Use of TLM derived models to estimate toxicity of weathered MC252 oil based on conventional chemical data and the potential impact of unresolved polar components

Abstract Target lipid model (TLM) and toxic unit (TU) approaches were applied to ecotoxicity and chemistry data from low-energy WAFs (LE-WAFs) of source and weathered crude oils originating from the Deepwater Horizon oil spill. The weathered oils included artificially weathered oils and naturally weathered samples collected in the Gulf of Mexico after the spill. Oil weathering greatly reduced the concentrations of identified LE-WAF components, however, the mass of uncharacterized polar material (UPC) in the LE-WAFs remained largely unchanged during the weathering process. While the TLM-derived calculations displayed a significant decrease in toxicity (TUs) for the heavily weathered oils, copepod toxicity, expressed as LC10-based TUs, were comparable between LE-WAFs of fresh and weathered oils. The discrepancy between observed and predicted toxicity for the LE-WAFs of artificially weathered oils may be related to limitations by the chemical analyses or increased toxicity due to generation of new unknown compounds during the weathering process.


Introduction
low-energy water accommodated fractions (le-WaFs) prepared from crude oils comprise a complex mixture of water-soluble petrogenic components, and components of environmental concern are typically limited to monocyclic aromatic hydrocarbons (Mahs) and polycyclic aromatic hydrocarbons (Pahs).acute toxicity levels for these components vary but are correlated to the octanol-water partition coefficient (K OW ) such that lc 50 decreases with increasing K OW .K OW is assumed to be proportional to the partitioning between the target tissue in the organism (presumed lipid) and the surrounding water at equilibrium.Mccarty et al. (1992Mccarty et al. ( , 1993)), Di toro et al. (2000Di toro et al. ( , 2007)), and others have shown that there is a linear negative relationship between the log lc 50 to aquatic organisms and the log K OW of organic chemicals that exhibit toxicity by nonspecific narcotic action.thus, the potential for environmental impact is the result of a balance between bioavailability (exposure) and toxicity once exposed.Because the acute toxicity of petroleum hydrocarbons to aquatic organisms is by nonspecific narcotic mode of action (Mccarty et al. 1992), the toxicities of individual hydrocarbons or hydrocarbon fractions are assumed to be additive, excluding potential antagonistic or synergistic effects (e.g.Di toro et al. 2000;French Mccay 2002).
the target lipid model (tlM) is a quantitative structure-activity relationship that describes the relationship between a chemical's acute toxicity and the octanol-water partition coefficient, K OW (Di toro et al. 2000) and was originally developed to derive water and sediment guidance.the tlM has been validated for predicting the toxicity of petroleum hydrocarbons (McGrath et al. 2005;McGrath and Di toro 2009).the tlM has also been used to derive hazardous concentration to 5% of species (hc5), a statistical extrapolation procedure commonly used in risk assessment by the european Union (McGrath et al. 2004).the hc5 methodology was applied to residual petroleum constituents focusing on the Mahs and Pahs (McGrath and Di toro 2009).For the tU calculations, the selected water effect concentration is the hc5 derived to protect 95% of the potentially exposed species from adverse effect.hc5 represents the 5 th percentile of the species sensitivity distribution of all test organisms that were used for the derivation of tlM and is considered a conservative threshold used for general purpose risk assessments (Redman et al. 2014).McGrath et al. (2018McGrath et al. ( , 2021) ) expanded the acute and chronic toxicity databases to include more species and a broader range of hydrocarbons representative of petroleum substances.tlM was recalibrated with these expanded databases and the hc5 equation was revised to include covarying model parameters.Faksness et al. (2015Faksness et al. ( , 2020) ) performed studies of the low energy water accommodated fractions (le-WaFs) of fresh and artificial weathered Macondo, and two field weathered Macondo oils, with special emphasis on chemistry and acute biological effects (algae (Skeletonema pseudocostatum clone NiVa Bac-1, formerly known as S. costatum) and copepods (Acartia tonsa)).Different weathering degrees were used in preparing the le-WaFs to illustrate 'snapshots' in the dynamic process of weathering and dissolution occurring during a spill situation.since a rather large data set on the Macondo oil has been available from these studies, an attempt to reveal which of the characterized component groups of the oils contribute the most to the le-WaF's acute toxicity was possible.this included the significance of dissolved bulk fraction of Unresolved complex Mixture (UcM), here referred to as the Unresolved Polar components (UPc), which is the total mass of dissolved components extracted by dichloromethane (quantified by gas chromatograph with flame ionization detector (Gc-FiD)) minus the mass of resolved components.Detailed compositional information for the le-WaFs of the oils provides the information needed to identify the relative contribution of different organic chemicals to the marine toxicity of the crude oils and petroleum products predicting toxicity using the K OW by tlM and hc5 approaches (McGrath and Di toro 2009;McGrath et al. 2018).For complex le-WaFs with known composition, the estimated lc 50 of individual components was used for calculating the toxicity for whole mixtures by the method of additive toxicity.this method assumes similar toxic mode of action so that the contribution of the individual constituents is additive.the acute toxicity measurements by A. tonsa and S. pseudocostatum were compared with estimated toxicities.estimated acute toxicities of the le-WaFs were calculated based on chemical composition of the le-WaFs and the log K ow for the components analyzed by comparing the estimated lc 50 of the individual components in the le-WaFs to their measured exposure concentrations in the 100% le-WaFs (i.e. a saturated le-WaF), and were done following the recommendations given in e.g.McGrath et al. (2018, 2021), Di toro et al. (2007), and McGrath and Di toro (2009).
the objective of the current work was to evaluate theoretical tlM based calculations of toxicity based on conventional chemical analyses of le-WaFs and the impact of increasing fractions of unresolved dissolved components caused by oil weathering.toxicity data from tlM based calculations have been compared to acute toxicity test results for the copepod Acartia tonsa and the marine microalgae Skeletonema pseudocostatum for le-WaFs from fresh and weathered Mc252 oil.

