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. Author manuscript; available in PMC: 2022 Nov 30.
Published in final edited form as: Environ Toxicol Chem. 2022 Oct 11;41(11):2822–2834. doi: 10.1002/etc.5472

The Zebrafish (Danio rerio) Embryo Model as a Tool to Assess Drinking Water Treatment Efficacy for Freshwater Impacted by Crude Oil Spill

Monika A Roy a,b,1, Aarthi Mohan c,*,1, Yankel Karasik a, John E Tobiason c, David A Reckhow c, Alicia R Timme-Laragy a
PMCID: PMC9711864  NIHMSID: NIHMS1843649  PMID: 36040130

Abstract

Traditional approaches toward evaluating oil spill mitigation effectiveness in drinking water supplies using analytical chemistry can overlook residual hydrocarbons and treatment byproducts of unknown toxicity. Zebrafish (Danio rerio) were used to address this limitation by evaluating the reduction in toxicity to fish exposed to laboratory solutions of dissolved crude oil constituents treated with 3 mg/L ozone (O3) with or without a peroxone-based advanced oxidation process using 0.5 M H2O2/M O3 or 1 M H2O2/M O3. Crude oil water mixtures (OWMs) were generated using three mixing protocols—orbital (OWM-Orb), rapid (OWM-Rap), and impeller (OWM-Imp) and contained dissolved total aromatic concentrations of 106–1019 μg/L. In a first experiment, embryos were exposed at 24 h post fertilization (hpf) to OWM-Orb or OWM-Rap diluted to 25%–50% of full-strength samples and in a second experiment, to untreated or treated OWM-Imp mixtures at 50% dilutions. Toxicity profiles included body length, pericardial area, and swim bladder inflation, and these varied depending on the OWM preparation, with OWM-Rap resulting in the most toxicity, followed by OWM-Imp and then OWM-Orb. Zebrafish exposed to a 50% dilution of OWM-Imp resulted in 6% shorter body length, 83% increased pericardial area, and no swim bladder inflation, but exposure to a 50% dilution of OWM-Imp treated with O3 alone or with 0.5 M H2O2/M O3 resulted in normal zebrafish development and average total aromatic destruction of 54%–57%. Additional aromatic removal occurred with O3 + 1 M H2O2/M O3 but without further attenuation of toxicity to zebrafish. This study demonstrates using zebrafish as an additional evaluation component for modeling the effectiveness of freshwater oil spill treatment methods.

Keywords: Advanced oxidation processes, analytical chemistry, crude oil spill treatment, Danio rerio, development toxicity, freshwater toxicology, oil spills, toxic effects

INTRODUCTION

Crude oil spills in freshwater pose serious challenges to treatment utilities, government agencies, the public, and the environment. The severity of spill depends on the crude oil type, spill volume, freshwater body size, and flow characteristics. Once released into a water body, other factors such as spreading, mousse formation, sedimentation, evaporation, sorption, photolysis, and dissolution can impact the transport, persistence, and ultimate weathering of the crude oil (Green & Trett, 1989). In general, no more than 5% of crude oil from accidental spills dissolves in water (Irwin et al., 1997). However, this water-soluble fraction, composed of mostly polar and easily assimilable organics, is responsible for the majority of the acute toxicity impacts to aquatic organisms (Gladyshev, 2021; Marigómez, 2014), and potentially to human health.

Crude oils are dominated by structurally diverse hydrocarbons, constituting 50%–98% of oil by weight and ranging in boiling points of up to 600 °C (Irwin et al., 1997). The monoaromatic fraction in crude oil is the most water soluble, as shown by the low octanol:water partition coefficient (KOW) indicating high polarity. Among the aromatics, benzene, toluene, ethylbenzene, and xylene (BTEX) are crude oil toxicants that are known to persist in groundwater and are regulated in drinking water supplies in the United States (Total Petroleum Hydrocarbon Criteria Working Group, 1998; Wilbur & Bosch, 2004). Dissolved total petroleum hydrocarbon (TPH) concentrations of up to 58 mg/L have been measured in water, and these levels are usually influenced by both oil loading and background composition (Shiu et al., 1990). Other identified hydrocarbon classes include paraffins, isoparaffins, naphthenes, polyaromatic hydrocarbons (PAHs), naphthenic acids, trace amounts of sulfur, and nitrogen- or oxygen-containing hydrocarbons (Gad, 2014; Hsu et al., 2000; Simanzhenkov & Idem, 2003).

Analyses of one or more of these compound groups in whole crude or the water-soluble fractions of crude oil involve gas chromatography–based methods combined with either a flame ionization detector or mass spectrometer (Forth et al., 2017; Kanan et al., 2012). However, due to matrix complexity, none of the published methods capture complete compositional information. In addition, unresolved polar and nonpolar complex mixtures in oil spill samples account for most of the observed toxicity (Farrington & Quinn, 2015; Melbye et al., 2009). Two-dimensional techniques improve analytical resolution to a certain extent, yet do not provide a comprehensive idea of matrix constituents (Coutinho et al., 2018; Melbye et al., 2009; Patterson et al., 2020; Striebich et al., 2014). Therefore, researchers have traditionally studied either individual or groups of target compounds for quantification, or have conducted nonspecific TPH fingerprinting for qualitative analysis indicative of the potential for human exposure and toxicity outcomes (Barron et al., 2018; Kristanto, 2012).

