A simple protocol for estimating the acute toxicity of unresolved polar compounds from field-weathered oils

Abstract Crude oil spilled at sea is chemically altered through environmental processes such as dissolution, biodegradation, and photodegradation. Transformation of hydrocarbons to oxygenated species increases water-solubility. Metabolites and oxidation products largely remain uncharacterized by common analytical methods but may be more bioavailable to aquatic organisms. Studies have shown that unresolved (i.e. unidentified) polar compounds (‘UPCs’) may constitute > 90% of the water-accommodated fraction (WAF) of heavily weathered crude oils, but still there is a paucity of information characterizing their toxicological significance in relation to other oil-derived toxicants. In this study, low-energy WAFs (no droplets) were generated from two field-weathered oils (collected during the 2010 Deepwater Horizon incident) and their polar fractions were isolated through fractionation. To allow establishment of thresholds for acute toxicity (LC50) of the dissolved and polar fraction of field collected oils, we concentrated both WAFs and polar fractions to beyond field-documented concentrations, and the acute toxicity of both to the marine copepod Acartia tonsa was measured and compared to the toxicity of the native WAF (non-concentrated). The difference in toxic units (TUs) between the total of the mixture and of identified compounds of known toxicity (polycyclic aromatic hydrocarbons [PAHs] and alkyl phenols) in both WAF and polar fractions was used to estimate the contribution of the UPC to overall toxicity. This approach identified that UPC had a similar contribution to toxicity as identified compounds within the WAFs of the field-weathered oils. This signifies the relative importance of polar compounds when assessing environmental impacts of spilled and weathered oil.