Oils used
source oil B was collected aboard the enterprise Discover on May 22, 2010, directly from the Mc252 well via the riser insertion tube downstream of the separators (oil, gas, water), and has been used as a basis for the bench scale artificial weathering.the oils studied are described in table s1 in supporting information (si1).
the artificial weathering of source oil B was performed to mimic the natural weathering of the oil at sea. evaporation of the lighter compounds from the fresh oil was carried out as a simple one-step distillation (stiver and Mackay 1984) heating the oil to vapor temperatures of 150, 200, 250, 275 and 300 °c. this resulted in residues with an evaporation loss corresponding to approximately <1 h, <0.5 day, 0.5-2 days, 2-3 days, and 2-5 days of weathering on the sea surface (Faksness et al. 2020).these residues are referred to as 150 °c+, 200 °c+, 250 °c+, 275 °c+, and 300 °c+, respectively.estimated time on sea for the two field collected oils were 3 to 5 days for the ctc oil and more than 5 days for the Juniper oil.artificial photo-oxidation was performed by irradiating a 2 mm layer of oil on water with a sunlight simulator (Faksness et al. 2020).two photo-oxidized samples were prepared; irradiated for 18 and 40 h respectively.additional tests were performed with an artificially weathered Macondo oil, submitted to siNteF from aecOM. the artificial weathering was performed by B & B laboratories and was based on evaporative loss.this oil is called 'NOaa weathered' in tables and figures.

Preparation of low energy water accommodated fraction (LE-WAF)
le-WaFs were prepared under controlled conditions following the guidelines established by cROseRF (aurand and coelho 2005).le-WaFs can be defined as a water solution of dissolved oil components prepared in closed vessels, with calm mixing of oil and water without formation of any vortex.in this study, the le-WaFs were chosen in order to avoid generation of oil droplets.a volume (9.25 l) of sterile filtered (0.2 µm) natural seawater collected from 90 m depth in trondheimsfjord was added to 10 l bottles giving water to air headspace ratio of 4 to 1. an oil-to-water ratio (OWR) of 1 to 10000 (100 mg oil/l seawater) were used for all le-WaFs.the preparation was carried out in darkness in room temperature (approximately 22 °c) using a mixing time of 48 h.samples for chemical analysis and toxicity testing were collected in glass vials and bottles without headspace to minimize the loss of volatiles.samples for chemical analysis were acidified (hydrochloric acid to ph < 2) immediately after sampling to avoid biodegradation during storage.

