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. Author manuscript; available in PMC: 2022 Apr 1.
Published in final edited form as: Arch Environ Contam Toxicol. 2021 Mar 17;80(3):579–586. doi: 10.1007/s00244-021-00824-4

Bioaccumulation of bis-(2-ethylhexyl)-3,4,5,6-tetrabromophthalate and mono-(2-ethylhexyl)-3,4,5,6-tetrabromophthalate by Lumbriculus variegatus

Lawrence P Burkhard 1,*, Tylor J Lahren 1, Terry L Highland 1, James R Hockett 1, David R Mount 1, Teresa J Norberg-King 1
PMCID: PMC8168711  NIHMSID: NIHMS1699404  PMID: 33730174

Abstract

The brominated flame retardant bis(2-ethylhexyl)-3,4,5,6-tetrabromophthalate (TBPH) is used widely in consumer items including polyurethane foam used in furniture. Information on its bioaccumulation in aquatic species is limited and in the current study, sediment bioaccumulation tests with the oligochaete Lumbriculus variegatus were performed on a spiked natural sediment equilibrated for 14.5 months. Analysis showed the TBPH used to spike the sediment contained a small amount (0.046% by mass) of mono-(2-ethylhexyl)-3,4,5,6-tetrabromophthalate (TBMEHP), a potential biotransformation product of the parent chemical. Steady-state biota-sediment accumulation factors (BSAFs) of 0.254 and 1.50 (kg organic carbon/kg lipid) were derived for TBPH and TBMEHP, respectively. TBPH had biphasic elimination behavior where 94% percent of the body burden was depleted within the first 12 hours of elimination (i.e., half-life of 1.2 hours or less), and the remaining 6% eliminated very slowly thereafter (half-life of 15 days). There was little evidence for biotransformation of either chemical by L. variegatus. This investigation confirms the extremely hydrophobic behavior of TBPH and its impact on its bioavailability.

Keywords: Bioaccumulation, Lumbriculus variegatus, oligochaetes, sediment, TBPH

Numerous brominated flame-retardants are used in commerce for decreasing risks associated with fires. One of the brominated flame retardants is bis(2-ethylhexyl)-3,4,5,6-tetrabromophthalate (TBPH) and within the United States, its production volume in 2012 ranged between 1 and 10 million pounds (US-EPA 2015). TBPH is a component of flame retardants formulations such as Firemaster® products (i.e., 550, 600, BZ-54 (Stapleton et al. 2012)) and DP-45 (Great Lakes Solutions 2010), often used to treat polyurethane foam used in a wide range of furniture. Resultantly, TBPH has been found across the globe in indoor and outdoor dust (Ali et al. 2016; Fromme et al. 2016; McGrath et al. 2018; Newton et al. 2015; Stapleton et al. 2008). The ultimate environmental sink besides landfills for TBPH is soils and sediments and numerous investigators have detected and quantified the levels of TBPH in these media (La Guardia et al. 2012; Newton et al. 2015). Once in the sediments, aquatic organisms may bioaccumulate TBPH (Houde et al. 2014; La Guardia et al. 2012; Zhu et al. 2014), and potentially present risks to wildlife and humans that consume them.

Information on the bioaccumulation of TBPH by aquatic species is limited. La Guardia et al. (2012) detected TBPH in Corbicula fluminea and Elimia proxima below a wastewater treatment plant outfall and reported biota-sediment accumulation factors (BSAFs) of 0.071 and 0.010 for C. fluminea, and 0.020 and 0.028 for E. proima. Bearr et al (2010) performed a dietary study with Pimephales promelas and reported limited uptake of 0.59% of the daily dosage (wet weight basis). de Jordan (2014) studied P. promelas in outdoor mesocosms; TBPH was detected in one fish on day 7 of a 42-day exposure, after which TBPH was not detect and several brominated transformation products were detected. The biotransformation products could not be identified due to their limited response on the mass spectrometry instrumentation. Guo et al. (2017) did not detect TBPH in Great Lake fishes even though the chemical was detected in water and air from the Great Lakes.