Introduction
Crude oil spilled at sea rapidly changes composition through a combination of physical, chemical, and biological weathering processes (Daling and Strøm 1999).Immediate changes are the loss of volatile and soluble compounds through spreading, evaporation, dispersion, emulsification, and dissolution, while processes such as biodegradation and photodegradation alter the chemical composition of the residual oil.Degradation typically causes transformation of hydrocarbons into oxygenated and thus more polar compounds (Aeppli et al. 2012;Hall et al. 2013), further increasing the mass fraction of water-soluble compounds.Over time, this leads to decreasing mass of the oil residue in the marine environment through facilitated further degradation and finally mineralization.
Due to its bioavailability, the soluble fraction of crude oils is regarded the most important driver for toxicity (Carls et al. 2008;Olsvik et al. 2011).Toxicity testing of oils is usually performed using water-accommodated fractions (WAFs), and the WAF composition depends on the weathering degree of the oil.WAFs of fresh crude oils are dominated by highly soluble and volatile compounds, such as the low molecular weight monoaromatic hydrocarbons, mainly benzene, toluene, ethylbenzene, and xylenes (BTEX), whereas WAFs of weathered oils are dominated by polycyclic aromatic hydrocarbons (PAHs) (French-McCay 2002;Di Toro et al. 2007;Adams et al. 2014).In heavily weathered crude oils, a large fraction of the most soluble PAHs is depleted and the dissolved fraction is minimal compared to the total mass of weathered oil residue (Faksness et al. 2020(Faksness et al. , 2015)), and this mass is dominated by gas chromatographically (GC) unresolved polar compounds ('UPCs') (Brakstad et al. 2014;Daling et al. 2014;Faksness et al. 2015).Well-studied examples of heavily weathered crude oil residues are the field collected slick samples from the 2010 Deepwater Horizon spill in the Gulf of Mexico.These surface slicks had been subjected to loss of soluble hydrocarbons during the rise from the wellhead, rapid degradation processes due to high temperature and UV exposure at the sea surface (Aeppli et al. 2012;Hall et al. 2013;Brakstad et al. 2015;Faksness et al. 2015).
Despite the chemical complexity of WAFs, most toxicity tests report exposure concentrations based on quantification of a limited number of analytes, usually BTEX and a varying number of PAHs, as suggested by official recommendations (Singer et al. 2000).Fully deconvoluting the composition of crude oil WAFs can hardly be accomplished owing to the cost and the complexity of such analyses (Hodson et al. 2019).Due to available analytical methods, calculations of oil toxicity thresholds (e.g.effect or lethal concentrations (EC x , LC x )) and toxic units (TUs) are usually based on only a small fraction of a quite complex oil mixture of potential toxicants (Meador and Nahrgang 2019).Using for instance summed PAH concentrations (tPAH) as nomination for the toxicity of a WAF is problematic and may result in misleading conclusions as PAHs constitute a small fraction of the total fresh oil and its dissolved concentration (Meador and Nahrgang 2019).Unresolved constituents of WAFs of weathered crude oil, including oxygenated degradation products, are rarely considered in toxicity tests, and some studies have highlighted the potential influence and contribution of UPCs to toxicity (Barron et al. 1999;Carls and Meador 2009;Melbye et al. 2009;Landrum et al. 2012;Meador and Nahrgang 2019).
Traditionally referred to as the unresolved complex mixture (UCM) (Farrington and Quinn 2015), the GC unresolved fraction of crude oil, contains a mixture of both aliphatic and aromatic hydrocarbons, as well as more polar, functionalized compounds.Constituents of non-polar UCMs, such as larger alkyl-substituted monoaromatic hydrocarbons, have been shown to bioaccumulate and cause health impairments in mussels (Booth et al. 2008(Booth et al. , 2007;;Scarlett et al. 2008) and developmental effects on fish early life stages (Sørensen, Hansen, et al. 2019).In contrast, the polar fraction of UCMs (i.e.UPCs) has been given less attention, and only few studies have focused on the toxicity of this fraction (Melbye et al. 2009;Rial et al. 2013;Katz et al. 2022), a fraction expected to increase in relative abundance and complexity with weathering.While studies have demonstrated negligible toxicity of weathered oils (Faksness et al. 2020(Faksness et al. , 2015)), other studies have suggested that polar fractions could account for a significant portion of the total WAF toxicity when isolated and present at high loadings (Melbye et al. 2009).More recent studies have directly attended to the modulation of WAF toxicity by different degradation mechanisms.One study showed that biodegradation caused an expected and significant reduction of dissolved PAH concentrations in a WAF.However, this was not accompanied by a reduction in WAF toxicity to early life stages of fish (Hansen et al. 2018).Similarly, photooxidation caused PAH depletion in oil residues, however, the WAFs of these residues were more toxic to copepods than WAFs of non-degraded oil (Katz et al. 2022).Resolved compounds (e.g.PAHs) may not be the only compounds contributing to WAF toxicity (Meador and Nahrgang 2019).
Acute toxicity thresholds (e.g.concentrations causing 50% mortality; LC 50 ), are routinely used to predict chronic toxicity, mixture effects and hazards concentrations of petroleum substances, acute to chronic ratios (ACRs), mixture toxicity models, and species sensitivity distributions.Heavily fieldweathered oils, such as the slicks sampled in the Gulf of Mexico during the Deepwater Horizon spill, were unable to provoke a sufficient toxic response in standard acute toxicity tests using the marine algae Skeletonema pseudocostatum and copepod Acartia tonsa to reliably calculate LC 50 s (Faksness et al. 2015).Yet, exposure to (high-energy) WAFs of the same field-weathered oils cause chronic and sub-lethal effects in early life stages of fish in laboratory tests (Incardona et al. 2014;Mager et al. 2014;Esbaugh et al. 2016).
Theoretical methods such as the target lipid model (TLM) (McGrath et al. 2005;McGrath and Di Toro 2009) are widely used as a basis for calculating chronic TUs and hazard concentrations of oils (McGrath et al. 2018).If sufficient chemical information of a given crude oil is available, its toxicity may be estimated by separating the whole oil fraction into hydrocarbon blocks with assigned toxicity (Redman, McGrath, et al. 2012, 2017).Unfortunately, historical data and standard chemical characterization do not provide sufficient analytical data to support this approach.To predict the potential environmental toxicity of heavily weathered crude oils, where UPC typically is the largest contribution (both by mass and number of chemicals) it is necessary to estimate the toxicity of this fraction.
The aim of this study was to characterize the relative contribution of UPCs to WAF toxicity.To achieve this, a protocol for isolating the polar WAF fraction, and for concentrating both the WAF and associated UPC of weathered oils was developed and used to generate exposure solutions for toxicity testing.The difference in TUs between resolved and quantified compounds of known toxicity and measured TUs from toxicity tests was used to estimate the toxicological contribution of the UPCs.This approach allowed a toxicity evaluation of the isolated UPC fraction and the total WAF generated from two fields collected weathered oil residues from the Deepwater Horizon oil spill toward nauplii of the marine copepod Acartia tonsa.