Chemical composition of the LE-WAFs
the samples were analyzed for semi-volatile organic compounds (sVOc (decalins, Pahs, phenols)) using gas chromatography with mass spectrometry (Gc-Ms), volatile compounds (VOc, c5-c9, including Btex) by use of Purge and trap Gc-Ms (P&t Gc-Ms), and for total petroleum hydrocarbons (tPh) using gas chromatography with flame ionization detector (Gc-FiD).sample preparation and chemical analyses have previously been published in Faksness et al. (2015) but are also supplied in supporting information (si2) including a list of all target analytes (table s2).this is a typical standard list for recommended analytes used during post-spill damage assessments (singer et al. 2000).

Acute toxicity of the LE-WAFs
the unicellular marine algae Skeletonema pseudocostatum was used to represent primary producers (international Organization for standardization (isO) 2006a, 2006b) and the potential effects on primary consumers were assessed with the marine pelagic copepod Acartia tonsa (isO 1999).the original protocols were not designed for testing of solutions containing volatiles and were adapted as described in the previously published test procedures in Faksness et al. (2015).the methods are also supplied in supporting information (si3.1 and si3.2).

Calculation of toxic units (TU)
a regression model is used to express the relationship between the acute toxicity and K ow of target chemicals to estimate the threshold toxic concentration of each chemical (Mccarty et al. (1992(Mccarty et al. ( , 1993) ) and Di toro et al. (2000Di toro et al. ( , 2007))). the relationship is described by a linear regression of log lc 50 (mmol/l) against log K ow for each organic compound: where m is the slope and b is the intercept of the regression line.the slope is related to the partition behavior of the chemical and should therefore be constant from species to species.the y-intercept b can be interpreted as the lipid-normalized critical body burden corresponding to the observed endpoint, such as 50% mortality for the lc 50 for the specific organism being considered.it is variable by species and likely life stage and condition (Di toro et al. 2007).the tlM is an extension of the critical body burden theory in which adverse effects occur when the total body burden within the test organism reaches a critical concentration (McGrath and Di toro 2009).the tlM considers the hypothetical lipid content of the organism and defines the threshold level of effect as the critical target lipid body burden (ctlBB) (McGrath et al. 2004).the ctlBB may vary for each species depending on sensitivity, and for an individual species, the tlM estimates lc 50 values for Mahs and Pahs as a function of a ctlBB according to the equation described in eq.(s1) (si3.3).
to compute a water effect concentration for a specific chemical and species, the log K OW of the chemical and the ctlBB for the species are needed.the chemical class corrections for some chemical classes to account for class-specific differences in partition behavior to the target lipid have existed for years for aliphatics, Mahs and Pahs (e.  (2021) for heterocyclic aromatics (hacs).the quality and quantity of the available toxicity data are often very variable.For some of the individual petroleum components there are no acute toxicity data available for marine organisms.here, the ePisuite estimation Program (Us ePa 2022) has then been used to calculate lc 50 for chemicals lacking toxicity data.the input data for calculations of lc 50 s are detailed in table s4 (si3.3).species specific ctlBB was given for S. costatum in McGrath et al. (2018) but were not available for Acartia tonsa.therefore, the marine mysid (or Americamysis bahia (formerly known as Mysidopsis bahia) was selected as a surrogate for A. tonsa.
the tlM framework was used to compute a hazard concentration for 95% species protection (hc5) based on the distribution and uncertainty of the ctlBBs (c l *) and acute to chronic Ratios (acRs) in the tlM database (McGrath et al. 2018).they presented the parameters for the hc5 equation based on the 2018 acute ctlBB and acR databases.the application of the hc5 equation to compute chronic protection values has been reevaluated by McGrath et al. (2018), and their revised equation has been used to calculate a chronic hc5 as described in eq.(s2) (si3.3).
McGrath et al. ( 2018) also described an alternative approach for computing chronic hc5s using the chronic toxicity directly in tlM calibration.the tlM universal slope and chemical class corrections were applied to the chronic toxicity data and the eq.(s1) (si3.3) was used for the calculations, but with chronic ctlBBs of 0.629 and 1.502 (mmol/g octanol) for A. bahia and S. pseudocostatum, respectively.toxic unit (tU) based on ctlBB for all individual organic chemicals in the le-WaF can be summed to produce a toxic unit, which is equivalent to an estimate of the acute toxicity of each le-WaF.the tU for each component (i) was calculated for each le-WaF system: (2) c i is the concentration of component i in the le-WaF and lc50 i is the estimated acute toxicity for component i (table s4, si3) calculated from eq. (si1) and eq.(s2s) in si3.3.the estimated toxicity of the total WaF is determined by the sum of the tUs of all component groups.a value of tU > 1 implies toxicity, i.e. the le-WaF is expected to cause 50% mortality in the test organisms.the analytes measured in the le-WaFs (table s2, si2) are classified structurally as type i narcotics.the additivity of the toxicity of narcotic chemicals and the use of toxic units has been demonstrated by several authors (e.g.Mccarty et al. 1993;Neff et al. 2000;French Mccay 2002; McGrath and Di toro2009).here, tUs have been calculated following the instructions given in McGrath et al. (2018), all based on the target lipid model (tlM), both acute and chronic.
in addition, acute tU for A. tonsa derived from the toxicity tests are calculated: (3)