Bioaccumulation of TBPH is also potentially influenced by biotransformation processes of the organisms, e.g., de-esterification of the phthalate. Roberts et al. (2012) reported no biotransformation with in vitro experiments with liver and intestinal subcellular fractions from human and rat tissues, but did observe biotransformation via the loss of one of the esters linkages using purified porcine carboxylesterase. Bearr et al. (2012) observed loss of parent chemical with in-vitro incubations using S9, cytosol and microsomal subcellular fractions from P. promelas and Cyprinus carpio.

The primary objective of this study was to determine the bioaccumulation of TBPH by the oligochaete Lumbriculus variegatus from spiked sediment. Uptake of persistent chemicals from sediment by benthic invertebrates is a key point of entry into the aquatic food, and quantitative data on this process are needed to inform models predicting chemical accumulation throughout aquatic food webs as well as for piscivorous birds and other aquatic-dependent wildlife. The secondary objective was to determine if L. variegatus biotransforms TBPH into the mono-ester form of the chemical, i.e., mono(2-ethylhexyl)-3,4,5,6-tetrabromophthalate (TBMEHP).

Materials and Methods

Chemicals and Analytical Materials

TBPH (95%+ purity) for sediment spiking was obtained from ARK Pharm, Inc (Arlington Heights, IL, USA). Analytical calibration standards were obtained from Accustandard (New Haven, CT, USA; TBPH and tetradecachloro-o-terphenyl), Wellington Laboratories (Guelph, ON, Canada; mass labeled TBPH [TBPH-L; bis(2-Ethylhexyl-d17)-tetrabromo[13C6]phthalate]), and Toronto Research Chemicals (North York, ON, Canada; TBMEHP and mass labeled TBMEHP [TBMEHP-L; mono(2-ethylhexyl-d17)-tetrabromophthalate]). Chloroform (HPLC grade), methanol (HPLC grade), toluene (Optima™ grade), glacial acetic acid (certified ACS grade), acetonitrile (HPLC grade), and acetone (Optima™ grade) were obtained from Fisher Scientific (Pittsburgh, PA, USA). Phenomenex Novum 3 mL SLE Cartridges (Phenomenex; Torrance, CA, USA) and sodium sulfate (ACS grade; 80-200 mesh) were used in sample processing. All glassware was ashed in a muffle furnace at 450 °C for 8 h prior to use. Ashing was needed because brominated flame retardants including TBPH are in use commercially and they commonly appear in laboratory background/procedural blank samples. For TBPH, laboratory blanks were very low relative to the exposure levels. For TBMEPH, blanks were low but impinged upon the elimination samples because of the very low level of TBMEPH in the exposure.

Sediments

Sediment collected from West Bearskin (WBS) Lake (Minnesota, USA; 48°03'50"N 90°24'13"W) in 2005 with TOC of 9.88% and moisture content of 84.34% was used. Sediments from WBS Lake have been used extensively and successfully in the development of U.S. EPA’s sediment toxicity and bioaccumulation testing methods (US-EPA 2000) and as control sediment in numerous studies (Burkhard et al. 2015; 2016; 2019; Phipps et al. 1993). A 20 mL aliquot of acetone containing 260 μg/mL of TBPH was added to a one-gallon (3.78 L) glass jar containing 100 g of 50–70 mesh white quartz silica (Sigma–Aldrich). Once spiked, the jar was fitted with a lid with a rotatable vacuum hose connection. The jar was then connected to house vacuum with a liquid nitrogen solvent trap in-line and rolled on its side on a roller mill at approximately 10 rpm until the solvent was evaporated. A separate solvent control was prepared by repeating this process with another jar using clean solvent containing 100 g of 50–70 mesh white quartz silica.

