Bogerd, al., 2018).Although not statistically significant, transcript abundance of insl3 was decreased by 1.5-and 2.3fold in the low and high treatment groups, respectively.In addition, transcript abundance of igf3 was decreased by 2.8 and 2.1-fold in testes of males from the low and high treatment groups, respectively, but these values are not statistically significant.Based on these findings, a decrease in the number of Type A und spermatogonia differentiating into Type A diff spermatogonia, thus decreasing the number of later developmental stages, would be expected, but was not observed.The function of anti-Müllerian hormone (AMH) in the testis is to inhibit the action of INSL3 and 11-KT in promoting differentiation/ proliferation of spermatogonia (Safian et al., 2019;Skaar et al., 2011).The transcript abundance of amh was not different in zebrafish exposed to UV-P.Previous studies have shown that knockdown of expression of the IGF3 gene in common carp (Cyprinus carpio) increased concentrations of 11-KT in blood plasma, increased the proportion of earlier stages of sperm development present in testis, and led to a lesser number of spermatids (Song et al., 2021).This is consistent with the decreased abundance of spermatids and increased concentrations of 11-KT in UV-P-exposed fish, relative to the control, observed in the present study.Type B spermatogonia represent the most proliferated stage prior to meiosis (Schulz et al., 2010).Abundance of the RNA-binding protein, DAZL, is increased in Type B spermatogonia relative to abundance in the early stages of primary spermatocytes (Chen et al., 2013;Oakes et al., 2019;Saunders et al., 2003).Previous research has determined that expression of DAZL is critical for the entry of germ cells into meiosis during germ cell development (Bertho et al., 2021;Chen et al., 2013;Li et al., 2016;Saunders et al., 2003).In mice, knockout of dazl inhibited progression of germ cell development following meiosis (Saunders et al., 2003).In the present study, the transcript abundance of dazl was significantly decreased by 3-and 3.1-fold in the testes of males from the low and high treatment groups, respectively.However, there was no change in the abundance of Type B spermatogonia in the testes of males exposed to UV-P, relative to the control.This suggests that embryonic exposure to UV-P could be impairing spermatogenesis by preventing entry into meiosis, which could be reflected by decreased abundance of later developmental stages, including primary and secondary spermatocytes, as well as spermatids.In the present study, the abundance of primary spermatocytes was nonsignificantly decreased by 1.4-fold in both treatment groups, the abundance of secondary spermatocytes was significantly increased by 1.6-fold, and the abundance of spermatids was nonsignificantly decreased by 1.5-fold in fish exposed as embryos to the high dose of UV-P.The decrease in primary spermatocytes could potentially be due to decreases in dazl expression, thus preventing Type B spermatogonia from entering meiosis and forming primary spermatocytes 11-KT is critical for the maturation of gametes because it ultimately signals the formation of spermatids (Schulz et al., 2010).The present study observed a small increase in blood plasma concentrations of 11-KT in fish exposed to UV-P, but this change was not consistent with the observed decrease in the abundance of spermatids, therefore the results suggest this impairment is likely not due to changes in 11-KT synthesis.Consequently, the production of secondary spermatocytes might be increased to compensate for the decrease in primary spermatocytes and spermatids.