Data treatment and data sources
Unless otherwise stated, statistics and fitted curves as well as computation of lc/ec x values were prepared by GraphPad Prism version 9.3.1 (GraphPad, Usa) and Microsoft excel (Microsoft Usa).Unless otherwise noted, all statistical variations are presented as standard deviations and significant levels are set to p = 0.05.chemical analyses and acute toxicity data for A. tonsa and S. pseudocostatum originate from Faksness et al. (2020Faksness et al. ( , 2015)).to compare acute tU lc50 from A. tonsa with acute tlM based tUs maximum mortality rates in the range 21 -49% have been extrapolated to 50% based on the average slope of the observed dose-response curves (Figure 6).

Chemical composition of LE-WAFs
the chemical composition of the oils and the chemical characterization and toxicity testing of their le-WaFs have previously been presented and discussed in Faksness et al. (2015Faksness et al. ( , 2020)).a summary of the results is given in table 1. in table 1, tU are calculated using eq.( si1), (si3.3). the regression coefficients for the tlM expressing ctlBB relative to A. bahia and S. costatum are from McGrath et al. (2018).the chemical composition of the oils and le-WaFs and the toxicity results are shown in supplementary information (Figure s1 (si2) and table s3 (si3)).the total le-WaF concentrations were given as the sum of tPh (consisting of the sum of sVOc and the unresolved polar components (UPc)) and volatiles (VOc). in the le-WaFs with oil to water ratio of 1 to 10000, the total le-WaF concentrations were in the range from 0.1 mg/l (Juniper) to 4 mg/l (fresh source oil B).le-WaF of Juniper contained also the lowest total Pah (tPah) concentration (0.004 mg/l), and the le-WaF of the 150 °c + residue the highest, with 0.173 mg/l tPahs.the VOcs constitute a major part of the le-WaF from the source oils and the less weathered oils (Figure s1B, si2), and the naphthalenes and alkylated naphthalenes were the dominating sVOc components.the total le-WaF concentration of the dissolved fraction decreased as a function of increased weathering degree.the relative contribution from the UPc (estimated by subtracting the sVOcs from the tPh concentration) increased from approximately 5% in le-WaF from source oil to more than 90% in le-WaFs of the two field collected oils ctc and Juniper (Faksness et al. 2020).the relations between total le-WaF concentration and the fraction of unresolved polar components in le-WaFs from fresh and artificially weathered oils are illustrated in Figure 1.there is a defined increase in in the fraction of UPc in le-WaFs of residues beyond 200 °c + and these are referred to as 'heavily weathered oils' whereas the fresh oils (source oil B and Mass fresh) and the 150 and 200 °c + residues are referred to as 'fresh and moderately weathered' .