Once the solvent was evaporated, 3.20 kg (wet weight) of WBS sediment was added to each jar. The jars were sealed with PTFE lined caps, taped shut, and then rolled overnight in a walk-in cooler at ca. 4 °C and 4 rpm to ensure thorough mixing. After the initial mixing, the jars were equilibrated at ca. 4 °C and rolled in the cooler three times a week (Monday, Wednesday, and Friday) for 15 min over the equilibration period of 441 days (14.5 months).

Sediment Bioaccumulation Exposure

The sediment exposure lasted a total of 56 days; some oligochaetes received continuous exposure for 56 days, while a subset was exposed to spiked sediments for only 28 days, followed by 28 days in clean sediment to assess depuration. Sediments were tested using USEPA test method 100.3, Lumbriculus variegatus Bioaccumulation Test for Sediments (US-EPA 2000), with oligochaetes obtained from in-house cultures. Performance of the test is described in detail in the Supplementary Materials: Section S1. In brief, weighed masses (approximately 200 mg wwt) of oligochaetes were added to each 300-mL screened tall-form beakers containing 100 mL of test sediment and 175 ml of overlying water (total of 53 beakers containing spiked sediment and 22 beakers containing unspiked WBS sediment). Beakers were held in a water renewal system like that described by Benoit et al. (1993) at 23°C and received 2 volume additions/day of fresh water (see SI Section S2 for water chemistry and monitoring information).

To assess uptake, beakers of spiked and unspiked sediment (3 and 2 replicates, respectively) were removed from the system after 3, 7, 14, 21, 28, 42 and 56 days of exposure, and oligochaetes were sieved from the samples, allowed to purge in clean water for 6 hours (Mount et al. 1999), and transferred to 8 mL amber glass vials and frozen immediately. After 28 days of exposure, a subset of 24 beakers were removed to assess elimination rate. Oligochaetes from these beakers were sieved from the sediment, allowed to purge in clean water for 6 hours (Mount et al. 1999) then transferred to fresh beakers containing unspiked WBS sediment. Tissue samples were collected in triplicate as described above on days 28.5, 29, 30, 31, 33, 35, 42 and 56, which correspond to 0.5, 1, 2, 3, 5, 7, 14, and 28 days of elimination. Elimination controls were collected in duplicate at days 30, 33, 42 and 56.

Chemical concentration in the spiked sediment was monitored in 8 beakers that were set up exactly like the oligochaete exposure samples. They were sampled in duplicate at days 7, 14, 28 and 56. The worms were not sieved from the sediment prior to analysis. Day 0 sediment was sampled directly from the jar prior to starting the exposure experiment.

Analysis of Sediment and Oligochaete Samples

Samples of L. variegatus were randomly ordered into sets of 8 samples containing two procedural blanks and two matrix spike (MS) samples per set for internal QC monitoring. They were spiked with mass labeled surrogates and extracted via ultrasonic probe 3 times with CHCl3:MeOH (1:1 v/v) and then once with an ACN:DCM (1:1 v/v):glacial acetic acid (20:1 v/v) solvent system of Bradley et al. (2013a; 2013b) to ensure recovery of TBMEHP. The CHCl3:MeOH extracts were combined and concentrated to 2.0 ml, and a subsample of the extract was taken for lipids analysis via a microcolorimetric method of Van Handel (1985). Subsequently, the ACN:DCM and CHCl3:MeOH extracts were combined, evaporated to dryness, and reconstituted in 30 μl of acetonitrile. After addition of 30 μl of an aqueous 1% formic acid solution, the extract was transferred to a 3 mL SLE cartridge and eluted sequentially with hexane and ethyl acetate for TBPH and TBMEHP, respectively. A similar method was used for extracting sediment samples and differed only in solvent type (i.e., acetone/hexane and acetonitrile/dichloromethane mixtures), due to high moisture content in the samples, and surrogate amount.