The same analytical approach is true for treatment byproducts, which are formed during water treatment processes typically incorporated at water utilities like ozonation and ozone-based advanced oxidation processes (AOPs; Andreozzi et al., 2000; Chen et al., 2006; Gamal El-Din et al., 2011; Garoma et al., 2008; Klamerth et al., 2015; Pérez-Estrada et al., 2011; Scott et al., 2008; Ziabari et al., 2016). AOPs are tested treatment methods for oil-type constituents in different types of waters (e.g., wastewater and produced water) and therefore, are potential freshwater oil spill treatment strategies. Previous research demonstrates the successful removal of PAHs, TPHs, and BTEX (Garoma et al., 2008), although sometimes with incomplete oxidation of naphthenic acids (Meshref et al., 2017; Scott et al., 2008). The intermediates and byproducts of complete or incomplete oxidation and AOPs are usually a combination of identified and unidentified chemical species that may result in higher exposure to toxicants; however, few studies have addressed this research area (Gamal El-Din et al., 2011; Klamerth et al., 2015).

Identifying candidate hydrocarbons and byproducts may not be the most productive route to determining the toxicity of samples contaminated by a crude oil spill, because the analytical characterization is not only time and resource intensive, but often the target chemical analytes may not necessarily be the most toxic in the mixture. For instance, polar photo-oxidation intermediates and products generated from oil hydrocarbons such as phenols, acids, and carbonyls may contribute to the overall toxicity potential (Barron et al., 2018; Maki et al., 2001) but are not always targeted in analyses. Furthermore, known and unknown soluble hydrocarbons that are toxic at levels below the method or instrumental detection limits are easily overlooked. Although understanding the constituents of these mixtures is essential, there is a great deal of variability in predicting toxicity impacts by sheer chemical measurements of hydrocarbons (Klerks et al., 2004). This necessitates incorporation of parallel toxicity studies that can offer a more comprehensive approach to evaluating the treatment of complex mixtures (Farrington & Quinn, 2015; Gough & Rowland, 1990; Irwin et al., 1998).

Zebrafish (Danio rerio) embryo assays are useful for the investigation of overall toxicity impacts for scenarios such as freshwater oil spills. Zebrafish are a freshwater species with transparent embryos that develop externally to the mother and grow relatively fast. Numerous validated assays have been developed with them to assess aquatic toxicity and endpoints relevant to human health (Strahle et al., 2012), including assays to assess contaminants that interact with the aryl hydrocarbon receptor (AhR) pathway such as PAHs, which are ubiquitous in crude oils. The AhR is a basic-helix-loop-helix/per-Arnt-sim (bHLH/PAS) transcription factor family member that can be activated by naturally occurring substances like PAHs and dioxin-like anthropogenic constituents such as polychlorinated biphenyls (PCBs; Coumailleau et al., 1995). Briefly, when AhR ligand binding occurs, the AhR translocates to the nucleus and binds to the xenobiotic response element to upregulate metabolism-related genes like cytochrome P450 1a (cyp1a), which is then translated into its enzyme counterpart, Cyp1a, to potentially metabolize the activating compound (Kuhnert et al., 2017). The AhR pathway activation for zebrafish and humans is similar, although due to genome duplication in zebrafish, AhR pathway effects associated with toxicity are mediated by the ahr2 gene isoform (Hahn et al., 2006). The Cyp1a enzyme in zebrafish is orthologous to the human Cyp1A1 enzyme (Goldstone et al., 2010), which is generally the most highly inducible Cyp1 member in both zebrafish and humans (Jonsson et al., 2007; Nebert et al., 2004). Several studies have shown that disruption of Cyp1a activity is associated with toxicity outcomes. Therefore, studying Cyp1a function in zebrafish during environmental chemical exposures can inform the human response to these chemicals (Billiard et al., 2006; Roy et al., 2019; Timme-Laragy et al., 2007).

The goal of our study was to illustrate an in vivo toxicity investigation using the zebrafish model to more comprehensively evaluate chemical treatment methods such as ozonation alone or in combination with a peroxone-based AOP of ozone (O3) + peroxide (H2O2) in removing dissolved crude oil hydrocarbons. Comparative toxicity assessments and Cyp1a activity measurements in conjunction with targeted analytical measurements were performed on untreated and chemically treated laboratory mixtures of dissolved crude oil hydrocarbons in raw water to demonstrate treatment efficacy.

MATERIALS AND METHODS

Chemicals

West Texas Intermediate (WTI) crude oil was purchased from Texas Raw Crude, a vendor in the Texas Permian region, for oil-water mixture (OWM) preparations. With a density of 0.85 g/cm3 (15 °C), WTI is classified by the American Petroleum Institute (API) as a “light” crude oil (API gravity: 36–41), which is moderately volatile and toxic (Lee et al., 2015). Prior toxicity testing of its water-soluble fractions on Daphnia magna have indicated a 48-h median effect concentration (EC50) of 12.71 mg/L (Environment Canada, 1994). The volatile organic content in WTI is 160 mg/L and 14.8% by weight aromatics (Wang et al., 2003; Environment Canada, 1994). An analytical standard mixture containing 38 petroleum aromatics (Part #44587) was used in targeted chemical screening. This component mixture was part of a larger kit of petroleum hydrocarbon standards (#44594-U) that was purchased from Millipore Sigma. Sodium chloride for salting was purchased from Fisher Scientific. Methanol and hexane were purchased from Fisher Scientific. For initiating AOPs, concentrated H2O2 stock solutions (ACS grade, 30% w/v) were purchased from Fisher Scientific. Standard mixture for aldehyde analysis was prepared from individual solutions, all purchased from Sigma Aldrich.