Toxicity tests
the toxicity tests were designed to characterize the toxicity of the water soluble fractions (le-WaFs) resulting from similar oil to water ratio (100 mg oil/l) of oils with different degrees of weathering.thus, each le-WaF was subjected to parallel dilution with four replicate series assuming that the relative composition was similar at all dilutions (no oil droplets present).the test organisms S. pseudocostatum and A. tonsa were exposed to determine effect concentration (ec 50 and lc 50 ).acute toxicity can be presented as specific toxicity, expressed from ec 50 or lc 50 values and normalized to mass-based concentration data, or relative toxicity, expressed as ec 50 and lc 50 values calculated from % le-WaF.Due to the low OWR (1:10 000) used, none of the diluted le-WaFs caused 50% growth reduction in the algae and most le-WaFs caused less than 50% mortality for A. tonsa (supporting information (si3), table s3). the further discussion and comparisons made with theoretical calculations are therefore mainly based on lc/ ec 10 .the results showed that A. tonsa was more sensitive to exposure than S. pseudocostatum, and on average the lc 10 s for A. tonsa were a factor of two lower than ec 10 for growth reduction in S. pseudocostatum.When toxicity is reported on mass-basis, the toxicity appears to increase during oil weathering (e.g.Faksness et al. 2020).Based only on the lc/ ec-values, this may lead to the false conclusion that environmental toxicity of crude oils significantly increases with increasing degree of oil weathering.as shown by Faksness et al. (2020), acute toxicity (ec 10 and lc 10 ) for A. tonsa and S. pseudocostatum decreased in parallel with the decreased solubility of the oil at increasing degrees of weathering.Using the concentrations of the undiluted le-WaFs to present the toxicity as tU show that the toxicity of the WaFs did not significantly change as the total le-WaF concentration decreased to about 4% (300 °c+) of that of the source oil B.
total mortality observed at the undiluted le-WaFs at the end of the tests with A. tonsa was highest in the two fresh oils source oil B (89%) and Mass oil (74%), whereas the mortalities in the remaining le-WaFs of artificially weathered oils ranged from 21 to 52% (table 1). the le-WaFs of the two field collected oils were less toxic with no mortality observed after ctc exposure and less than 9% increase in mortality caused by Juniper.as shown in Figure 2a, the tU for A. tonsa varied between 1.1 and 3.6, indicating no clear relationship between toxicity and tWaF concentration.comparing fresh and moderately weathered oils (≤200 °c + residue, >1 mg/l tWaF) with the more weathered oils (>200 °c + residue, <1 mg/l tWaF) the average tUs were 2.06 (± 0.92) and 2.01 (± 0.84), respectively.this shows that the composition and not only the total concentration of the dissolved phase is crucial in predicting the toxicities of le-WaFs.Due to the limited number of ec 10 values obtained for S. pseudocostatum, no conclusion is made for this species (Figure 2B). it should be noted that the le-WaF systems seek to approach an equilibrium between oil and water and as such is considered a worst-case situation.the current results also represent the toxicity related of a specific and constant oil loading and should not be confused with the progression of toxicity in a field situation (Maloney et al. 2021) where there is a constant loss of oil mass during weathering.Furthermore, constant dilution in the open sea will keep the water concentration well below the equilibrium due to the dissolution kinetics which is expected to have a higher impact on the weathered oils with a larger fraction of less soluble components.

Calculation of acute TUs
acute tUs for A. bahia and S. costatum were computed from the measured concentrations of identified hydrocarbons in 100% le-WaF of the various oils according to eq. (s1) (si3.3) using input data given in table s4 (si3) and are shown in table 1. Phenols and UPc are not included in the initial tU calculations, but the potential impact of including UPc fraction is discussed in section 3.6.Results are summarized in Figure 3 with more details given in supplementary information (Figure s2, si3).For S. costatum the acute tUs were below 1 in all le-WaFs when applying the species specific ctlBB (table 1), suggesting that less than 50% growth reduction is expected to occur from exposure to these le-WaFs.the estimated acute tUs of the le-WaFs from fresh and artificially weathered oils for A. bahia ranged 0.28-1.29,whereas the field weathered oils had tUs of 0.09 (Juniper) and 0.17 (ctc). in contrast to the results from the toxicity tests with A. tonsa there was a significant difference (p < 0.005) in the average tUs between the fresh to moderately weathered oils (>1 mg/l tWaF) of 0.95 (± 0.25) and the artificially heavily weathered oils (<1 mg/l tWaF) with average tU of 0.42 (± 0.09).a similar pattern was seen for S. pseudocostatum displaying average values of 0.61 (± 0.16) and 0.27 (± 0.06), respectively.the slopes of the linear regression lines were 0.23 (95% conf, int.= 0.17-0.29)for A. tonsa and 0.15 (95% conf, int.= 0.11-0.19)for S. pseudocostatum and significantly different from zero for both species (p < 0.0001).
the contributions of different compound groups to tU in the 100% le-WaFs are shown in Figure 4 for A. bahia and in Figure s3 (supporting info) for S. costatum.For source oil B, Mass and 150 °c+, the volatile components accounted for 73, 64 and 55% of the tU, respectively.in the more weathered oils, the contents of volatile components are decreasing, thus contributing less to the tU.these observations indicate that the loss of the more soluble, low-molecular weight components in the weathered oils decreases the toxicity of the  le-WaFs as weathering progresses, consistent with e.g.Faksness et al. (2020), Di toro et al. (2007), and Maloney et al. (2021).