TBPH fraction was analyzed using an isotopic dilution quantitation method on a Q-Exactive High Resolution Accurate Mass (HRAM) Mass Spectrometer (MS) coupled with a Trace 1310 Gas Chromatograph (GC) (Thermo Scientific; Waltham, Massachusetts, USA) in positive electron ionization (EI) mode. TBMEHP fraction was analyzed using isotopic dilution quantitation on an Agilent 1200/6410 High Performance Liquid Chromatograph with Triple Quadrupole Mass Spectrometer (LC-QQQ) using Electrospray Ionization (ESI) in negative ion mode. Complete details on sample preparation and mass spectrometry analysis are provided in the Supplemental Materials.

Data Analysis

Biota-sediment accumulation factors (BSAFs) were computed using the equation of Ankley et al. (1992):

BSAF=CO∕fℓCS∕fTOC (1)

where Co is the chemical concentration in the organism (μg/kg of wet weight),fℓ is the lipid fraction of the organism (kg lipid/kg wet weight), Cs is the chemical concentration in surficial sediment (μg/kg of dry weight), and fTOC is the fraction of total organic carbon in the sediment (kg organic carbon/kg dry weight). BSAFs were computed using measured residues on days 28 and 56 of the exposure.

Two sets of kinetic models were used in the analysis of the uptake and elimination data. The first model was a first-order one-compartment model for chemical uptake and elimination (Landrum et al. 1992; Spacie and Hamelink 1982). To estimate the uptake and elimination rate constants, elimination exposure data were fitted to a first-order one-compartment model (Equation 3) and with estimated ke parameter from Equation 3 regressions, Equation 2 was fitted to the uptake data:

Uptake model:CO,ℓ=(ks∕ke)×(Cs∕fTOC)×(1−e−ket) (2)
Elimination Model:CO,ℓ=CA×(e−ke(t−28)) (3)

ks is the uptake rate constant (kg OC/[kg lipid x d]), ke is the first-order elimination rate constant (1/d), CA is the residue in the L. variegatus on day 28 on a lipid normalized basis (μg/kg-lipid), and t is time from the start of the test (d). The steady-state BSAFs were determined using the first-order one-compartment kinetic approach (Landrum et al. 1992):

BSAFSS=kske (4)

The second model was a two-compartment first-order model in each compartment for chemical uptake and elimination (Spacie and Hamelink 1982):

CO,ℓ=(ks−α∕∝)×(Cs∕fTOC)×(1−e−∝t)+(ks−β∕β)×(Cs∕fTOC)×(1−e−βt) (5)
CO,ℓ=CA×(e−α(t−28))+CB×(e−β(t−28)) (6)

where CA and CB are the concentrations in the independent organism lipid compartments A and B (ug/kg lipid); ks-α and ks-β are the uptake rate constants (kg OC/[kg lipid x d]) for compartments A and B; t is time from the start of the test (d); and α and β are the elimination rate coefficients (1/d) for lipid compartments A and B, respectively. For the two-compartment first-order model, the steady-state BSAFs can be determined using the equation:

BSAFSS=ks−αα+ks−ββ (7)

Statistical analyses and regressions were performed using RStudio software (version 0.98.507) with the nlxb regression within the nlmrt package (version 2016.3.2) (Nash 2016; RStudio 2018).

For each sample, concentrations are reported as amount and its associated minimum detection limit (MDL) in Supplemental Materials, Table 3. Data were not left-censored (Antweiler et al. 2008) and for samples with no chromatographic peak, ½ of MDL was plotted in Figures 1 and 2. In fitting the models to the uptake and elimination data, the only instance of using ½ of the MDL was for the day 0 TBPH residue in the oligochaetes. For TBPH, MDLs were calculated from an estimated IDL (Instrument Detection Limit) (Supplementary Materials: Section S3). For TBMEHP, MDLs were estimated by use of three times the noise, found with Agilent LC-QQQ signal-to-noise algorithm.

Figure 1.

Figure 1.