For the zebrafish assays, 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126) was acquired from Ultra Scientific, dissolved in 100% dimethyl sulfoxide (DMSO; Thermo Fisher Scientific). Stock solutions were stored at −20 °C in glass amber vials and were fully thawed and vortexed before use.

OWM preparation

The WTI crude oil was used for the preparation of laboratory OWMs using an uncontaminated raw water collected from a reservoir in the northeast United States with an average dissolved organic carbon (DOC) of 1.85 ± 0.08 mg/L. Unless otherwise specified, all pHs were near-neutral. In all cases of OWM preparation, the ratio of oil to water was held constant at 0.28% by volume. This ratio was selected to generate a mixture with an additional DOC of 1–2 mg/L above the raw water DOC content. Changes were introduced only to the type of mixing and scale of mixture preparation. The OWMs were prepared using three different mixing protocols—orbital, rapid, and impeller—with final mixtures designated as OWM-Orb, OWM-Rap, and OWM-Imp. The OWM-Orb and OWM-Rap mixtures were both prepared at a small scale in 2-L Erlenmeyer flasks sealed with laboratory film (0.28% v/v oil:water; 2 L total volume; 24-h oil to water contact). The OWM-Orb mixture was prepared at 100-rpm, low-intensity orbital shaking. The OWM-Rap was prepared at a relatively higher intensity of 350 rpm using magnetic stirring, which resulted in a vortex greater than one-third of the water column depth. These mixtures were subjected to vacuum filtration using a glass fiber filter (GF/F, 125 mm; GE Whatman) prior to analysis or exposures. The OWM-Imp was prepared on a larger scale (~23 L total) for use in multiple smaller treatment experiments not presented here. The raw water with 0.28% v/v oil:water (~23 L total volume) and minimal headspace was continuously stirred for approximately 24 h at approximately 450 rpm in a 6-gallon polyethylene terephthalate jar fitted with a spout (CP Lab Safety) using an overhead impeller (Tline Laboratory Stirrer, model 102; Talboys Engineering). There was no vortex formation for OWM-Imp, and the mixture generally appeared homogenous. Over time, oil emulsions in raw water were formed. After 24 h of mixing, the mixture was allowed to undergo phase separation for approximately 15 min. The raw water (now containing dissolved oil constituents) was collected using the spout and filtered using nitrogen-driven pressure filtration through a coarse glass fiber filter (125 mm; GE Whatman) followed by a fine-pore filter (GF/F; GE Whatman). Such modifications in the filtration protocol were introduced to minimize handling losses for large-volume OWMs and early breakthrough of oil micelles during filtration. Modifications to OWM preparations were attempted due to the lack of a standardized protocol for freshwater spill scenarios while also keeping in mind the varying volume requirements often imposed by bench and pilot-scale treatment methods.

Chemical treatment of OWMs

Ozone was generated using a corona-type generator (Welsbach model T-408 Laboratory Ozone Generator; Welsbach Ozone Systems) that is fed pure oxygen. This was bubbled into a slightly acidified laboratory-grade water to prepare aqueous O3 stock solutions. Ozone stock concentrations and residuals were measured through ultraviolet (UV) absorbance measurements at 260 nm. Ozone was dosed at 3 mg/L (1 h O3 contact) in batch mode for all treatment cases. For the AOPs, H2O2 and O3 (together referred to as peroxone), were applied at two doses such that the final molar ratios were 0.5 and 1 M H2O2/M O3. Standardization of H2O2 stock and residual H2O2 measurements were performed using a horseradish peroxidase method that involves measuring ABTS+ which is formed as a result of a reaction between azino-di-[3-ethylbenzthiazoline sulfonate (ABTS) and H2O2 (Gordon et al., 1992). All treatment experiments were performed at 20 °C. The treated and untreated OWMs were stored headspace free in 100-ml amber glass beakers at 4 °C and were used in zebrafish experiments within 3 days.

General characterization of OWMs

The DOC analysis was performed using a total organic carbon analyzer (Shimadzu, TOC-VCPH). Absorbance scans (UV-Vis) were acquired using a UV-Vis spectrophotometer (Agilent Technologies). Absorbance at 254 nm (a routinely tracked water quality parameter) was obtained as a surrogate measurement of dissolved aromatics. Preconcentration of dissolved aromatic constituents was performed using a headspace solid-phase microextraction technique previously used for analysis of benzene, toluene, and ethyl benzene, and briefly described elsewhere (Jeznach et al., 2021). A concise description of this method extrapolated to more than 30 aromatic hydrocarbons (C6-C12) is presented in the Supporting Information (Supplemental Methods S1 and Table S1; Supplemental Methods S2 and Table S2). For aldehyde analysis, 20-ml samples were subjected to derivatization with pentafluorobenzylhydroxyl amine followed by extraction of the resulting oxime into hexane and analysis with gas chromatography and electron capture detection.

Animal care

Adult wild-type AB zebrafish (D. rerio) were housed in a 14:10-h light:dark cycle in a recirculating Aquaneering system maintained at 28.5 °C. Embryos were collected at 1 h post fertilization (hpf), rinsed, and stored at low density in 0.3× Danieau’s media [17 mM NaCl, 2mM KCl, 0.12 mM MgSO4, 1.8 mM Ca(NO3)2, 1.5 mM HEPES, pH 7.2] in an incubator with the same temperature and light conditions as the adult fish. At 24 hpf, embryos were manually dechorionated using Watchmakers forceps and screened for normal development before use in experiments. All animal care and experiments were conducted in accordance with protocols approved by the University of Massachusetts Amherst Institutional Animal Care and Use Committee (Animal Welfare Assurance Number 2019-0067). Animals were treated humanely with due consideration to the alleviation of stress and discomfort.

