Evaluating the sensitivity of a chronic plant bioassay relative to an independently derived predicted no effect thresholds to support risk assessment of very hydrophobic organic chemicals

doi:10.21203/rs.3.rs-4473046/v1

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Evaluating the sensitivity of a chronic plant bioassay relative to an independently derived predicted no effect thresholds to support risk assessment of very hydrophobic organic chemicals

https://doi.org/10.21203/rs.3.rs-4473046/v1

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Environmental risk assessments require high quality toxicity data to establish protective thresholds. The chronic effects of very hydrophobic organic compounds (VHOCs) in soils are often difficult to determine because multiple processes (e.g. sorption, volatilization, biodegradation) can complicate the interpretation of results. We have developed a standardized soil dosing and aging procedure for assessing bioavailability of high logK_ow VHOCs in a synthetic soil surrogate, and then used it to evaluate the toxicity of high logK_ow VHOCs across a range of test substance concentrations and soil organic carbon content. The soil preparation protocol resulted in relatively stable freely dissolved concentrations of test substance compared to bulk soil concentrations with some losses likely due to volatility and biodegradation. This dosing method wasused in a chronic terrestrial plant toxicity bioassay to evaluate the potential toxicity of VHOCs on complex reproductive endpoints like inflorescence and seed bud formation. Testing included common hydrocarbons and three very hydrophobic lubricant substances (logKow > 10). The toxicity data were used to evaluate existing predicted no effect concentrations (PNEC) that had originally been derived without these higher order chronic plant endpoints. The initial exposure concentrations were set at the independently-derived PNECs to provide an independent validation of the PNEC framework. This evaluation was performed to expand the domain of applicability of the PNEC to VHOCs and for the chronic terrestrial plant endpoints. We saw no effects on plant biomass or inflorescence production at these low exposure concentrations, demonstrating that the established PNEC is protective of long term plant health. The results of the present study confirm that the new dosing method is fit for purpose, and that the existing PNEC framework can be extended to chronic plant endpoints for VHOCs.

Soil is an important environmental compartment that supports food production, habitat, and biodiversity (Warkentin 1995, Brussaard, De Ruiter et al. 2007, Voroney and Heck 2007). Chemical management programs under different jurisdictions require environmental and human health risk evaluations of substance exposure via soil (EC 2006, Turle, Nason et al. 2007). The evaluation of chemicals in soil require toxicological characterization of soil invertebrate and plant species to establish toxicity limits that are used in risk assessments (ECHA 2008).

Soils are complex environments encompassing a wide variety of physical and (bio)chemical properties and processes (e.g., organic carbon content, biodegradation, etc.) that control the fate and bioavailability of chemicals (Council 2003, Semple, Morriss et al. 2003). Additionally, the change in bioavailability during soil aging processes can dictate test chemicals’ toxicity in soil (Alexander 2000, Sverdrup, Jensen et al. 2002, Hiki, Watanabe et al. 2021). Therefore, consideration for the loss of test chemicals through irreversible binding to soil components, volatilization, or biodegradation is required when developing meaningful hazard data from studies using amended soils (Alexander 2000, Northcott and Jones 2001). The combination of properties provide unique challenges during preparation of test soils, particularly when environmentally realistic exposures are the goal (Sijm, Kraaij et al. 2000, Lanno, Wells et al. 2004). For example, a lack of sufficient equilibration and aging can substantially impact toxicity test results producing toxicity data that are not representative of typical environmental exposures (Reid, Northcott et al. 1998). Finally, when testing hydrophobic chemicals at elevated concentrations there is potential to introduce free phase material into the test system, thereby adding a potential stressor that would further complicate interpretation of test results (Redman, Parkerton et al. 2014). The present paper focuses on optimizing soil spiking variables (spiking method, mixing times, wet vs. dry equilibration) in artificial (OECD 2008) soil and subsequently applying these learnings to definitive terrestrial toxicity testing.

A second goal of this work was to evaluate the predicted no effect concentrations (PNEC) for hydrocarbons (Redman, Parkerton et al. 2014) for application to chronic plant endpoints. This PNEC was originally developed for hydrocarbons and other nonpolar organic chemicals, and is reasonably comprehensive in it’s derivation. For example, in McGrath and Di Toro (McGrath and Di Toro 2009) compiled acute and chronic toxicity data for 47 different aquatic test organisms including algae, fish, and invertebrates for more than 50 chemicals, analysed with the target lipid model (TLM) framework. The TLM framework provides a method for comparing toxicity data from different chemicals and organisms on the basis of a computed critical target lipid body burden. This method is flexible and is generally applicable to most test species and organic chemicals. The goal of the present study is to evaluate the toxicity of higher order plant endpoints (e.g., flowering, etc). the original derivation of the PNEC included toxicity data for several species of algae, but no higher plant species, and the chemicals used in that study typically ranged from logKow 2 to 6.