Oil samples
Field-weathered samples from the Deepwater Horizon oil spill were collected by skimming from surface slicks at two sites, referred to as Juniper and CTC, in the Gulf of Mexico sea surface where they had surfaced from 1500 m depth.These oils had undergone an extensive weathering process and formed stable water-in-oil emulsions with water contents of about 50% in the Juniper oil and 60% in the CTC oil.The estimated weathering time on sea was estimated to be 3-5 d for CTC and more than 5 d for Juniper (Faksness et al. 2015) when compared with the laboratory weathered Macondo oils discussed by Daling et al. (2014).No volatile components were detected in the oils, and the concentrations naphthalenes were very low (Supporting Information [SI], Table S2).Further information of the sampling and composition of the field collected oils are given by others (Daling et al. 2014;Stout 2015;Stefansson et al. 2016;Forth et al. 2017).Upon collection, the oils were clearly labeled and subject to strict chain-of-custody.All samples were stored dark and cool (<5 � C).More details describing the oils and their origin are given in SI1, Table S1.

Preparation of water accommodated fraction (WAF)
Preparation of low energy WAFs of the different oils was performed under controlled conditions following the guidelines established by the Chemical Response to Oil Spills: Ecological Effects Research Forum (CROSERF) (Aurand and Coelho 2005).Photos of WAF systems are shown in SI2 (Figure S1).Sterile filtered (0.2 mm) natural seawater collected from 90 m depth in Trondheimsfjord was added to 10 L or 50 L borosilicate glass bottles giving a water to air headspace ratio of 5 to 1. Oil was carefully applied to the water surface to achieve an oil to water loading of 1-100 (10 g oil/L seawater).The water was stirred gently using a magnetic stirrer (<200 rpm) assuring that the oil film rested on the water surface without creating a vortex and without dispersing visible oil droplets into the water.The preparation was carried out in darkness at room temperature (20 ± 2 � C) using a mixing time of 72 h.Samples for extraction, chemical analysis, and toxicity testing were collected in glass vials and bottles with Teflon lined caps, allowing no headspace to minimize the loss of volatiles.Samples for chemical analysis were acidified (HCl, pH <2) and stored dark and cool prior to further handling.

Extraction and fractionation of WAFs
Water samples (42.95 L (CTC), 15.325 L (Juniper), 6.6 L (MASS), or 7.065 L (seawater procedural blank)) from the prepared WAFs were serially extracted using dichloromethane (DCM) following a modification of EPA method 3510 C. The combined extracts were dried over sodium sulfate and concentrated to 10 mL by gentle evaporation (Turbovap V R 500).Half of the total extract was used for chemical analysis and toxicity testing of the total WAF.The other half was concentrated to approximately 1 mL prior to fractionation of the polar fraction.The polar fraction was isolated using an adaptation of a previously published protocol (Sørensen, McCormack, et al. 2019).Silica solid phase extraction (SPE) columns (Agilent Bond Elut SI, 500 mg) were conditioned with n-hexane and a sub-sample of the WAF extracts were transferred to the columns.The sample vials were rinsed with n-hexane (3 mL), which was also transferred to the columns.The less polar compounds (mainly aliphatic and aromatic hydrocarbons) were rinsed off the columns by elution with two column volumes of n-hexane and DCM and discarded.The more polar fraction was collected by gradient elution using increasing fraction of methanol in DCM (5-20%).A schematic overview of the experiments and analyses is given in Figure 1.

Toxicity testing using Acartia tonsa
Toxicity testing was performed using the marine copepod A. tonsa in accordance with the ISO guideline (ISO 1999) with some modifications described in detail in Supporting Information (SI4).Briefly, for each exposure media (reconstituted extract or WAF), nine serial dilutions were prepared by diluting the prepared media with seawater.All nine dilutions were distributed into four replicate exposure vessels (N ¼ 4) used for toxicity testing.Each exposure vessel was added 20 or 12 animals for nauplii and adult copepods, respectively.Negative controls (N ¼ 8) contained only sea water, and as positive controls, 3,5-dichlorophenol (3,5-DCP, 1.0 mg/L) was used as reference toxicant (N ¼ 4).For most of the studies we used nauplii instead of adults.This was done to allow testing of reconstituted WAFs and polar fractions in a small water volume (4.5 mL for nauplii and 100 mL for adults).A table showing all tests performed with recorded values for pH, O 2 saturation, and temperature are given in SI4 (Table S4).