Comparison between TLM for A. bahia and toxicity tests with A. tonsa
Due to the low OWR used in the le-WaF preparation, the lc 50 s for A. tonsa ranged from 21 to 89% in the undiluted le-WaFs.to provide the acute tUs for A. tonsa the lc 50 s of groups with a total mortality between 21 and 49% were estimated based on the average ratio of 0.71 (± 0.09) between lc 10 and lc 50 based on 10 toxicity tests performed at the same loading of the same oils.Figure 5 show the acute tUs based on ctlBB for A. bahia relative to acute tUs derived from A. tonsa toxicity tests for corresponding le-WaFs (eq.( 3)). the figure shows that the A. bahia model gives a good approximation for the A. tonsa acute toxicity for the fresh and moderately weathered oils (≤ 200 °c + residue, >1 mg/l tWaF).For the two fresh oils, the average acute tU was 1.30 whereas the corresponding calculated value based on parameters from A. bahia was 1.18.however, for the heavily weathered oils the tlM model produces tUs that is consistently lower and about one third of those derived from the A. tonsa acute test.this may be caused by a lower proportion of characterized components in the weathered oils.the number of components detected above lOQ in the le-WaFs ranged from 36 to 77 with consistently lower numbers in le-WaFs of the most weathered oils.the disappearing components during weathering are mostly volatiles as the number of semi volatiles are consistently in the range 36 -43.
an additional explanation may be that the UPc fraction of the oil changes and become more complex during the weathering process through microbial degradation and photooxidation.intense solar radiation causes photooxidation of surface slicks resulting in production of oxygenated hydrocarbons and sulfur compounds (lee 2003).the complexity of the UPc fraction may increase during the weathering process as indicated by mass spectra from le-WaFs of the field weathered oils Juniper and ctc (sørensen et al. 2024).these oxidized compounds may be more easily biodegraded but can also be more toxic than the mother compounds because they can be more reactive (lee 2003).artificial weathering using photooxidation may also generate components that contribute to toxicity, but, to our knowledge there is no information on whether extensive heating of the oil used in artificial weathering will have a similar effect.

Chronic toxicity and HC5
a comparison between observed and predicted chronic toxicity cannot be done as chronic toxicity tests were not performed with A. tonsa, but the methodology described in McGrath et al. (2018) has been used to predict chronic hc5 values.McGrath et al. (2018) discusses two approaches to estimate chronic toxicity.One was a revision of the hc5 equation given in eq.(s2) (si3.3) and the other applied the tlM universal slope and chemical class corrections to the chronic toxicity data and computed ctlBBs for the species in the chronic toxicity database.according to McGrath et al. (2018), the two methods provided nearly identical hc5s for hydrocarbons, however, this is not in accordance with the calculations done in the present work, as the hc5 using eq.(s2) (si3.3)gives approximately 4 times lower hc5 compared with the tlM approach for chronic toxicity using chronic ctlBB for A. bahia (table 2).this results in a chronic tU for the total le-WaFs that are approximately 4 times higher when eq. (si2) (si3.3) is used.chronic tU based on hc5 calculations were in the range from 2.7 for le-WaF of Juniper to 47 for le-WaF of source oil B. Using the tlM approach to predict chronic ctlBB for A. bahia, the value for le-WaF of Juniper was 0.7 (and the only system with chronic tU < 1) and was 11 for le-WaF of source oil B. Performing the same calculations with chronic ctlBB for S. costatum gave lower values, with three le-WaF systems with chronic tU > 1 (source oil B, its 150 °c + residue, and Mass fresh oil).Table 2. calculation of predicted chronic toxicity in 100% le-WaF using the following approaches using equations in Mcgrath et al. (2018): ΣTU based on chronic hc5 (eq.(si2) (si3.3)) and ΣTU based on chronic critical target lipid body burdens (eq.(s1) (si3.3))relative to A. bahia (mysid) and S. costatum (marine algae).
Predicted chronic TU in 100% le-WaF le-WaF system hc5 chronic (eq.( s2))  2017) used the tlM-derived hc5 as critical effect level.they compared observed and predicted chronic effects and observed that in most cases observed chronic effects occurred at ΣtU_hc5 between 10 and 100, indicating that the tlM-derived hc5 is protective of chronic observed effects derived from toxicity tests with petroleum substances.By definition, the ΣtU = 1 for acute effects would represent a le-WaF exposure that is predicted to cause a 50% response at a given oil loading (Redman et al. 2017). in contrast, a ΣtU = 1 for chronic effects would represent a substance loading with no significant or limited acute effects (i.2021) reported an average acR of 3.66 in a heterocyclic acR data set.the acR between predicted acute and chronic using the ctlBB approach (table 2) in our calculations was approximately 8.2 for A. bahia and 1.8 for S. costatum, giving an average value of 5. as mentioned above, no chronic toxicity tests have been performed in our study, but the predictions indicated that A. bahia is more sensitive to the le-WaF exposures than S. costatum, also regarding chronic toxicity.
Figure 6 shows the tUs calculated for the various le-WaFs according to the tlM frameworks compared to acute tUs derived from tests with A. tonsa.the general trend is that compared to the A. tonsa acute test, the tlM data show consistently lower toxicity for the weathered oils relative to the fresh oils.the comparison shows a good correlation between the tlM based tUs and tUs from the A. tonsa acute test for the two fresh Macondo oils (Mass fresh and source oil B). if accepting that A. bahia is a good surrogate for a. tonsa for fresh and moderately weathered oils, the estimated average acR based on chronic tlM versus the A. tonsa test for the five least weathered oils is 5.49 (± 0.51).this is close to the geometric average of 5.22 reported by McGrath et al. (2018).however, for the six most weathered oils, the average acR is reduced to 2.26 (± 0.38).it is also evident that the hc5-method is significantly more conservative than the chronic ctlBB for A. bahia, which may be expected since this derived method is designed to protect 95% of plants, invertebrates, and fish (McGrath et al. 2018).For the fresh and moderately weathered oils, the hc5 to acute ratio for A. tonsa was 23.7 (±2.12).