The extension of thie TLM-derived aquatic PNEC to soils and sediments was performed by Redman et al (Redman, Parkerton et al. 2014) by using EqP to convert the aqueous PNECs to soil and sediment using organic carbon-water partition coefficients (Koc). This approach was validated against data for 28 additional test species from soil and sediment compartments including higher order plants, soil invertebrates, and soil microbial endpoints. The plant endpoints, however, only considered short term (e.g., 14d) germination studies due to data availability but it did demonstrate applicability of the TLM-derived PNEC to chemicals with logKow > 6, but highlighting challenges with the test systems related to aging and dosing procedures. The outcome of this prior (Redman, Parkerton et al. 2014) study demonstrated that the TLM-derived PNEC from McGrath and Di Toro (McGrath and Di Toro 2009) was protective of soil and sediment organisms due to similar species sensitivities. The results of this EqP study are generally consistent with several other EqP studies showing similar sensitivity between aquatic and soil, or sediment species for a wide range of chemical classes using PNECs based on relatively large collections of test species (Maund, Hamer et al. 2002, Golsteijn, van Zelm et al. 2013, ECETOC 2020, Fuchsman, Fetters et al. 2023).

Recently, the TLM-derived PNEC was updated to include many new data for more species, and the database was expanded for 79 individual species(McGrath and Di Toro 2009). With a similar collection of species types and chemicals as the 2009 version. Notably, the PNEC from McGrath et al (McGrath and Di Toro 2009) included acute and chronic data across eight major taxonomic groups, where algae was the main represenatitive from plant species along with data for other hydroponic plant species (e.g., Lemna gibba, etc). Algae is a meaningful assay because it represents multiple generations of growth, and is generally a robust bioassay. The hydroponic plants typically have endpoints that based on growth rates at 7 to 14d. The inclusion of these plant endpoints, and other data, results in a robust PNEC that supports general purpose risk assessments (Redman, Parkerton et al. 2014). However, this PNEC did not include complex endpoints observed in terrestrial plant assays such as flowering, or seed pod production (STANDARD and ISO 2005, Kalsch, Junker et al. 2006, Tarazona, Cesnaitis et al. 2013).

Therefore, the second objective of this work is to specifically compare the aqueous PNEC derived by McGrath and Di Toro (McGrath and Di Toro 2009) to the terrestrial chronic plant bioassay (Kalsch, Junker et al. 2006, Tarazona, Cesnaitis et al. 2013) using the EqP framework. The chronic plant assay can take more than 60d to conduct based on the growth rates and development of seed pods, or flowers, which is the key endpoint of this bioassay. The test can be labor intensive, sometime requiring manual pollination of the flowers as the test progresses. The long duration of the test could suffer from loss of test substance through volatilization or biodegradation. In the present study, the experimental design included development of multiple chronic plant tests for multiple chemicals that span a wide range in logKow (4 to > 10). This required application of an aging and dosing method described in the the Methods section. A single exposure concentration was chosen so that multiple chemicals could be tested to expand the domain of applicability of the PNEC and testing methods. The exposure concentration was calculated based on the aqueous PNEC as derived by McGrath and Di Toro (McGrath and Di Toro 2009) and EqP. Derivation of the PNEC did not include data from terrestrial chronic plant bioassays, therefore, this experimental design represents an independent test of the PNEC with respect to higher order plant endpoints.

The new experimental data are then compared to literature data (Sverdrup, Krogh et al. 2003, Redman, Parkerton et al. 2014, McGrath, Fanelli et al. 2018) to further evaluate sensitivity of this endpoint. A successful comparison was defined as the lack of toxic effects in the higher order chronic plant bioassay endpoints, which were tested at the PNEC, which was derived independently from this assay type.

The novel aspects of this study include a new dosing method, which was characterized bulk soil concentrations and porewater concentrations using solid phase microextraction (SPME) for a range of VHOC, some of which are well outside typical domain of applicability for terrestrial toxicity tests. Further, the modeling analysis makes use of available data on a higher order chronic plant toxicity test to validate application of a PNEC which was derived independently, including extension of the modeling domain into VHOC. These advancements provide a technical basis for testing and interpretation of toxicity data from difficult to test substances

2.1 Test materials

An overview of single substances (Heptamethylnonane (HMN), n-heptylcyclohexane (HCH), 1,2,3,4,4a,4b,5,6-octahydrophenanthrene (OHP), o-terphenyl (OTP), 2,7-di-isopropyl naphthalene (DiPN), and hexadecahydropyrene (HDHP)) used in this study, purities and relevant physicochemical properties is provided in Table S1. Additional testing was performed on synthetic lubricant substances, including mixtures of polymerized ethers (Lubricant #1), and alkyl (with an alkyl chain of > 12 carbons, C12+) naphthalenes in the form of monomers and dimers (Lubricant #2), or dimers and trimers (Lubricant #3). Representative physicochemical properties for major constituents in these complex substances are provided in Table S1. For practical reasons the experimental design focused on testing at a single dose to maximise the number of chemicals to be tested, and to avoid dose response testing at elevated concentrations that would induce toxicity through fouling or oiling of the plants. According to equilibrium parititoning theory C_free will increase linearly with loading, up until the water solubility limit, where free phase oil will form. The impact and process of fouling of biological material, such as seeds, is not a well understood process and impact downstream endpoints such as germination and growth and is not a relevant process for most typical use scenarios.