Chemical analysis
Oil samples and extracts of both the WAF and UPC fraction were analyzed for total extractable organic matter (TEOM) using GC-FID, and for target compounds (selection of saturate compounds, PAHs, alkylated PAHs, and phenols) using GC-MS according to published protocols (Faksness et al. 2015), but are also supplied in Supporting Info (SI3), including a list of target analytes (Table S3) recommended to be used during post-spill damage assessments (Singer et al. 2000).Volatile organic compounds (C 5 -C 9 hydrocarbons, including BTEX (benzene, toluene, ethylbenzenes, and xylenes) in the source oil and its WAF was analyzed using Purge and Trap GC-MS (P&T GC-MS).VOC analysis was only performed on the source oil and corresponding WAF, as volatiles have previously been demonstrated not to be present in the field weathered oils (Faksness et al. 2015).Here, the total WAF concentration is the sum of volatiles (from P&T GC-MS) and TEOM (from GC-FID).Additionally, the previously described samples were screened for polar constituents using direct injection electrospray ionization Time-of-Flight mass spectrometry (ESI ToF-MS).Details of the analytical procedures are given in Supporting Information SI3.The mass spectra were exported from Agilent Masshunter (2011, B.04.00, SP2, Agilent Technologies, Inc. 2011, Palo Alto, CA) in mz.xml format, which was imported by the mzR library in R (R-Core-Team 2014).In R, the data were binned to one decimal to produce the dataset that was used for the comparison.The mass range (m/z) imported was 50-1000.

Calculation of TU and LC 50 for UPC
The TLM considers the hypothetical critical target tissue (CTL) of the organism and defines the critical target body burden (CTLBB) as the threshold level for an effect to occur.The CTLBB may vary between species depending on sensitivity and species-specific CTLBB are not available for Acartia tonsa.Therefore, the CTLBB for the marine mysid Americamysis bahia was selected as a surrogate for A. tonsa.TUs were calculated based on quantified oil components and LC 50 s based on CTLBB for A. bahia using chemical class corrections from McGrath et al. (2021McGrath et al. ( , 2018)).The TU of the UPC fraction were then assumed to be the difference between the TU recorded in the toxicity test (TU mix ) and the TU calculated based on concentrations of identified oil components (TU ID ): where TU mix is the measured TU of the solution based on the toxicity test, and TU ID and TU UPC are the toxicities of the 49 identified hydrocarbons (Table S3) and corresponding unresolved (i.e.unidentified) fraction, respectively.The LC 50 of the UPC fraction was then calculated based on TU UPC : where LC 50-UPC is the LC 50 of the UPC fraction and C UPC the concentration of the UPC.

Statistical analyses
The calculated LC 10 and/or LC 50 values were corrected for mortality in the control series by subtraction, and the effect was calculated within the span of 0% and 100% by constraining the top and bottom of the concentration-effect curve to 100 and 0 using GraphPad Prism.All calculations were performed using GraphPad Prism or Microsoft Excel.Venn plots of mass spectra from CTC oils were created using R, and the ggplot library (Warnes 2012).