OWM exposures to zebrafish

In a preliminary experiment to optimize a dilution for OWM toxicity testing, zebrafish were exposed at 24 hpf to either 25% of full-strength dilutions (1:3 sample:Danieau’s) or 50% of full-strength dilutions (1:1 sample:Danieau’s) of raw water, OWM-Orb, or OWM-Rap in Danieau’s embryo media. For each experimental group, five zebrafish embryos were exposed in 10 ml of total solution in 20-ml glass scintillation vials. As a peripheral endpoint of interest, experiments were prepared to also capture Cyp1a enzymatic activity using the well-established ethoxyresorufin-O-deethylase (EROD) assay to monitor the in vivo response of Cyp1a. In this assay, AhR-mediated induction of Cyp1a activity cleaves 7-ethoxyresorufin to resorufin, which is then visible under a fluorescent microscope and is representative of Cyp1a activity (Kais et al., 2018; Nacci et al., 2005). For this assay, at 24 hpf, 7-ethoxyresorufin-O-deethylase (7-ER; MP Biomedicals) was dissolved into each vial of zebrafish embryos so that the final concentration was 0.5 μg/L.

Vials were placed in an incubator (28.5 °C, 14:10-h light:dark cycle, no vial agitation) until imaging at 96 hpf. In a subsequent experiment to test the efficacy of chemical treatment techniques, zebrafish were exposed at 24 hpf to a 50% dilution of either treated (O3, O3 + 0.5 M H2O2/M O3, or O3 + 1 M H2O2/M O3) or untreated raw water or OWM-Imp. For both experiments, each exposure group contained at least two technical replicates (vials), and each experiment was repeated three times with different clutches of embryos. For the second experiment, separate vials of zebrafish were also exposed to either DMSO as a negative control or 5 nM PCB-126 as a positive control, because PCB-126 is a well-known inducer of the same morphological deformities that exposure to crude oil causes in zebrafish (Rousseau et al., 2015; Roy et al., 2019), and these exposures took place in 100% Danieau’s embryo media as laboratory controls.

Microscopy and image analysis

At 96 hpf, live larvae were sedated by a 10-s exposure to 2% v/v tricaine mesylate solution (prepared as 4 mg/ml tricaine powder in water, pH buffered, and stored at −20 °C until thawed for use) before being mounted on individual 3% methylcellulose drops in a left-lateral orientation. The zebrafish were imaged under a brightfield setting on a Zeiss Stereo Axio Zoom.V16 equipped with a HXP 200 C light source (Carl Zeiss) at 20× total magnification to capture images for measurements of zebrafish body length, pericardial area, and swim bladder inflation. At 96 hpf, when fish were imaged for overall morphology, they were also imaged at 100× total magnification under a red fluorescence protein filter and fixed exposure time to capture EROD (Cyp1a activity) light intensity. Brightfield images were also captured at 100× total magnification to create overlay images of Cyp1a activity.

Body length, pericardial area, and EROD light intensity were measured with the Zen Lite program (Carl Zeiss). To quantify light intensity for EROD images, the software was used to find the maximum intensity out of the entire image. To calculate background intensity, the circle tool was used in a consistent place in each image, and the mean intensity was recorded. To compare fluorescent images between experimental replicates, both background light intensity and dark pixel intensity (a calculated constant; Pang et al., 2012) were subtracted from the recorded maximum light intensity for every image, and then this value was divided by the exposure time. Each experimental replicate was normalized to its respective control group, and then data from experimental replicates were merged. The EROD for the raw water control groups was set to 100% for comparison with the OWM groups for their respective dilutions. Swim bladder inflation was scored as 0 = not inflated and 1 = inflated; full inflation normally occurs by 120 hpf (Jonsson et al., 2012).

Statistical analyses

For the zebrafish experiments, data were analyzed for outliers (0.25 quartile tails) for body length, EROD, and pericardial area endpoints across all experimental replicates, and individual samples were removed completely from all analyses if they were flagged as outliers for at least one endpoint (on average one fish/exposure group across all experiments). Descriptive statistics were performed in Excel Ver 16.62 for the control group for the endpoints with continuous data (body length, EROD, and pericardial area) to aid in assessing normality. All three measured outcomes (body length, EROD, pericardial area, and swim bladder inflation) of fish at 96 hpf were averaged/vial so that each vial of up to five pooled fish was n = 1. A one-way analysis of variance with a Tukey–Kramer post hoc test was performed for body length, EROD and pericardial area endpoints with JMP® Pro software Ver 15.1.0. Statistical significance was considered using a 95% confidence interval (α = 0.05). Swim bladder inflation data are presented as averaged percentages, and no statistical test was performed.