Components for the artificial soil (sand, peat, clay) were obtained from US Silica, BFG Supply W15 Grand Rapids, and Standard Ceramic Supply Company. Three 54W blue (3000 °K) and two red fluorescent lamps (3000 °K) (Sun Systems, Vancouver WA) were used to supply the appropriate light intensity for toxicity testing.

2.2 Test Organisms

The test species included oat (Avena sativa) and wild turnip (Brassica rapa); the recommended monocot and dicot species in the International Standard Organization (ISO) 22030 test guideline (STANDARD and ISO 2005, Kalsch, Junker et al. 2006, Tarazona, Cesnaitis et al. 2013). A. sativa seeds were supplied by Johnny’s Selected Seeds (Winslow, ME) for tests with hydrocarbons and Lubricant #1, or from Select Seeds (Union CT) for Lubricant #2 and #3. B. rapa seeds were supplied by Carolina Biological Supply Co. (Burlington, NC). Seed germination and inhibition of growth and reproductive capability tests in control soils showed > 90% germination, consistent with the guideline recommendations.

2.3 Soil preparation

Artificial soil was a mixture of commercially available air dried quartz sand, kaolin clay, and sphagnum peat of approximately 70, 20, and 10% respectively, based on dry weight (ISO 22030 guideline recommendation). The peat was approximately 40% carbon. Adjustments to soil organic carbon (OC) content for the equilibration and aging experiments were achieved by varying the amount of peat added when preparing the soil. All constituents in the soil (i.e. peat, clay, sand) were passed through a 2 mm sieve to remove fragments prior to the addition of test substance. Calcium carbonate was used when needed to obtain an initial pH of 6.5 for the prepared soil.

2.4 Soil equilibration and aging

A preliminary equilibration and aging experiment was conducted to assess optimal pre-treatment conditions for consistent soil preparation. Past studies have shown terrestrial plant toxicity correlates to soil porewater concentrations (Sverdrup, Krogh et al. 2003, Chang, Ji et al. 2021). Additionally, fiber-water partition coefficients for these constituents were estimated from literature (Butler, Parkerton et al. 2016) and by QSAR (Ulrich, Endo et al. 2017) (Table S1). Analysis of bulk chemical concentrations with conventional, exhaustive extraction techniques was performed at discrete time points in the experiment.

Individual HOCs used in the method development were spiked into two separate batches of artificial soil at a high concentration near 250 mg/kg dry weight (dw) or a low concentration near 12.5 mg/kg dw, directly to the dry peat. The peat phase is assumed to control the partitioning and is analogous to passive dosing phases, which minimizes the introduction of carrier solvent into the test system, which is implicated as a possible source of variability for soil testing (Hiki, Watanabe et al. 2021). The spiked peat was allowed to mix for 4 hours on a roller table at 50 rpm. Subsequently, sand and clay were mixed into the spiked peat to yield an organic carbon content of 0.8% or 3% depending on its ratio to the sand and clay. Dry soil constituents were mixed for 3 days on a roller table at 50 rpm followed by water addition and 4 days of wet static equilibration (with gentle stirring once daily) at 23℃ with the soil being hydrated to 35% of its total saturation for a total of 7 days of aging. While other wet and dry aging scenarios were investigated, this option provided a biologically relevant peat pre-conditioning that allowed stabilization of pH, ammonia (Hiki, Fischer et al. 2021) and generation of microbial biomass, and provided reproducible analytical results.

Porewater measurements were collected to characterize equilibrium conditions using a single 2cm length of SPME fiber with film thickness of 100um (1.88uL PDMS), which was equilibrated with 4g (wet weight) of spiked soil inside a 10 mL glass vial for 14 days after the initial 7d of spiking and homogenization. The vials were then placed onto a vial roller at 10 rpm until the vial was randomly sampled for analytical characterization. Preliminary work indicated that soil-fiber equilibrium was reached after 7 days of mixing. Triplicate samples were collected on days 1, 2, 6, and 14 and the fibers after the 7d equilibration were added to the soil matrix promptly. The aging provided time for the test chemicals to equilibrate with the soil substrate, but also allowed for the cycling of nutrients (e.g., ammonia) to stabilize prior to initiation of the test (data not shown).

2.5 Terrestrial toxicity test

These preliminary experiments showed that the 3% OC soil provided the most homogenous exposure with consistent and reproducible analytical results. The mean percent OC of 4 replicates (± standard deviation) was 3.0 ± 0.2% determined by SGS North America (previously Accutest laboratories), Dayton, NJ. The experiments with Lubricants #1, 2, and 3 followed the artificial soil recipe in the ISO 22030 chronic test guideline, consisting of 10% peat. This resulted in soil percent OC of 3.37%, 3.36% and 3.57% respectively.