Chemical characterization of the WAFs and polar fractions
The oils source oil (MASS), the field-weathered residues (CTC and Juniper), and their corresponding WAFs have previously been chemically characterized using standard GC methodologies (Aurand and Coelho 2005).The compositional differences between the MASS and CTC/Juniper are caused by the weathering process occurring from the release at the wellhead, rising to the surface and surface-occurring weathering processes.The compositional differences between CTC and Juniper are mainly attributed to differences in residency time at sea surface (3-5 d for CTC and >5 d for Juniper) (Faksness et al. 2020(Faksness et al. , 2015)).
The WAFs and isolated polar fractions of the three oils were compared based on resolved compounds using targeted GC-MS analyses.Most hydrocarbons (decalins, PAHs, and alkylated PAHs) were effectively removed by the UPC isolation protocol (SPE), with only trace amounts remaining (Figure 2).Phenols, however, co-eluted with the polar fractions during SPE separation due to their polarity and are thus part of the polar fraction together with the UPCs.
Estimated by subtracting the concentration of identified compounds (decalins, PAHs, and alkyl phenols quantified by GC-MS) from the TEOM (GC-FID), the WAFs of field-weathered CTC and Juniper oils contained �90 to �95% unresolved compounds, respectively, compared to �10% in the WAF of the non-weathered source oil (MASS) (Faksness et al. 2015).The slight increase in relative content of unresolved compounds in Juniper compared to CTC is attributed to the longer estimated residence time at sea (Faksness et al. 2015).
The weathering period at the surface would have led to increased influence of photo-oxidation and biodegradation on the composition of the CTC and Juniper residues.As such, it is expected that the polar content of Juniper is larger than that of CTC, but both being significantly more polar than MASS.
To understand the complexity of the UPC of the oil, direct injection electrospray (ESI, both positive (þ) and negative (-) mode) ToF-MS analysis of the WAF extracts was performed.
To study bulk oil complexity and changes in composition during weathering, ESI± ToF-MS offers a decent supplement to GC analysis, providing detection power targeting particularly the polar, ionizable compounds typically formed during oxidation of hydrocarbons.ToF-MS resolution does not hold the same molecular formulae assignment potential as high-resolution accurate mass spectrometry (HRAM-MS, typically Orbitrap and FT-ICR-MS), and will not be able to provide direct chemical composition insight.Still, the use of the ESI± ToF-MS can give an indication of differences, and in particular changes, in complexity of the very polar fraction of samples.Typical ESI-mass spectra from extracts of WAFs of the MASS source oil and the Juniper and CTC field weathered oils (Figure 3(A-C)) show that the WAF from the MASS source oil, as expected, displayed a high number of lower molecular mass compounds compared to the WAFs from the corresponding field weathered oils.The WAFs of the weathered oils displayed a greater number of masses detected at higher mass-to-charge ratios (m/z) (i.e. higher molecular mass compounds).In addition to the shift in molecular size distribution, the number of unique mass-to-charge values detected provided information on the complexity of the WAFs as a function of degree of oil weathering.The Venn diagrams (Figure 3(D,E) made based on detected masses in the spectra showed that while WAF prepared from MASS oil had 131 (ESIþ) and 373 (ESI-) unique masses that were not detected in the CTC and Juniper WAFs, the WAFs from the weathered oils had 357 (ESIþ) and 451 (ESI-) unique masses that were not detected in the MASS WAF extract.For both detection modes, WAF prepared from the most weathered oil, Juniper, displayed the highest number of unique masses compared to the fresh oil.This further confirms that weathering results in significant changes of the chemical composition toward more polar compounds that are detectable by ESI± ToF-MS, and although the surface collected CTC and Juniper oils share a significant number of detected masses with the source oil, an increased number of unique masses present in the ESI± mass spectra was observed in the WAFs from the weathered oils.

Reconstituted WAF and UPC
To provoke a toxic response in acute toxicity tests, isolation and concentration of both WAF and polar isolate extracts to generate an artificially high exposure concentration was necessary.The laboratory protocol for extraction, fractionation, and concentration has previously been validated and applied to studying effects of the apolar and polar fractions of offshore produced waters containing oil residues and degradation products (Hansen et al. 2018;Sørensen, McCormack, et al. 2019).In this study, the concentration factor of the 'reconstituted WAF' (RE-WAF) respective to the native WAF was in the range 2-4, and no resulting concentrations of individual quantified compounds exceeded their theoretical solubility.The procedure (liquid-liquid extraction followed by evaporation of the solvent) removed the bulk of volatile compounds in the exposure media, resulting in lower concentrations of volatiles and semi-volatiles (e.g.naphthalene) in RE-WAF than in the native WAFs.This artifact had only a minor impact on the comparability of chemical composition of the field weathered oils, as these were already depleted of volatiles.However, the effect on the volatiles-rich MASS source oil was so pronounced that the protocol was deemed unsuitable for this oil.The native WAF from MASS had a total WAF concentration of 21.1 mg/L, which included 18.2 mg/L volatile components.However, the volatiles were lost during the reconstitution of the WAF extract, resulting in a decrease in the WAF concentration to 2.45 mg/L after 'concentration'.On the other hand, WAFs prepared from the field weathered CTC and Juniper oils were concentrated from their original total WAF concentration of 0.47-1.13mg/L for CTC, and from 0.49 to 1.47 mg/L for Juniper.Alkyl phenols constituted a significant fraction of target compounds quantified in the WAFs and isolated polar fractions, however, when comparing the  S3.
For the CTC, the effect of the fractionation and re-constitution on the mass balance of total polar fractions content was studied by determining the number of common observable masses in the WAF, polar fraction and samples from the re-dissolution steps (Supporting Information S4).The percentage of common observable masses remained 62-63% throughout both steps.This indicates that both observable and unobservable loss of unresolved compounds can occur during the sample preparation steps, and that there could still be compounds in the WAF where the contribution to toxicity is unknown.As such, this study gives new data on the toxicity of UPC of WAFs, but there is still a large fraction of the total UPC that is unaccounted for with respect to toxicity.
The observations from the chemical analysis confirm that toxicity testing using reconstituted WAFs (Sørensen, McCormack, et al. 2019) is limited to weathered oil residues due to the extensive loss of volatile compounds during sample preparation.The use of ToF-MS in this study helped to show that a relatively larger number of UPC constituents become available in the water phase when preparing WAFs from weathered oils compared to their corresponding source oil.This demonstrates that there are a greater number of polar compounds present in the weathered oils as compared to the source oil, which makes the determination of the toxicity of UPCs of both native and weathered oil WAFs important.As of currently, the chemical composition of the UPC has not been determined, and this is an important challenge to resolve in future work.Using modern analytical tools such as multi-dimensional gas and liquid chromatography techniques in combination with high resolution mass spectrometry and data analysis pipelines can resolve these compounds and allow putative chemical formulae to be determined.Upon structure verification, one can envision developing fitfor-purpose quantitation methods.However, the longevity and bioaccumulation potential of such compounds are also factor to consider when determining their risk to aquatic organisms.Biomimetic extraction techniques applied to polar WAFs may offer one solution along this path (Katz et al. 2022).