RESULTS

Chemical screening of OWMs

When the oil to water ratio was held constant at 0.28% v/v, the OWM preparation methods yielded DOC values measured at 1–2 mg/L greater than raw water background DOC due to dissolution of crude oil hydrocarbons. In addition, the UV absorbance at 254 nm increased by approximately 0.02 cm−1 over the value of the raw water background. Because DOC values in the same range might not mean similar dissolution of hydrocarbons across OWM mixing methods, the type of aromatic constituents that the zebrafish were exposed to were quantitatively and qualitatively screened for a range of mono-aromatics. Targeted analysis was performed for both the regulated BTEX compounds and a set of other compounds that are not regulated in drinking water but can constitute an important fraction of the dissolved organics (Supporting Information, Figure S1). Twenty-six compounds out of the targeted 35 were detected consistently in the three OWM solutions, all of which had similar aromatic fingerprints (Supporting Information, Figure S2). However, quantitative differences resulting from variations in mixing protocols were observed. The concentration of tert-butylbenzene was the highest among the aromatics analyzed in all three OWMs, with a range of 93–332 μg/L. The concentrations of BTEX and other measured aromatics are presented in Table 1 along with available information regarding aromatic content in the original WTI crude oil. Based on the added mass of oil for each OWM preparation scenario and known masses of BTEX/gram of WTI oil, the total dissolved BTEX in water would be approximately 55 mg/L if there were a 100% dissolution of oil into water. On a mass basis, the measured concentrations were only 0.006%–0.7% of the dissolved concentration expected at 100% oil dissolution. The overall mass of dissolved aromatics decreased in the order: OWM-Rap > OWM-Imp > OWM-Orb.

TABLE 1:

Summary of water quality parameters for the raw water and oil–water mixtures prepared in raw water (before dilutions in zebrafish media)

Aromatic WTI (ppm)a WTI (μg/g oil)b OWM-Orb (ppb) OWM-Rap (ppb) OWM-Imp (ppb) Solubility (ppm)c
Benzene 1380 4026 bdl 15.82 2.02 1780
Toluene 2860 7395 bdl 135.94 8.68 515
Ethylbenzene 1120 4845 bdl 52.78 6.27 152
Xylene (m-, p-) 4290 7105 2.16 158.42 9.25 170d
Xylene (o-) 1.16 47.07 22.8 175
BTEX (total) 9650 23 371 3.80 410 49
ΣTargeted aromatics 106 1019 220
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c

Solubility data are for distilled water measured at 20–25 °C; Clark & MacLeod (1977); Green & Trett (1989).

d

US Environmental Protection Agency (1996).

bdl = below detection limit; BTEX = benzene, toluene, ethylbenzene, and xylene; m- = meta; o- = ortho; p- = para; OWM = oil–water mixture; Orb = orbital; Rap = rapid; Imp = impeller; ppm = parts per million; ppb = parts per billion; WTI = West Texas intermediate.

The zebrafish embryo model is useful for assessing the toxicity of OWMs at 50% dilutions

An initial experiment assessed toxicity effects to zebrafish from exposures to 25% or 50% of full-strength dilutions of either raw water, OWM-Orb, or OWM-Rap between 24 and 96 hpf. Zebrafish control groups exposed to either 25% or 50% of full-strength dilutions of raw water developed normally in terms of body length (Figure 1A) and pericardial area (Figure 1B), with 40%–60% swim bladder inflation (Figure 1C). Zebrafish exposed to 25% dilutions of OWM-Orb or OWM-Rap developed normally, although fish exposed to OWM-Rap only had 15% swim bladder inflation (Figure 1A–D). Zebrafish exposed to 50% dilutions of OWM-Orb resulted in normal body length and pericardial area, but only 11% with swim bladder inflation (Figure 1A–D). Zebrafish exposed to 50% dilutions of OWM-Rap experienced significant toxicity, with a 7% decrease in length (Figure 1A), a 226% increase in pericardial area (Figure 1B), and no swim bladder inflation (Figure 1C). Representative images of whole fish can be seen in Figure 1D.

FIGURE 1:

FIGURE 1:

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Zebrafish were exposed between 24 and 96 hours post fertilization (hpf) to 25% or 50% of full-strength dilutions of raw water (RW) or oil–water mixtures (OWMs) and at 96 hpf measured for (A) body length and (B) pericardial area (mean ± SEM, n = 4–8 vials of fish/exposure group across three experiments, one-way analysis of variance with a Tukey post hoc test, p < 0.05). (C) Swim bladder (SB) inflation (not statistically assessed). (D) Representative images of fish from each exposure group. Orb = orbital; Rap = rapid.

Toxicity to zebrafish embryos varies depending on the protocol used for OWM preparation

Based on the initial zebrafish experiment, a 50% of full-strength dilution was deemed sufficient to see toxicity at measurable levels. A new OWM was prepared, OWM-Imp, to test chemical treatments, and all resulting mixtures were used at 50% dilutions in zebrafish exposures to assess toxicological outcomes. Table 2 summarizes the toxicity effects to zebrafish presented in Figure 1 and compares them with the toxicity parameters of zebrafish exposed to a 50% dilution of OWM-Imp (results from OWM-Imp are further expanded in Figure 2). The ratio of oil to water was consistent across all OWMs but the mixing methods and volumes were varied to suit the experiment goals. Exposures to a 50% dilution of OWM-Rap produced the most severe toxicological outcomes in zebrafish, followed by exposures to a 50% dilution of OWM-Imp. The observed morphological toxicity trends closely aligned with the total dissolved aromatic measurements in each OWM (Table 1).

TABLE 2:

Comparison of zebrafish toxicity parameters for 50% dilutions of oil–water mixtures compared with their 50% dilution raw water controls

Parameter OWM-Orb OWM-Rap OWM-Imp
Body length ↓ 1.27% ↓ 7.12% ↓ 5.59%
Pericardial area ↑ 12.6% ↑ 226% ↑ 83.1%
SB inflation ↓ 81.4% ↓ 100% ↓ 100%
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Bolded results indicate significance compared with each experiment’s raw water control group. Swim bladder inflation was not assessed for significance and is shown in italics as a simple percentage decrease.