Fiberglass wicks (~ 1 cm in diameter and 5–10 mm in length) were inserted into a predrilled hole in the bottom of each replicate pot prior to soil addition in order to water the plants (See Image 1 in SI). Fiberglass wicks were inserted allowing ~ 1 cm of wick to remain in the soil to avoid excess water in the lower soil layers. Each replicate was placed into an individual reservoir with underlying deionized water for capillary action to occur through the wick. In addition to wick watering, manual watering (slowly pouring or spraying water needed onto the soil surface daily) deionized water was added to maintain a nominal moisture content of 60% by weight.

As determined in the equilibrating and aging experiments, spiked, dry soil was allowed to equilibrate and age for 3 to 4 days while rolling at 50 RPM, moistened and allowed to equilibrate for 4 additional days. Environmental chamber humidity was maintained using deionized water in a humidifier within the testing space. Initial pH ranged from 6.34–6.97 for the control soil and 5.60–6.97 for the treatments. The aging procedure appears to limit the need for calcium carbonate addition to control the pH. The final moisture content was taken by composite of replicates at study termination and was determined gravimetrically to range from 24–54% for the control and 26–50% for the treatments.

Chronic toxicity tests with A. sativa were conducted in a plant growth chamber, with the chemicals split into two sets due to space constraints in the test chamber. The first set consisted of individual tests for HMN, OTP and HCH, which were conducted at a single test concentration with a corresponding control. The second set of individual tests consisted of another set of single concentration tests with OHP and HDHP and a corresponding control. The single test concentrations were selected to be at or slightly above the predicted no effect concentration for the tested hydrocarbons (Redman, Parkerton et al. 2014). Additional testing at concentrations approaching solubility was performed on Lubricant #1, #2, and #3 for both the oat and wild turnip with individual controls for each test.

All exposures took place in chambers with the following environmental conditions: temperature 23 ± 1°C, 16:8 hour illumination:darkness with light intensity of 13,000 lux ± 2,000 lux, and humidity ≥ 70%. Test substrates were prepared in polypropylene pots (about 10 cm high by 12 cm in diameter at the soil surface and 10 cm in diameter at the bottom) and contained ~ 400 g (wet weight) of treatment or control soil. Tests were performed using 4 replicates per concentration and a corresponding control. For Test 1, six replicates were prepared so that two could be randomly sacrificed midway through the test for analytical measurements and not used for endpoint analyses. Samples were taken in duplicate. Nominal concentrations were 75, 35, 15, 50, and 13mg/kg dry wt for OHP, HDHP, HMN, OTP and HCH, respectively, and around 101 (nominal), 106 to 109 (average A. sativa, B. rapa), and 41 to 42 (average A. sativa, B. rapa) mg/kg for Lubricants #1, #2, #3 respectively.

Ten seeds were randomly sown in each pot, avoiding the edges with allowance for equal spacing (ISO 22030 guideline recommendation). Shortly after 50% control emergence, the number of plants was randomly reduced to 8 per pot. At about fourteen days post emergence, 4 seedlings (randomly selected) per replicate per treatment and control were cut off at the soil surface and immediately weighed (fresh and dry weight). The remaining seedlings were held under test conditions until the tests were terminated between day 48 and day 72. Pollination of B. rapa was assisted with the application of cotton swabs two times per week following flowering. The duration of the test varied depending on the performance of the control and the species tested. The guideline specifies that the assay terminates after inflorescences in the control emerge (A. sativa) or seed pods have developed (B. rapa).

2.6 Test substance analysis

2.6.1 Fiber analysis sample preparation

During the soil aging experiment, SPME fibers were removed from their respective sample vials or test chambers, rinsed with glass-distilled water to remove particulate matter, wiped clean with a damp lint-free wipe and then placed in individual amber glass GC vials containing a 0.5 mL insert. For the respective low and high soil concentration, 250 µL and 500 µL of 1:1 methylene chloride/acetone mixture was added. The vials were placed on a wrist action shaker for 60 minutes. Following the 60 minute solvent extraction, the entire GC vial (solvent and fibers) was analyzed using large volume injection (LVI) gas chromatography with flame ionization detection with the instrument conditions noted below (section 2.6.3).

2.6.2 Conventional analysis sample preparation

During the definitive, in-life phase of this study, samples for conventional analytical determination of exposure concentrations were collected in vials, mixed with Hydromatrix and allowed to dry for 5–10 hours, depending on the relative volatility of the material being extracted. The dried soil sample/Hydromatrix mixtures were packed into 10 mL accelerated solvent extraction (ASE) cells containing a cellulose filter. Oven dried Hydromatrix was added to the ASE cell to minimize dead volume. The sampling vials were rinsed with approximately 2 mL of 1:1 methylene chloride/acetone and added to the ASE cell. Extraction conditions were as follows (2 cycles): Preheat − 0 minutes, Heat for 5 minutes, Static for 5 minutes, Flush 30%, Purge for 60 seconds. All at a pressure of 1500 psi and an extraction temperature of 100ºC. The solvent was 1:1 methylene chloride:acetone. The soil sample extract was transferred to a graduated cylinder and volume adjusted to 18 to 20 mL with 1:1 methylene chloride/acetone and stored in a GC vial for GC analysis per instrument conditions noted below. Lubricant #1 has very low solubility and was not amenable to analytical measurements so the results of the exposures are presented as nominal concentrations. Only Lubricant #2 had measureable concentrations in the controls, which are considered background from the soil matrix (~ 20 mg/kg dw). These concentrations were subtracted from the measurements in the treatment (e.g., 148 mg/kg dw total in the treatment, and 124 mg/kg dw after subtraction).