Acute toxicity of native WAFs and reconstituted extracts
Available water column monitoring data from Deepwater Horizon analyzed based on total petroleum hydrocarbons measured by GC showed that 84% of the samples collected during the Deepwater Horizon incident were <1 mg/L and 5% of the samples were >250 mg/L (Wade et al. 2016).The generated WAF of the MASS oil herein (21.2 mg/L) was comparable only to concentrations found in very few samples collected in relation to the Deepwater Horizon.Even WAF concentrations in the 100% WAFs were in the high end (472 and 488 mg/L for CTC and Juniper, respectively) compared to concentrations in field samples reported for Deepwater Horizon (Wade et al. 2016).Since the relative fraction of unresolved components in WAFs can vary from low in fresh oils and condensates to more than 90% in highly weathered or biodegraded oils, such as CTC and Juniper (Faksness et al. 2015), it is necessary to include the contribution of unresolved compounds when assessing toxicity of highly weathered oils.To establish thresholds for acute toxicity of the dissolved fraction of field collected oils, we prepared WAFs concentrated to beyond documented field concentrations.The rationale for this approach was to use the acute toxicity thresholds to estimate potential for chronic effects from WAFs from highly weathered oils dominated by uncharacterized compounds.
Acute toxicity tests performed with native MASS WAFs showed comparable sensitivities for nauplii and adult copepods, with LC 10 of 6.14 (5.71-6.60)mg/L and 6.65 (6.05-7.32)mg/L, and LC 50 of 8.3 (7.90-8.71)mg/L and 11.1 (10.7-11.6)mg/L for adult and nauplii, respectively.Exposure to the WAFs from the field collected oils caused limited mortality to A. tonsa.For the CTC WAF, 100% WAFs caused 11.5% mortality for adults and 7.3% mortality for nauplii, and mortality was even lower (0% mortality for adults and 1.1% mortality) for the 100% native WAF of Juniper (Figure 4), which is in line with the results previously reported (Faksness et al. 2015).
The increase in total concentration for the reconstituted WAFs ('RE-WAF'), from 0.47 to 1.11 mg/L (2.4� concentrated) and from 0.49 to 1.47 mg/L (3� concentrated) for CTC and Juniper, respectively, was able to provoke a toxic response in the nauplii test.The isolated and reconstituted polar fractions (RE-POL) for the different oils were concentrated accordingly, achieving 1.22 and 1.28 mg/L for CTC and Juniper, respectively, which also provoked measurable LC 50 s in the nauplii test.See SI5 for more details.
Triplicate tests were performed for the reconstituted CTC WAFs and the overall reproducibility between the parallels was good, both in terms of achieved concentrations of RE-WAF and RE-POL, and observed mortality for the nauplii (SI5, Table S5).For CTC, an LC 50 of 0.86 mg/L (range 0.765-1.01mg/L) was estimated for the RE-POL fraction compared to an LC 50 of 0.59 mg/L (range 0.547-0.608mg/L) for the corresponding concentrated RE-WAF (Figure 4(A) and SI5).For Juniper the LC 50 was 1.27 mg/L (range 1.22-1.32mg/L) for the RE-POL fraction and 0.797 mg/L (range 0.769-0.827)for the RE-WAF (Figure 4(B)).Solvent controls with concentrated DCM did not induce mortality (SI5, Table S5), thus confirming that the toxicity was attributed to the presence of oil compounds without contribution from the solvent.Furthermore, positive control experiments using 3,5-dichlorophenol (DCP) confirmed that the sensitivity of the nauplii was comparable between tests (SI5, Table S5).
In addition to the field-weathered oils, the test assay with nauplii and RE-WAF were performed using the fresh MASS source oil.The results showed that the relative toxicity of the native WAF (50% mortality (LC 50 ) occurred at WAF diluted to 39%) with a total concentration of 21.1 mg/L, including 18.2 mg/L volatiles, is higher than the RE-WAF (LC 50 of 74% WAF), which contained no volatiles (total concentration 2.45 mg/L), indicating that the loss of volatile compounds reduces toxicity, which is not surprising.The results also indicate that the LC 50 concentration of the native WAFs are 2 − 2.5 times higher than the maximum WAF concentration (100% WAF) possible to obtain using a conventional WAF set-up with the oils at a high oil loading (oil water ratio 1:1000), and 4-8 times higher than the reported corresponding WAF concentrations of CTC and Juniper oils at more environmentally realistic concentrations, but still high oilwater-ratios of 1:10 000 (100 mg oil/L) of the same oils.Overview of all experimental results, including LC 50 values, is found in Supporting Information (SI5, Table S5).