OWM = oil–water mixture; Orb = orbital; Rap = rapid; Imp = impeller; SB = swim bladder.

FIGURE 2:

FIGURE 2:

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Effect of 3 mg O3 alone or with either 0.5 M or 1 M H2O2/M O3 on the oil–water mixture (OWM) 3 matrix. The results show (A) measured removal of different aromatic constituents and (B) the formation of select aldehydes after treatment.

Chemical screening demonstrates treatment impacts

The OWM-Imp was subjected to chemical treatment using ozonation alone or with a peroxone AOP. For ozonation alone, the decrease in BTEX concentration was 46%–67% compared with the untreated OWM-Imp control (Figure 2A). This did not significantly improve with O3 + 0.5 M H2O2/M O3. However, for O3 + 1 M H2O2/M O3, BTEX removal was slightly enhanced to 56%–75%. Similar trends were exhibited among the other hydrocarbons besides BTEX; ozonation resulted in an average destruction of approximately 58%, which improved to approximately 72% with O3 + 1 M H2O2/M O3 (Figure 2A).

Aldehydes were the only group of ozonation byproducts measured. Dissolved crude oil hydrocarbons after oxidation using ozone yielded aldehydes in the range of not detected to 61 μg/L in the order: formaldehyde > methyl glyoxal > acetaldehyde > glyoxal > propionaldehyde (Figure 2B). After application of peroxone at the lowest dose tested (O3 + 0.5 M H2O2/M O3), most aldehydes decreased by 23%–94%. With O3 + 1 M H2O2/M O3, acetaldehyde was destroyed completely whereas formaldehyde slightly increased compared with O3 + 0.5 M H2O2/M O3. Glyoxal and methylglyoxal decreased by approximately 50% compared with ozonation alone. Such decreasing trends have been noted with aldehydes in raw waters at very high ozone doses, which was also observed in the present study when a peroxone AOP was used (Nawrocki & Kalkowska, 1998).

Zebrafish embryos exposed to an OWM treated with O3 alone or with a peroxone-based AOP develop normally

Chemical treatments were conducted on OWM-Imp and its associated raw water, and all mixtures were used for zebrafish exposures at 50% of full-strength dilutions. Zebrafish exposed to laboratory controls DMSO or 5 nM PCB-126 in 100% embryo media were also included, and at 96 hpf, zebrafish exposed to PCB-126 displayed significant toxicity, with an 11% decrease in body length (Figure 3A), a 125% increase in pericardial area (Figure 3B), and no swim bladder inflation (Figure 3C), compared with zebrafish exposed to DMSO. Zebrafish exposed to OWM-Imp alone experienced a significant 6% decrease in body length (Figure 3A), a significant 83% increase in pericardial area (Figure 3B), and no swim bladder inflation (Figure 3C), compared with zebrafish exposed to raw water alone. Zebrafish exposed to raw water or OWM-Imp treated with either O3 alone, O3 + 0.5 M H2O2/M O3, or O3 + 1 M H2O2/M O3 developed normally, compared with the raw water control group (Figure 3A–C). Representative images of whole fish can be seen in Figure 3D.

FIGURE 3:

FIGURE 3:

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Zebrafish were exposed between 24 and 96 hours post fertilization (hpf) to 50% of full-strength dilutions of raw water (RW) or oil–water mixture impeller (OWM-Imp) untreated or treated with O3, O3 + 0.5 M H2O2/M O3, or O3 + 1 M H2O2/M O3 and compared with laboratory controls (CTLs) dimethyl sulfoxide (DMSO) and polychlorinated biphenyl (PCB)-126 (exposed in 100% embryo media). At 96 hpf, zebrafish were observed for (A) body length and (B) pericardial area (mean ± SEM, n = 5–6 vials of fish/exposure group across three experiments, one-way analysis of variance with a Tukey post hoc test, p < 0.05). (C) Swim bladder (SB) inflation (not statistically assessed). (D) Representative images of fish from each exposure group.

DISCUSSION

The bulk water chemistries of three differently prepared OWMs were compared, and their toxicity effects to zebrafish were evaluated. Of the three OWMs, OWM-Imp was treated with different ozone and peroxone-based AOPs and was then evaluated using analytical chemistry. Zebrafish were also used to observe the toxicity attenuation of the different treatments. Many types of crude oil exist, varying in sourcing, processing, density, and hydrocarbon content. The WTI crude oil, which is sourced primarily from the United States, was used to prepare the OWMs in the present study based on its potential to contaminate drinking water supplies in the United States. Using analytical chemistry, we demonstrated that similar qualitative aromatic profiles occurred in all OWMs regardless of preparation methods, but with significant quantitative differences, suggesting that OWM preparation protocols can influence the extent of toxicity. Such differences can be attributed to the mode of transfer of oil hydrocarbons into water. For instance, from visual observation it appeared that for OWM-Rap and OWM-Imp, this transfer mechanism was dominated by oil droplets, whereas for OWM-Orb it was through slick formation, indicating a lower extent of dissolution (Mackay & Leinonen, 1977; Mackay et al., 1980).