2.6.3 Analytical procedure for soil fiber/ASE extracts

Calibration standards and individual solvent extracts were analyzed by GC-FID on a Perkin-Elmer Autosystem XL gas chromatograph using a 15 m x 0.53 mm id capillary column with 1.5 µm Rtx-5 stationary phase. Integration of chromatograms was performed using Perkin-Elmer Totalchrom (v6.3.1) software. Instrument conditions for fiber extract, ASE extracts and corresponding standards were as follows: the injection volumes were 5µL for fiber analysis and 10µL for ASE analysis; column flow was 15 mL/min; inlet temperature program was 60°C for 3 minutes with a ramp to 250°C at 200°C/min with a hold for 10 minutes; GC temperature program was 50°C for 1 minute with a ramp to 200°C at 10°C/min with a hold for 1 minute. Detector temperature was 275°C. Air and hydrogen flow rates were at 450 and 45 mL/min, respectively.

2.7 Data analysis

Statistical analysis was performed with data collected at day 14 post-emergence and at test termination using SAS JMP 11.2. The 14d data are comparable to data from short-term plant toxicity tests (OECD.). The quantitative endpoints collected at Day 14 post-emergence consisted of fresh plant mass (measured immediately after cutting the plant) and the proportion of live plants compared to dead plants. The quantitative endpoints collected during the final harvest consisted of total number of flowers or fertile seed pods per plant, both fresh and dry mass of the shoots, and of inflorescences or seed pods. Shapiro-Wilk’s and Bartlett’s tests were used to determine data normality and variance. Analysis of variances (ANOVA) was performed for no- or low-effect concentrations relative to controls. Depending on the homogeneity and/or variance of the dataset the type of ANOVA selected was either comparison of means (Dunnett’s Test) or non-parametric (Steels rank test).

2.8 Target Lipid Model -Equlibrium Partitioning framework

The Target Lipid Model (TLM) has been used extensively to predict acute and chronic toxicity of organic chemicals to aquatic organisms (McGrath and Di Toro 2009). The TLM is a critical body burden model which relates baseline chemical toxicity to the external concentration of the parent chemical using the following relationship

\(log\text{L}\text{C}50=-0.94*logKow+log{\text{C}}_{L}^{*}\) (Eq. 1)

where the effect level (e.g., LC50, or NOEC, mM) is related to lipid-water partitioning using − 0.94 logKow (L/kg), and the inferred internal body burden \(log{\text{C}}_{L}^{*}\) (mmol/kg lipid), which is also know as the critical target lipid body burden. This approach has been widely applied to predict acute and chronic toxicity for fish, invertebrates, miocrobes, algae and plants for aquatic, soil and sediment compartments (Di Toro, McGrath et al. 2000, McGrath, Parkerton et al. 2004, Redman, McGrath et al. 2007, McGrath and Di Toro 2009, Redman, Mihaich et al. 2012, Redman, Parkerton et al. 2014, McGrath, Fanelli et al. 2018, McGrath, Joshua et al. 2019). Our present work will provide additional validation for unique chronic terrestrial plant endpoints.

The aquatic effect levels from the TLM are converted to soil values using the measured organic carbon content and the estimated organic carbon-water partition coefficient (logKoc) (Di Toro and McGrath 2000, Redman, Parkerton et al. 2014)

\(log\text{K}\text{o}\text{c}=0.981*logKow- 0.000283\) (Eq. 2)

The combination of Eqs. 1 and 2 are used to predict effect concentrations in soil, or sediment (LC50ss, or NOECss).

\(log\text{L}\text{C}50ss=0.045*logKow+log{\text{C}}_{L}^{*}\) (Eq. 3)

The slope constant (subtracting Eq. 2 from Eq. 1) becomes a small positive number (0.045 logKow) since the toxicity relationship (Eq. 1) and logKoc relationships (Eq. 2) essentially cancel. The toxicity threshold, \(log{\text{C}}_{L}^{*}\), can represent acute, chronic endpoints, or a PNEC which is a metric designed to be protective of the environmental compartment. In this case the \({\text{C}}_{L}^{*}\) associated with the PNEC is approximately 2.6 mmol/kg target lipid. Target lipid is a theoretical phase like those found in biological membranes of sensitive tissues. Lipid normalization is common practice for evaluating toxicity of different chemicals, and their mixtures, on the basis of species sensitivity (McCarty, Dixon et al. 1992, McCarty, Dixon et al. 1992). Assuming a MW of 200 and a logKow of 6, Eq. 3 provides an LC50 of 980 mmol/kg OC (logPNEC = 0.045*6 + 0.42, Eq. 3), which converts to 30 mg/kg dw using OC content of 3%. In this study, the TLM-derived PNEC (approximately 30 mg/kg dw) was used as a target single test concentration for the plant experiment. As discussed in the Introduction, this PNEC was derived without terrestrial chronic plant toxicity data since they were unavailable. Therefore, comparison of this PNEC to the new experimental toxicity represents an independent validation, and extension of this modeling framework to these new endpoints.