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Measured concentrations of TBPH in Lumbriculus variegatus from dosed WBS sediments (circles uptake, diamonds elimination, symbols in red color: no instrument response and plotted using ½ of MDL). The solid line is the regression fit using a simple empirical one-compartment first-order kinetic model for chemical, and the dashed line is the regression fit of a two-compartment first-order kinetic elimination model.

Figure 2.

Figure 2.

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Measured concentrations of TBMEHP in Lumbriculus variegatus from dosed WBS sediments (circles uptake, diamond elimination, symbols in red color: no instrument response and plotted using ½ of MDL). The solid line is the regression fit using a simple empirical one-compartment first-order kinetic model for chemical uptake using data for days 0 through day 28, and the dashed line is the regression fit of a first-order kinetic elimination model using data for days 28 through day 30.

Results and Discussion

After dosing the sediments with the TBPH chemical, we discovered that there was a small amount of TBMEHP, mean of 0.0464% (standard deviation of ±0.0219%) by mass, in the stock material. After equilibrating the dosed sediments for 441 days (14.5 months), average concentrations in the sediment were 0.00976 (±0.00216) mg TBMEHP/kg-dw and 12.34 (±0.16) mg TBPH/kg-dw, representing a fractional value of 0.0791% (±0.0176%) TBMEHP. Given the similarities in fractional values of the stock material and equilibrated sediment, we believe the TBMEHP in the sediment, after equilibration, was primarily from the original spike, rather than hydrolysis during equilibration. During the sediment bioaccumulation test, concentrations of TBPH and TBMEHP remained constant in the sediments over the 56-day test (Supplementary Materials: Figure S1, Table S1). Both of these findings are consistent with the mesocosm study of de Jourdan et al (2013), which estimated a median dissipation time (Boesten et al. 2005)/half-life in excess of 9,000 days for TBPH.

The oligochaetes in both control and dosed sediments showed similar patterns of declining weight (aggregate mass of recovered oligochaetes) and lipid during the experiment (Supplementary Data: Table S2 and Figure S1). Weights of organisms showed little change for the first 28 days (circa 3% weight loss), then showed a larger decline (to an aggregate loss of about 15%) between days 28 and 56. Lipid content showed a different pattern, with a decline of about 50% up through the day 28 of the exposure, but then remained largely constant through days 28-56. Patterns of weight and lipid change were generally similar between spiked and unspiked sediment, and between oligochaetes exposed continuously and those used for depuration, and were also similar to prior tests using common methods (Burkhard et al. 2013; 2015; 2016; 2019). Over the test, the oligochaetes looked and behaved normally, and weight and lipid data showed no evidence that TBPH caused overt toxicity to the oligochaetes.

Uptake of TBPH, showed first order kinetics, with tissue concentrations rising to about 1, 6, and 12 mg TBPH/g lipid by days 3, 28, and 56, respectively (Figure 1 and SI Table S3). Failure to reach steady state in 28 days is consistent with several other experiments using highly hydrophobic chemicals (Burkhard et al. 2019; Leppänen and Kukkonen 2004; Tian and Zhu 2011). Elimination kinetics were strongly biphasic; tissue concentrations dropped by more than an order of magnitude within the first half day, but declined much more slowly over the ensuing four weeks (Figure 1 and SI Table S3). In a prior study, we observed biphasic elimination with oligochaetes for highly hydrophobic chemicals, i.e., decabromodiphenyl ether, perchloro-p-terphenyl, perchloro-m-terphenyl, decabromodiphenyl ethane and perchloro-p-quaterphenyl (Burkhard et al. 2019) and others have made similar observations (Leppänen and Kukkonen 2004; Zhang et al. 2015). In this study, the first elimination sample was collected after 12 hours and that point resides with the slow elimination kinetics data, suggesting elimination from Compartment A (fast depuration) was nearly complete in 12 hours or less. Leppanen and Kukkonen (2004) applied a two-compartment model for elimination of brominated diphenyl ethers by oligochaetes and reported that Compartment A retained a large fraction of the chemical and eliminated the chemical very rapidly (life-lives: 10.5-47.5 h), and Compartment B contained less of the chemical and eliminated chemical slowly. TBPH followed this behavior where 94% of the chemical in the organisms was eliminated within 12 hours, resulting in a maximum half-life of 1.2 hours. The remaining 6% was eliminated slowly resulting in a slow elimination rate constant of 0.0461 (±0.00485) 1/days (Table 1).