Several abiotic and biotic factors can influence the bioavailability of toxic substances to aquatic organisms, such as temperature, pH, the water chemistry, the toxicant chemistry, and the aquatic organism’s diet and physiology, among other factors (Katagi, 2010). Many of these factors were consistent across the zebrafish toxicity experiments conducted, with the primary variable being the preparation conditions of the OWMs. Each of the differently prepared OWMs resulted in varying degrees of toxicity to zebrafish, with OWM-Rap inducing the most severe morphological toxicity outcomes (Table 2). Pericardial edema was evaluated as a toxicity endpoint because it is a characteristic outcome of fish embryos exposed to both halogenated aromatic compounds like PCB-126 (Liu et al., 2016) and PAH mixtures found in crude oil (Incardona et al., 2005). This toxicity endpoint is often associated with activation and/or partial inhibition of the AhR pathway and downstream enzymes such as Cyp1a, which are important for the biotransformation of the compound, although this depends on the environmental contaminants; crude oil and associated constituents have been found to induce toxicity both dependently and independently of the AhR pathway (Incardona et al., 2005; Incardona et al., 2011). It is relatively easier to study how individual compounds interact with the AhR pathway, but much more complicated with complex mixtures such as the OWMs used in the present study. As a peripheral endpoint, Cyp1a activity was evaluated alongside the other zebrafish endpoints, with the overall finding that all three OWMs decreased Cyp1a activity (Supporting Information, Figures S3 and S4). These results point toward constituents in the OWMs that both activate the AhR pathway and suppress Cyp1a or other hepatic enzyme activity, as has been observed in previous studies (Brown et al., 2016; Roy et al., 2019). However, the focus of this study was not to determine whether the pericardial edema observed in the OWM control groups was AhR mediated. Instead, the focus was to verify that the OWMs do induce toxicity and then to evaluate whether reductions in toxicity occur after exposures to the treated OWMs.

Research groups have used both zebrafish (Perrichon et al., 2016; Raimondo et al., 2014) and other marine (Incardona et al., 2013; Karam et al., 2014; Olsen et al., 2013) and freshwater fish species (Pollino & Holdway, 2002) to characterize toxicological outcomes after exposure to different types of crude oil, confirming pericardial abnormalities similar to the ones reported here. It is also clear that crude oil hydrocarbons impact other freshwater meiofauna and nematode abundance, diversity, and species composition (Monteiro et al., 2019). Oil spill treatment strategies in situations not involving freshwaters have been assessed using some of these indicator species. Most of these studies, although related to oil sands process-affected water (OSPW), confirm the attenuation of toxicity in rainbow trout (Oncorhynchus mykiss), fathead minnows (Pimephales promelas), goldfish (Carassius auratus L.), Vibrio fischeri bacteria, the H295R cell line, and other larval zebrafish (D. rerio) studies after exposure to ozonated OSPW (Hagen et al., 2014; He et al., 2010; He et al., 2012; Reichert et al., 2017; Scarlett et al., 2013; Sun et al., 2014; Wang et al., 2013; Wiseman et al., 2013). Sensory, behavioral, motility, developmental, and immune response are some of the endpoints evaluated in these studies. Processes other than ozonation, such as UV irradiation-based AOPs, have also been noted to decrease the toxicological outcomes of V. fischeri after exposure to OSPW (Fang et al., 2019). However, to our knowledge, no research has been conducted to assess the efficacy of ozonation or other treatment processes widely used in drinking water utilities from a freshwater oil spill perspective using a fish model, which is a gap this study attempts to fill. In this study, ozonation with or without a peroxone-based treatment process was observed to mitigate toxicity imparted by the original OWMs in zebrafish, which is in agreement with the studies on OSPW just cited.

The dissolved phase hydrocarbons in the laboratory-generated OWMs contain myriad co-occurring hydrocarbons with varying levels of toxicity (Singer et al., 2000). Most studies demonstrating the effectiveness of ozonation on OSPW provide an analytical characterization of naphthenic acids. On the other hand, most mixture toxicity studies related to marine oil spills have analytically characterized PAHs and/or TPHs (Li et al., 2019; Price & Mager, 2020; Price et al., 2022). The PAHs are known to be toxic to fish embryos with EC50s in the range of 0.3–60 μg/L (Billiard et al., 2008; Hodson, 2017; Incardona et al., 2005). However, a recent study questioned prior conclusions by others that PAHs are the primary toxicity-inducing constituents in oil mixtures (Meador & Nahrgang, 2019). There is no consensus regarding the minimal analytical screening required for oil constituents in a freshwater matrix during a spill event. The expectation due to salinity differences is that more of the oil constituents will dissolve in freshwater than marine (Green & Trett, 1989). Acknowledging the differences in approach opted by others and the fact that a matrix like OWM contains 90%–98% unresolved components within the overall TPH content (Faksness et al., 2008), the goal of the present study was more refined; it was not to identify what compounds or groups of compounds induce the most toxicity in zebrafish. Instead, the focus was to investigate whether this bulk mixture toxicity to zebrafish induced by untreated OWMs is eliminated as a result of oxidation using ozone with or without a peroxone-based AOP.