3.1 Soil equilibration and aging experiments

3.1.1 Freely dissolved concentrations of single constituents

The mixing regime applied the test substance to the organic carbon phase first, followed by equilibration with the soil matrix for 3 days before the soil was hydrated and was allowed to equilibrate for an additional 4 days. Porewater concentrations stabilized within 2–5 days from initial mixing, and represents the combined kinetics of sorption to the SPME fiber and soil components.

After mixing, the freely dissolved concentrations (C_W) in the soil solution were measured using PDMS-coated SPME fibers. The concentration-time series are given in Fig. 1 and reported in Table S2. The time series show concentration profiles at 3.0% and 0.8% organic carbon at high (~ 250 mg/kg dw) and low (~ 15 mg/kg dw) test substance concentrations. Some of the six tested single chemicals show little, or negligible, depletion over time (e.g., o-Terphenyl and hexadecahydropyrene), while others decline substantially such as heptamethylnonane and heptylcyclohexane.

The kinetics of the present study are relatively fast, ~ 7-14d, for a subset of the study chemicals, which is generally consistent with other SPME applications where near equilibrium conditions were achieved within 2 weeks of exposure, SI Fig. 4 in Jonker et al (Jonker, Van Der Heijden et al. 2018). The freely dissolved concentrations appeared stable for most chemicals, suggesting the SPME contact times were sufficient to achieve equilibrium. In the present study artificial soils were prepared using peat, which can be different from field sediments where the presence of black carbon phases may complicate the kinetics. As an independent evaluation of the system, the concentrations of chemicals in the SPME PDMS fibers were evaluated against the organic carbon content of the soil preparations.

The soil organic carbon was set at 0.8% and 3%, which is a factor of 3.75 and a similar change in the PDMS concentrations was expected. The concentrations of some chemicals like HCH and HMN declined substantially through the test (Table S2) and were not evaluated here. Data for the other chemicals (OHP, OTP) showed relatively higher concentrations in the PDMS for tests using 0.8% soil organic carbon. The ratios of concentrations in the PDMS concentrations in the low to highorganic carbon tests varied from 1.5 to 3.9 with an average of 2.8 for high (250 mg/kg dw) and 3.4 for the low (15 mg/kg dw) dose preprations. The higher dose preparations had lower ratios, probably due to the concentrations being nearer the solubility limits of the freely dissolved chemicals (Fig. 1). These PDMS concentration ratios were reasonably similar to the ratio of soil organic carbons in the two different preparations (0.8% and 3%, ratio fo 3.75) indicating that the system was near equilibrium which supports the analysis presented below.

The SPME approach provides a method for evaluating exposures relative to water solubility limits, which is an important consideration for designing toxicity testing to limit confounding factors such as physical fouling, which is particularly important for high (> 5) logK_OW chemicals. For most of the chemicals, the experimental design resulted in predicted dissolved concentrations below the estimated water solubility limit (horizontal lines in Fig. 1) except for a few preparations of octahydrophenanthrene (open symbols), which were within 3-fold of the estimated solubility limit, suggesting that the latter may be an underestimate.

While the dataset is limited to 6 test chemicals the observed half-lives in soil (Table S2) for the present study are comparable to the mean observed half-lives for these same chemicals in aquatic media (Prosser, Redman et al. 2016, Davis, Camenzuli et al. 2022) (Table S1, Figure S1). The half lives estimated in the present study are not solely a result of biodegradation since the experimental design was focused on development of soil equilibration methods and and terrestrial plant toxicity test protocols. The use of non-radiolabeled test substances did not allow for complete mass balances to be determined. The half-lives are considered a simplification of the physical and biological processes that affect chemical concentrations in soil, but they do indicate potential for biodegradation. In these aerobic soils the observed half-lives are generally similar in magnitude to those observed in aerobic aquatic media (Prosser, Redman et al. 2016). The exception is OHP where the aqueous half-life was 3.9 d and the soil HLs were > 70 d. This discrepancy is not fully understood, but it is observed for both the high and low OC systems and assumed to be predominantly related to low bioavailability due to sorption (Kickham, Otton et al. 2012, Ortega-Calvo, Harmsen et al. 2015) or perhaps variations in the function of the microbial communities in the different test systems.

3.1.2 Bioavailability

In the present study bioavailability is defined as the concentration of freely dissolved chemicals in the porewater. This is reflected in the variation in organic carbon partitioning coefficient (logK_OC) which is the ratio of the concentration of absorbed chemical to the concentration of freely dissolved chemical. The calculated logK_OC values for each of the test systems increase with increasing logK_OW (Figure S2), but are offset by approximately 0.8 log units reflecting a relatively lower affinity for the peat in the artificial soil used in the present study. Relatively constant freely dissolved concentrations were maintained for many chemicals.