Potential impact of unresolved polar components (UPC)
the sum tU computed represents the tUs from all characterized and identified hydrocarbons in the exposure system.Phenols were not included in the calculations, as they are not considered type i narcotic chemicals.UPc, which contributed up to more than 90% of the total le-WaF concentration (see Figure s1, supplementary info), is another component group that has not been included in tU calculations reported above.sørensen et al. ( 2024) estimated an lc 50 for UPc of 1.01 (± sD = 0.24) mg/l for the copepod A. tonsa.By using the concentrations of UPc in the various oils (supplementary info, Figure s1c), it is possible to include UPc as an additional component with an assigned toxicity threshold.Figure 7 shows the impact of including UPc with an assumed lc 50 of 1 mg/l (sørensen et al. 2024).the figure shows that the increase in tU is approximately the same (≈ 0.21 ± sD = 0.04) for all le-WaFs except for those of the most weathered oils (300 °c + and the field weathered oils).although the relative mass fraction of UPc in the le-WaFs varies from 5% up to more than 90%, the actual mass is little affected by the weathering (Figure 1 and Figure s1c (si2)).thus, since the assigned toxicity is fixed, the contribution to tU will be in the same range for all weathering degrees originating from the same source oil. the inclusion of UPc reduces the difference between the tU derived from acute tests with A. tonsa and tUs based on ctlBB for A. bahia, but the pattern indicates an underestimation of toxicity for the le-WaFs of the most weathered oils.