In the present study, analytical measurements were only performed for target mono-aromatics, which are relatively polar and quickly dissolve in water. The role of mono-aromatics and their potential combined toxicity with PAHs has been pointed out in a recent study on larva and fish (Sørensen et al., 2019). Among these, BTEX are regulated by the US Environmental Protection Agency (2022) at 0.005–10 mg/L in drinking water. Therefore, we measured these along with other soluble monoaromatics that are ubiquitous in crude oil water-soluble fractions before and after ozonation-based treatment. We also measured aldehydes, a class of ozonation byproducts that most existing studies evaluating ozonation for toxicity mitigation in oil spills matrices do not address. For the ozone and peroxone doses used in the present study, an average aromatic destruction in the range of 54%–71% was noted, accompanied by increases in aldehyde concentrations in the low μg/L range. To the best of our knowledge, information regarding the general toxicity of aldehydes relative to aromatics is unavailable. Therefore, these targeted measurements do not clarify whether in fact by exposing OWMs to a peroxone-based treatment, there is an unintended enhancement of toxicity from byproducts. Note that aromatics and aldehydes are only a subset of crude oil hydrocarbons or treatment byproducts expected in a complex matrix of this nature. For instance, other hydrocarbons following monoaromatics in the order of solubility include cycloalkanes, branched alkanes, and n-alkanes, which were not measured in the present study (Clark & MacLeod, 1977; Wheeler, 1978; Green & Trett, 1989). Regarding byproducts, keto acids and carboxylic acids are some of the many ozonation byproducts expected to persist (Liu et al., 2022; Nawrocki et al., 2003; Wang et al., 2022). Any effort directed at identifying and quantifying all residual hydrocarbons and byproducts is not feasible. Assessment of relative morphological toxicity of untreated OWMs and chemically treated OWMs overcomes this limitation and is unbiased by residual hydrocarbon measurements, byproduct screening, and OWM preparation methods. Therefore, differences in toxicity observed between the untreated and treated OWMs are purely the result of the efficacy of the treatment process in consideration.

No standardization in OWM preparation protocol was performed in the present study. However, future efforts should be directed toward standardization, especially for oil spill treatment studies related to freshwater. This is not a new challenge in marine spill research; the lack of standardization is well acknowledged and some effort has been put into formulating protocols to facilitate reproducibility (Singer et al., 2000). In a closely related follow-up study, we discuss a protocol for OWM generation involving dilutions from high-concentration OWM stocks (Barron & Ka’aihue, 2003; Boehm & Page, 2007; Landrum et al., 2012) that will enable researchers to perform treatment experiments at both bench and pilot scales, a requirement that is not usually a driving factor otherwise in typical toxicity studies. Furthermore, the dilution optimization presented for the zebrafish experiments is specific to the raw water and OWM preparations executed in the present study. It is recommended that future studies conduct initial dilution testing on test mixtures to ensure that measurable morphological deformities or other targeted toxicity endpoints can be observed for zebrafish exposed to an OWM, as well as that zebrafish develop normally for that same dilution of raw water (control). Therefore, the approach presented in our study can be adapted for the environment of concern.

CONCLUSIONS

Toxicity studies involving in vivo aquatic species provide the ability to effectively evaluate drinking water treatment processes for target and non-target contaminant removal by also considering impacts from known and unknown treatment byproducts. In the present study, toxicity outcomes were observed for zebrafish exposed to 50% dilutions of three differently prepared OWMs from 24 to 96 hpf; zebrafish exposed to 50% dilutions of an OWM treated with O3, O3 + 0.5 M H2O2/M O3, or O3 + 1 M H2O2/M O3 developed normally. Aromatic removal was not enhanced for O3 + 0.5 M H2O2/M O3 compared with only O3, and some additional removal was observed for select aromatics with O3 + 1 M H2O2/M O3. These results have implications for treatment strategies that can be employed following accidental oil spills in drinking water bodies to minimize toxic exposures to humans. This study also highlights the importance of employing toxicity characterization in drinking water treatment studies coupled with chemical analyses, especially for complex freshwater matrices such as oil spill mixtures.

Supplementary Material

Supplemental Materials

Acknowledgments—

We would like to acknowledge the members of the Timme-Laragy laboratory for providing excellent zebrafish care at University of Massachusetts Amherst. Funding for the present study was provided in part by the National Institutes of Health to Alicia R. Timme-Laragy (R01 ES025748) and to Monika A. Roy (National Research Service Awards T32 GM108556 and F31 ES030975). Funding was also provided in part to Aarthi Mohan, David Reckhow, and John Tobiason through the Massachusetts Water Resources Authority.

Footnotes

This article includes online-only Supporting Information.

Supporting Information—The Supporting information are available on the Wiley Online Library at https://doi.org/10.1002/etc.5472.

Disclaimer—The authors declare they have no actual or potential competing conflicts of interest.

Data Availability Statement—

The data supporting the findings of this study are available within the article and/or its Supporting Information. All raw data were generated at the University of Massachusetts Amherst labs. The nature of this work did not generate large datasets requiring the use of data repositories. However, the author Monika A. Roy (monikaaroy@gmail.com) may be contacted for further inquiries regarding the data, associated metadata, or calculation methods associated with the toxicity data, and the author Aarthi Mohan (aarthi1391@gmail.com) may be contacted regarding the chemical analysis data and the oil preparation methods used.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Materials
NIHMS1843649-supplement-Supplemental_Materials.pdf (334.3KB, pdf)

Data Availability Statement

The data supporting the findings of this study are available within the article and/or its Supporting Information. All raw data were generated at the University of Massachusetts Amherst labs. The nature of this work did not generate large datasets requiring the use of data repositories. However, the author Monika A. Roy (monikaaroy@gmail.com) may be contacted for further inquiries regarding the data, associated metadata, or calculation methods associated with the toxicity data, and the author Aarthi Mohan (aarthi1391@gmail.com) may be contacted regarding the chemical analysis data and the oil preparation methods used.

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