Table 1:

Uptake and elimination rate constants and BSAFs for TBPH

Average Standard Error Units
Regression coefficients for Compartment B: Slow first order elimination kinetics
CB 382.84 10.72 ug/kg-lipid
kβ 0.04614 0.004845 1/day
Regression coefficients for Compartment A: Fast first order elimination kineticsa
CA 5763.5 423.3 ug/kg-lipid
kα 13.45 90.67 1/day
Pseudo-one-compartment first order uptake (ks) and elimination (ke) rate constants
ks 0.001480 0.0003388 g OC/[g lipid x day]
ke 0.005838 0.01051 1/day
BSAF 0.254 ±0.460b kg OC/kg-lipid
Measured BSAFs Day 28 Day 56
0.0401 ±0.0169c 0.0761 ±0.0132c kg OC/kg-lipid
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a

Compartment A parameters determined by nonlinear regression of equation 6 with parameters CB and kβ determined from the nonlinear regression of Compartment B with slow elimination kinetics.

b

mean ± standard error

c

mean ± standard deviations

The mechanism for biphasic uptake and elimination kinetics of highly hydrophobic chemicals in L. variegatus is unknown. Causes might be residual sediment within the organisms, chemical associated with GI tract membranes/mucous, and possibly, by physical displacement by clean sediment (Burkhard et al. 2019). In order to understand and parse between the above alternatives in future studies, collection of oligochaetes with even shorter elimination times, e.g., 1, 2, 3, … 11 hours might be informative.

Attempts to fit a two compartment first order model (Equation 5) to the uptake data for TBPH failed. For example, the nonlinear regression solution provided a negative uptake rate constant for one of the compartments. However, a one-compartment first-order uptake model could be fit to the data (Table 1); but this is not technically correct, because theory demands that the same model be used for both uptake and depuration. Because of the model mismatch, we have labeled the results as being pseudo-first-order rate constants. With the pseudo-first-order rate constants, a steady-state BSAF of 0.254 is determined, and measured day 28 and 56 BSAFs are 0.0401 and 0.0761, respectively (Table 1). Clearly, steady-state conditions were not obtained in the test. TBPH has a measured log KOW of 9.21 (Hanson et al. 2019), and its BSAFSS (0.254 ±0.460) from this study aligns reasonably well with the BSAFSS for decabromodiphenyl ether (with a log KOW = 8.37 (Hanson et al. 2019)) reported by Li et al. (2014); Zhang et al. (2013); and Burkhard et al. (2013) of 0.12±0.021 and 0.079±0.013; 0.103±0.025; and 0.279±0.176 for L. variegatus, respectively.

For TBMEHP, the exposure concentration of 7.62 μg/kg-dw was 3 orders of magnitude lower than TBPH. Analytically, measurements for TBMEHP were challenging, especially during the elimination phase of the test and many of the quantifications were just above their MDLs (Figure 2, Supplementary Materials: Table S3). During uptake, TBMEHP was detectable in oligochaetes at the first sampling (day 3), but increased slowly after through day 28 (Figure 2). After day 28, concentrations decreased in the oligochaetes, and we do not have an explanation for the decrease in concentrations. With the concentrations of TBPH increasing throughout the test for the oligochaetes, the decrease in concentrations of TBMEHP after day 28 does not appear to be related to the health of the oligochaetes. A first-order one-compartment uptake model (Equation 2) was fit to the data for days 0 – 28 and for days 0 – 56 (Figure 2, Table 2). The estimated BSAFss for the two nonlinear regressions were 1.50 (±0.547) and 1.17 (±0.929), respectively. With the unusual behavior of declining residues after day 28 in the uptake portion of the test, we feel that the BSAFSS from the regression using days 0 – 28 is more representative of the bioaccumulation behavior for the TBMEHP. In evaluating the elimination data, residues drop quickly from day 28 to day 30 and then, plateau to values slightly above the samples respective MDLs. Resultantly, a first order elimination model (Equation 3) was fit to the residues from day 28 through day 30 (Table 2, Figure 2). The elimination rate is fast, i.e., ke=1.01 (±0.212) (1/d) and half-life of 0.676 days. In contrast, the half-life for TBPH is 15.0 days for Compartment B.