Conclusions and suggestions for future research
the present study concerns the toxicity of the water-soluble fractions of a series of fresh and artificially weathered Macondo oil at an oil to water ratio of 1:10 000 (100 mg oil/l).the toxicity tests with the copepod A. tonsa indicated only minor differences in the toxicity (tUs) of the various le-WaFs despite a large span in total petroleum concentration in the collected water fractions from the fresh and weathered oils.this slight decrease of lc/ec 50 in the le-WaFs at higher weathering degrees is due to an increased fraction of larger and more toxic components such as Pahs.calculation of oil toxicity based partial characterization of the oil according to previous recommendations (singer et al. 2000) appears to match well with the current toxicity test on fresh oils.however, for the most weathered oil qualities, the tlM based tUs is about a quarter of those of the toxicity tests, suggesting a toxic contribution from the UPc not accounted for in our tlM based tU calculations.including the UPc fraction as a single component with a fixed toxicity threshold (lc 50 of 1.01 mg/l (sørensen et al. 2024)) in the calculations only partially compensated for the observed differences (Figure 7).since the mass of the measured soluble concentration of the UPc remains within the same range independent of weathering degree, the tU contribution of the UPc was also almost constant at about 0.2 tU units when assigned the fixed lc 50 .the results indicate that tlM derived methods applied to a standard list of analytes may to some extent underestimate the toxicity of artificially heavily weathered oils.to improve the tlM based calculations of tU, extended analyses of water-soluble fractions of weathered oils by high resolution analytical methods (e.g.Gc × Gc) should be considered (Redman et al. 2012).the UcP fraction has yet to be structurally resolved.their composition and contribution to toxicity may change as the oil is continuously weathered, and it is important to explore their toxicological significance and also if they display other modes of action than narcosis.Furthermore, as our studies focused primarily on copepods, a wider range of taxa should also be considered in future studies.More detailed chemical and toxicological analyses of UPc will benefit future toxicity calculations and elaborate on the reasons behind the observed discrepancy between observed and predicted toxicity, especially for the artificially weathered oils.
g. McGrath and Di toro 2009), but was introduced in McGrath et al.

Figure 1 .
Figure 1.content unresolved polar components (UPc) versus total dissolved material (tWaF) in le-WaFs generated with oil-to-water ratios of 1: 10000 (100 mg/l oil) and subjected to toxicity testing.a; fraction of UPc relatve to tWaF.B; concentrations of UPc arranged in increasing order of tWaF concentrations.Red dots represent le-WaFs of field collected weathered oils whereas black dots represent le-WaFs of fresh and artificially weathered oils.

Figure 2 .
Figure 2. TUs based on acute toxicity tests relating lc 10 for A. tonsa (a) and ec 10 for growth in S. pseudocostatum (B) to total le-WaF concentration of fresh and artificially weathered oils.For S. pseudocostatum weathering by heating beyond 200 °c + caused less than 10% growth reduction and TUs could not be determined.

Figure 3 .
Figure 3. correlation between total le-WaF (oWR = 1: 10 000) concentrations of various oils with calculated TUs based on acute cTlBB for A. bahia (a) and S. costatum (B).solid black lines indicate linear regression (plotted on a log scale) between total le-WaF concentration and TU with ±95% confidence interval indicated by dotted lines.The two fields weathered oils (Juniper and cTc) marked with red were not included in the regressions.

Figure 5 .
Figure 5. comparison between acute TUs based on cTlBB for A. bahia (A.b.) and TUs derived from acute test with A. tonsa (A.t.) for le-WaFs with oWR = 1: 10 000.circles (weathered oils) and dots (fresh oils) indicate the TlM derived TUs for A. bahia relative of TUs from acute tests with A. tonsa for corresponding le-WaFs.solid line indicates linear regression (plotted on a log scale) between tWaF concentration and TU with ±95% confidence interval indicated by dotted lines.solid line indicates the linear regression line (slope = 0.17; significantly different from zero).
e. NOel or el10; 10% response) at or below this benchmark.Redman et al. (2017) used a default acute-tochronic ratio (acR) of 4.5, which is typical for nonpolar organics.McGrath et al. (2018) calculated a geometric average acR of 5.22 across species, and McGrath et al. (

Figure 6 .
Figure 6.comparison of TUs calculated for A. bahia by the TlM framework and TUs derived from acute toxicity tests with A. tonsa on le-WaFs of fresh oils and artificially weathered Macondo source oil (oWR = 1: 10 000).solid and broken lines are linear regression lines (plotted on a log scale) with 95% confidence interval indicated by dotted lines.

Figure 7 .
Figure 7.The impact on TU of including the presumed acute toxicity of 1 mg/l to UPc (sørensen et al. 2024) in the TU calculation.comparison between TUs with and without including UPc with linear regression line (solid line) with 95% confidence interval indicated (dotted lines).The slope of the regression line (excluding cTc and Juniper) is 1.08 (± se = 0.039).

Table 1 .
summary of toxicity test results expressed as % effect on the animals at test endpoint in 100% le-WaF and as predicted toxicity expressed as acute toxic units for S. pseudocostatum and for A. tonsa using A. bahia as a surrogate.
the input data for calculations of lc 50 s are detailed in table s4 (si3).Redman et al. (