The individual measurements of K_OC (SI Table 2) are generally consistent between treatments with a composite coefficient of variation < 0.1 log units. The CV for HCH declines by about 10-fold across approximately 100-fold variation in the initial soil concentration. This suggests that extensive biodegradation is taking place, which is expected to be a concentration-dependent process (Prince, Butler et al. 2017), where lower emprical half lives correlated with lower initial concentrations.

The relatively small variation in logK_OC between treatments suggests that the mixing protocol employed in the present study results in stable dissolved concentrations of test chemicals in the soil preparations. Testing at higher concentrations could introduce confounding factors from undissolved material, which is a chemical-specific consideration. OHP is the test substance with lowest logK_ow and highest water solubility tested in this study. There is only a slight decrease in variability in logK_OC with increasing initial soil concentrations, which indicates that the exposures with the highest C_W are not modified by the potential presence of free phase test chemical.

3.2 Plant tests

3.2.1 Exposure confirmation in toxicity tests

There was general agreement between nominal and measured concentrations at day 0 across test compounds (Table S3). Concentrations of both Lubricant # 2 (mono/dimer) and #3 (di/trimer) remained relatively stable over 60 days, exhibiting 30% and 15% loss respectively. HDHP was relatively stable for 60 days, consistent with its lack of volatility and resistance to biodegradation. OHP and OTP were relatively stable throughout the first 14 days of the exposure and remained within 35% of initial concentrations throughout the duration of the test. HMN and HCH were observed to decrease 63 and 96% respectively over the duration of the experiment. This could be due to volatilization and biodegradation for HMN and biodegradation for HCH (Table S2, Fig. 1) as discussed above. The mean measured concentration was used to express endpoint effect concentrations for all test chemicals. This demonstrates the repeatability of the mixing protocol used in the present study.

3.2.2 Evaluation of endpoints from toxicity tests

3.2.2.1 Mid-term seedling growth

Seedling emergence was 100% in the control and treatment groups, and seedling survival was 100% in the control and treatment groups at 14 days post-emergence and at test termination. There were no statistically significant effects on biomass on Day 14 in any of the exposures (Table S4, Fig. 2). This is true for both fresh and dry weight of the shoots.

The lack of effects is consistent with the low exposure concentrations near the PNEC and water solubility limits. The PNEC is expected to be a lower-bound threshold at which chemical-related effects would not be likely. Acute toxicity studies with seed emergence typically show effects around 1130–7600 mg/kg OC (Sverdrup, Krogh et al. 2003). The exposures in the present study were 400–3000 mg/kg OC, which are similar to the PNEC extrapolated to soil using EqP (see Methods, Eq. 1–3), and approximately 10-fold lower than exposures typically associated with toxicity on seedling emergence (Sverdrup, Krogh et al. 2003), (see Comparison to PNEC). This is consistent with the study design where test concentrations were chosen to independently validate the PNEC was that derived without data from the chronic plant assay.

3.2.2.2 Plant reproductive endpoints at test termination

The second objective of this work was to evaluate the protectiveness of the PNEC toward chronic plant toxicity to hydrophobic organic chemicals. The results show no statistically significant effects for any endpoint evaluated (Table S5, Fig. 3, Figure S3), including shoot mass (fresh and dry), inflorescence mass (fresh and dry) and the number of inflorescences for A. sativa, or the mass (fresh and dry) and number of seedpods for B. rapa.

The test data from the lubricant exposures, showed no statistical difference from the controls for any endpoint for either plant at the approximate solubility of the substance in the soil matrix.

There were thus no statistically significant (p = 0.05) effects for any endpoint for any of the tested chemicals. This is consistent with the chosen exposure concentration, which was near the PNEC value for the HOCs (see Methods).

3.2.3 Comparison to PNEC

Comparison of the test data to the PNEC derived for organic chemicals was performed by plotting the average test concentration (Table S2) against the logKow of the test chemical (Table S1), in Fig. 4. The test data from the present study are plotted as ‘>’ symbols to indicate that no toxicity was observed at these exposure concentrations at either the 14d mid-test shoot endpoints and the final endpoints on plant mass and inflorescence and seed pod development. The PNEC line (Eq. 1–3) is plotted as a solid line, which passes through all of the test data from the present study. This observation confirms that the independently-derived PNEC is protective of the chronic plant endpoint for a range of chemicals that span a wide range of physicochemical properties (logKow 5.2 to > 16). Nevertheless toxicity may be expected above the PNEC for some of these chemicals. For example, literature data for 14d seedling germination tests show EC20 for plant growth near 30 mmol/kg OC (or ~ 100 mg/kg dw) (Sverdrup, Krogh et al. 2003), grey circles in Fig. 4. So it is reasonable to expect that chronic plant toxicity may occur in this range for the lower log Kow HOCs. However, it is clear that the PNEC is protective of these effect concentrations.