The present study determined that embryonic exposure to UV-P decreased the reproductive performance of male zebrafish and therefore attempted to identify the potential mechanism(s) of this effect by investigating disruption of spermatogenesis via changes in steroidogenesis and expression of genes related to sperm development and quantity, but the precise mechanism remains unclear.The observed impairment in reproduction aligns with the small and often not statistically significant changes in gene expression and plasma hormone concentrations, thus making it difficult to elucidate a specific mechanism of effect.However, these small changes in gene expression and hormone concentrations are likely due to the use of doses that have been measured in biota in some locations, as well as the relatively small change in fertilization success observed.Thus, future studies are needed to pinpoint the mechanism(s) by which embryonic exposure to UV-P impairs zebrafish reproduction.As indicated by the present study, there is potential for UV-P to act on multiple pathways, all of which might contribute to impaired fertility.To elucidate the mechanism(s) by which UV-P acts, future research should explore the molecular regulation of postmitotic stages of spermatogenesis, such as quantifying luteinizing hormone and maturation-inducing hormone.Also, although it was observed that sperm quantity is likely decreasing (as indicated by decreased abundance of primary spermatocytes and spermatids), decreased sperm quality can also play a role in decreased fertility.The effects of embryonic exposure to UV-P on sperm quality parameters, such as sperm motility, sperm volume and concentration, seminal plasma pH, osmolality, membrane stability, and enzymatic activity should be measured in future studies (Kowalski & Cejko, 2019).Regardless of the precise mechanism(s), the present study demonstrates the potential for BUVSs to impair reproductive success in fishes via decreased fertility of male fish.However, the 9% to 15% decrease in fertilization success observed in the present study would be unlikely to cause declines in populations because 20% to 30% decreases in the number of offspring have been proposed as being required for population-level effects (Conolly et al., 2017).However, if concentrations of UV-P in aquatic systems continue to increase, there could be the potential for population-level effects via decreased fertility of males.Furthermore, other BUVSs, such as 2-(5-tert-butyl-2-hydroxyphenyl)benzotriazole, 2-[2-hydroxy-5-[2-(methacryloyloxy)ethyl]phenyl]-2H-benzotriazole, 2-[3,5-bis(1methyl-1-phenylethyl)-2-hydroxyphenyl]-2H-benzotriazole, 2-(2hydroxy-3-tert-butyl-5-methylphenyl)-5-chlorobenzotriazole, 2-(2-hydroxy-5-tert-octylphenyl)-benzotriazole, 2-(3-s-butyl-5-tertbutyl-2-hydroxyphenyl)benzotriazole, and UV-328 can act as anti-androgens (Sakuragi et al., 2021;Zhaung et al., 2017).Thus, compared with single compounds, exposure to multiple BUVSs might have a greater impact on the fertility of male fish.In addition, differences in sensitivity to endocrine disruption are known to exist among species of fishes, therefore species that are more sensitive than zebrafish to anti-androgenicity be more impacted by the doses used in the present study (Ankley & Johnson, 2004).Thus, assessments of the effects of UV-P on the reproductive performance of adult male fish following embryonic exposure should be extended to other fish species.Finally, the role of the epigenome in the observed decreases in fertilization success should be investigated.Specifically, the DNA methylome of zebrafish undergoes a period of remodelling following fertilization, where the maternal methylation pattern is erased and the paternal pattern is inherited (Potok et al., 2013;Wang & Bhandari, 2019).This reprogramming is sensitive to disruption by contaminants, and because changes in DNA methylation are mitotically stable, alterations to the methylome can have effects that extend across life stages and generations (Chen et al., 2019;Major et al., 2020;Xu et al., 2023).Future studies should also assess whether embryonic exposure to UV-P causes multigenerational or transgenerational effects via alterations to the DNA methylome.

CONCLUSION
Overall, embryonic exposure of zebrafish to UV-P impaired the reproductive performance of male zebrafish by decreasing fertilization success.Small but consistent decreases in abundances of transcripts that regulate sex steroid synthesis indicate disruption of the steroidogenic pathway.In addition, there was evidence that ELS exposure to UV-P disrupted spermatogenesis, potentially reducing the quantity of sperm produced.However, further research is needed to determine the mechanism by which embryonic exposure to UV-P impairs fertility, specifically, the molecular regulation of postmitotic stages of spermatogenesis, as well as the investigation of sperm quality.The present study is the first to demonstrate that embryonic exposure to an environmentally realistic dose of a BUVS impaired the reproductive performance of a fish species, the zebrafish.Because of the ubiquitous presence of BUVS in freshwater ecosystems, and the strong likelihood that concentrations of these chemicals will continue to increase, future research is needed to fully understand the risk BUVSs pose to fishes and other aquatic biota.
Supporting Information-The Supporting Information is available on the Wiley Online Library at https://doi.org/10.1002/etc.5790.