Contribution of unresolved compounds to toxicity of WAF
The reconstituted polar fractions (RE-POL) used as a proxy of the UPC contained traces of resolved compounds, e.g.phenols (Figure 2), which may contribute to the toxicity observed in the acute tests.To isolate the contribution from UPCs in the reconstituted extracts (WAF and polar fraction), the contribution from the identified compounds had to be deducted.To do this several assumptions had to be made: (i) The compounds in the reconstituted fractions are dissolved in the sample (no precipitation).There was no visual evidence of precipitation or droplet formation when the extracts were reconstituted into sea water prior to be used for toxicity testing on A. tonsa.This is important as particulates have a different uptake route in copepods than dissolved compounds (Hansen et al. 2018) and thus different kinetics and associated toxicity.(ii) The WAF concentrations (TEOM) measured by GC is representative of all compounds (identified and unresolved) in the extracts/mixtures.Please note that all peaks not included in the list of target compounds are here considered 'unresolved'.Not all compounds in a WAF will be detected by one instrument alone (Adams and Canadian Science Advisory Secretariat 2017;Hodson et al. 2019); however, GC has traditionally been the methodology of choice for quantification of oil sample mass and is still considered the best approximation of total oil, (iii) the contribution of resolved and unresolved compounds to acute toxicity (narcosis) are additive, being an assumption previously accepted by others (French-McCay 2002;Rial et al. 2013), and (iv) the theoretical model (TLM model using parameters for A. bahia (McGrath et al. 2021;McGrath and Di Toro 2009) used represents the sensitivity of A. tonsa nauplii.According to Faksness et al. (2024), the TLM model based on parameters from A. bahia can be used as a substitute for calculating TUs for adult A. tonsa for fresh and moderately weathered Deepwater Horizon oil with a large fraction of resolved and quantified material.This model was thus used to compare the theoretical TUs based on identified components in the RE-WAFs and their corresponding RE-POLs.Due to limited availability of the fractionated samples, the volume of the exposure solutions had to be reduced and A. tonsa nauplii rather than the adult stage were used in these tests.While the A. bahia model is representative for adult A. tonsa, there was a need to compare the sensitivity of the two developmental stages of A. tonsa.Exposure of both adult and nauplii stages of A. tonsa to WAF of the native MASS oil showed that the nauplii were slightly less sensitive than the adults with TUs of 1.9 and 2.54 for nauplii and adults, respectively.For another copepod species (Calanus finmarchicus), nauplii and adults displayed comparable toxicity thresholds when exposed to WAFs of crude oil (Jager et al. 2016).On the other hand, the positive control experiments in this study indicated that the nauplii were twice as sensitive as the adults to DCP.However, DCP is not a hydrocarbon, and since both Juniper and CTC originated from the MASS oil, we assumed that the nauplii would also be less sensitive than adults to the WAFs of the weathered oils.A correction factor of 0.745 (1,9/2,4) was therefore applied to the TUs from A. bahia to represent the toxicity of identified components to A. tonsa nauplii.The TUs based on the adjusted A. bahia model were then compared to the TU derived from the toxicity tests with reconstituted WAFs based on the total WAF concentration.The difference between the total of the two TU calculations (i e. TU not accounted for by the adjusted A. bahia model) was subsequently assumed to be toxicity caused by the UPC fraction (Figure 5).The average TU not accounted for by the adjusted A. bahia model was 0.98 (range 0.71 − 1.26) corresponding to an average LC 50 of 1.04 mg/L (range 0.74-1.40mg/L) for the UPC fraction.
For the field weathered oils, the calculated TUs for the identified compounds suggest that the main contributor to toxicity was 2-3 ring PAHs, accounting for 37% and 33% of the TUs in CTC RE-WAF and Juniper RE-WAF, respectively.The estimated contribution of the UPC to toxicity based on TU in the CTC and Juniper WAFs was 50% and 57%, respectively (Table 1).The contribution of UPCs was thus higher in the more weathered Juniper oil.As expected, the highest contribution from UPC was found in the RE-POL since the content of identified hydrocarbons is depleted by the fractionation procedure.The apparent and potentially significant contribution of the UPC to toxicity in highly weathered oils has implications for theoretical calculations of toxicity based exclusively on identified oil compounds.Calculating LC 50 s based on solely on the sum of resolved compounds will overestimate the toxicity of the whole mixture.
Standard analytical methodologies quantifying limited targets chemicals cover only a small fraction of the total oil mass and are blind when assessing degradation products.The PETROTOX model, utilizing the hydrocarbon block methodology based on two-dimensional GC (Redman, Parkerton, et al. 2012), is a promising step forward to separate compound classes and assigning chemical and toxicological properties, including TUs.Further work should also focus investigating appropriate analytical solutions to account the UPC on a block-inspired basis, as elucidation of individual targets will be too complex to be applicable to routine toxicity testing.