Further, acute and chronic toxicity data from aquatic plant toxicity data (Lemna gibba, Lactuca sativa, and Myriophyllum spicatum) were compared to the EqP framework to convert aquatic effect concentrations to bulk soil concentrations. These represent higher plant species and fill an important taxominic niche beyond the more common algal toxicity data. These endpoints are typically based on inhibition of growth rates, which are distinctly different than the chronic endpoints in the long term plant studies such as formation of flowers and seed pods. There appears to be internal consistency in these data, that acute and chronic plant toxicity occurs above 10 mmol/kg OC, and that no toxicity was observed near the PNEC (~ 3 mmol/kg OC). This comparison also suggests that the independently derived PNEC is protective of the chronic plant endpoints, and that the other plant assays (seedling emergence, growth rate) may be representative of the flowering, or seed pod development endpoints for the long term terrestrial assay.

Some of the test substances in the present study are very hydrophobic and may not have sufficient solubility to cause toxicity. Or they may show slow toxicokinetics. For example, the thin dashed line in Fig. 4 represents the water solubility limit converted to bulk soil concentrations using the logKoc, see Redman et al 2014 (Redman, Parkerton et al. 2014) for more details. This represents the upper limit for dissolved phase exposures before formation of oily phases, also known as a critical phase separation concentration. This is the point in the soil matrix where the sorptive capacity of the organic carbon is exhausted, and the porewater concentrations exceed their respective solubility limit. The Lubricant test substances (alkyl naphthalenes and ester dimers and trimers) are in a very hydrophobic chemical space (logKow > 10, Fig. 4, and Table S1) so that the test concentrations near the PNEC for the lubricant samples are approximately at saturation, and so were slightly lower than the PNEC to avoid the potential for oiling-related toxicity. Also, at logKow > 6 the PNEC is greater than water solubility limits for freely dissolved chemical (Fig. 4). Some classes of very hydrobhobic chemicals (e.g., hydrocarbons, esters, etc) show a lack of toxicity at logKow > 6 (McGrath and Di Toro 2009, Redman, Mihaich et al. 2012, Mackay, Powell et al. 2015, Redman, Parkerton et al. 2015, Parkerton, Letinski et al. 2021), which is consistent with the observed lack of toxicity for the lubricant substances in the present study. Especially for the Lubricant #1, which was tested at a relatively high concentration (approximately 1.3 mmol/ kg OC, or 107 mg/kd DW), similar to the other short term toxicity tests from literature (Fig. 4, circles).

Long term chronic toxicity tests like the plant bioassay evaluated here are difficult to perform due to the potential for artifacts in soil aging, dosing and potential losses during the test as well as the detailed number of endpoints that are evaluated as part of the test guideline. The use of spiking and aging protocols like the one presented in this study, combined with soil bioavailability measurements to properly characterize exposure, provided a relatively stable exposure system that avoided effects due to oiling because testing was at or below the estimated solubility limit. The dosing of the organic carbon phase represents a viable method for introducing hydrophobic liquid chemicals into a soil test system that results in repeatable exposures in the soil matrix. Additional validation may be required if testing at very high concentrations near the solubility limits of the test substances.

In the chronic plant toxicity tests, the exposure concentrations were set near the independent PNEC for HOC with the expectation that chronic toxicity would be limited. The goal of this approach was to demonstrate that the independent PNEC was protective of chronic plant reproduction and growth endpoints for HOCs with a wide range in physicochemical properties. The practical assumption is that the sensitivity of the terrestrial chronic plant assay is similar to the other endpoints and species used in the PNEC derivation and validation.

The results of the toxicity testing showed no effects, consistent with the protection goals of the PNEC and consistent with the observed plant toxicity data for PAH from literature. Based on the toxicity test result of this study, we can conclude that the TLM-derived PNEC is protective of the chronic plant toxicity endpoint and expands the technical basis of the PNEC for risk assessment. Further, the analysis in the present study demonstrates that the equilibrium partitioning approach to toxicity assessments can be extended to very hydrophobic chemicals with consideration for maximum test concentrations based on solubility limits to avoid artefactual effects due to oiling.

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Evaluating the sensitivity of a chronic plant bioassay relative to an independently derived predicted no effect thresholds to support risk assessment of very hydrophobic organic chemicals

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Abstract

Figures

1.0 Introduction

2.0 Methods and Materials

2.1 Test materials

2.2 Test Organisms

2.3 Soil preparation

2.4 Soil equilibration and aging

2.5 Terrestrial toxicity test

2.6 Test substance analysis

2.6.1 Fiber analysis sample preparation

2.6.2 Conventional analysis sample preparation

2.6.3 Analytical procedure for soil fiber/ASE extracts

2.7 Data analysis

2.8 Target Lipid Model -Equlibrium Partitioning framework

3.0 Results and Discussion

3.1 Soil equilibration and aging experiments

3.1.1 Freely dissolved concentrations of single constituents

3.1.2 Bioavailability

3.2 Plant tests

3.2.1 Exposure confirmation in toxicity tests

3.2.2 Evaluation of endpoints from toxicity tests

3.2.2.1 Mid-term seedling growth

3.2.2.2 Plant reproductive endpoints at test termination

3.2.3 Comparison to PNEC

4.0 Summary

References

Supplementary Files

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