Table 2:

Uptake and elimination rate constants and BSAFs for TBMEHP

Average Standard Error Units
One-compartment first order elimination (ke) rate constant: Days 28 through 30
CA 204.602 19.09 ug/kg-lipid
ke 1.0088 0.2115 1/day
One-compartment first order uptake (ks) and elimination (ke) rate constants, Days 0 through 28
ks 0.3004 0.07024 g OC/[g lipid x day]
ke 0.1991 0.05522 1/day
BSAF 1.50 ± 0.547b kg OC/kg-lipid
One-compartment first order uptake (ks) and elimination (ke) rate constants, Days 0 through 56
ks 0.5136 0.2804 g OC/[g lipid x day]
ke 0.4390 0.2532 1/day
BSAF 1.17 ± 0.929a kg OC/kg-lipid
Measured BSAFs Day 28 Day 56
2.45 ± 1.03b 0.672 ±0.232b kg OC/kg-lipid
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a

mean ± standard error

b

mean ± standard deviations

One of the objectives of this study was to determine if L. variegatus would biotransform TBPH by deesterification to TBMEHP. The presence of TBMEHP in the source material used to dose the sediments complicated our abilities to detect this biotransformation. The estimated log Kow of TBMEHP, i.e., 7.53 (US-EPA 2020), falls between the measured log Kows of 2,2',4,4',5,5'-hexachlorbiphenyl (PCB-153) of 6.34 and decachlorobiphenyl (PCB-209) of 8.27 (US-EPA 2020). If diffusional processes control the uptake of TBMEHP, its BSAFSS should be consistent/in agreement with the BSAFss values for PCB-153 & PCB-209. Sediment bioaccumulation tests with the same sediment yielded BSAFss values of 1.46, 1.26, and 1.27 for PCB-52 (2,2′,5,5′-tetrachlorobiphenyl, log KOW = 6.09), PCB-153, and PCB-209, respectively (Burkhard et al. 2019), and TBMEHP’s BSAFSS of 1.50 is consistent with these values. The PCBs are very slowly if at all biotransformed by L. vareigatus. The consistency/agreement of TBMEHP BSAFSS with the PCBs suggests that TBMEHP is not being biotransformed by L. variegatus nor is it being created from the deesterification of the TBPH parent chemical.

This study demonstrated that TBPH and TBMEHP are bioaccumulated by L. variegatus from sediment and TBPH was eliminated with biphasic behavior by the L. variegatus. The first measurement in the elimination phase of the test occurred at 12 hours and that data point resided with Compartment B with the slow depuration kinetics. Further testing with measurements occurring earlier than 12 hours are needed for understanding the mechanism causing the biphasic behavior of highly hydrophobic chemicals with L. variegatus. Although complicated by the presence of TBMEHP in the original stock of TBPH, we found no evidence to suggest that L. variegatus biotransform TBPH to the TBMEHP.

Supplementary Material

Supplement1

Acknowledgements

The authors thank Robert Burgess and Guilherme Lotufo for reviewing this manuscript. The information in this document has been funded wholly by the U.S. Environmental Protection Agency. It has been subjected to review by the Center for Computational Toxicology and Exposure (CCTE) and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

Footnotes

Conflicts of Interest

The authors declare they have no conflicts of interest.

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