Conclusion
This study offers a novel and simple methodology that, despite some limitations (e.g.assumptions for unresolved and polar compounds and loss of volatiles), allows a preliminary understanding of the contribution of water-soluble UPCs to weathered oil toxicity.Non-target ToF-MS profiling showed a relative increase in the complexity (number of peaks/masses) of UPCs with weathering.While considerable previous work has quantified the toxicity of target crude oil compounds (mainly BTEX and select PAHs), and efforts have been done to understand the toxicity of 'other' apolar compounds, e.g. the hydrocarbon block methods (Redman, McGrath, et al. 2012;Redman, Parkerton, et al. 2012), this study is one of the first attempts to quantify the contribution of UPCs to toxicity.While the overall acute toxicity of laboratory weathered oils is very low, it is noteworthy that a significant part of this (low) toxicity may be caused by UPCs which has yet to be structurally elucidated, and that both the relative content and toxic contribution of this fraction increases with increased weathering.More work is recommended to understand both the formation and transformation of these compounds.The current work suggests that estimates of the toxicity of oils with large unresolved mass fractions, including heavily weathered oils, may be improved by introducing a tentative toxicity threshold (and TU) for the unresolved fraction, with the limitation that working beyond environmentally observed concentrations is needed to provoke effects for estimation of these thresholds.

Figure 1 .
Figure 1.Overview of the experimental work.Low-energy WAFs (LE-WAFs) of the MASS oil, as well as the CTC and Juniper residues were subject to extraction and isolation of the polar fraction.Both extract and polar fraction were re-constituted in seawater (RE-WAF, RE-POL).The resulting solutions (WAF, RE-WAF, and RE-POL) were subject to toxicity tests and chemical analysis.

Figure 2 .
Figure 2. GC-MS target analytes quantified in WAFs from MASS, CTC and Juniper oils and their corresponding polar fractions.Please note the differences in scale between the figures.Abbreviations are explained in Supporting Information SI3, TableS3.

Figure 3 .
Figure 3. ESI-TOF mass spectra of WAFs from the MASS oil (A) and the field weathered oils CTC (B) and Juniper (C).The spectra show the increased complexity in chemical composition of the oil after weathering.More features with higher m/z ratios are observed.The Venn diagrams of distinct features counted in the binned spectra also show this trend for both positive (D) and negative mode (E) spectra.More distinct features are present in the WAFs from the weathered oils than in the source oil.