Deriving bioconcentration factors of constituents of essential oils - PowerPoint PPT Presentation

Deriving bioconcentration factors of constituents of essential oils using in-vivo benchmarked dietary exposure studies Roxana Sühring, Chang’er L. Chen, Gisela Horlitz, Michael McLachlan, Matthew MacLeod

Bioconcentration is important … and difficult to measure Water-based exposure? k M k 1 𝑫 𝒈𝒋𝒕𝒊 = 𝒍 𝟐 k G 𝑪𝑫𝑮 = k D 𝑫 𝒙𝒃𝒖𝒇𝒔 𝒍 𝑼 k 2 k E 𝑒𝐷 F Variability? 𝑒𝑢 = 𝑙 1 𝐷 W + 𝑙 𝐸 𝐷 𝐸 − 𝑙 2 + 𝑙 𝐹 + 𝑙 𝑁 + 𝑙 𝐻 𝐷 F Mixtures? 1 Arnot & Gobas 2004 2 Arnot & Gobas 2006

Using dietary exposure measured 𝑪𝑫𝑮 = 𝒍 𝟐 𝒍 𝑼 estimated Arnot & Gobas, QSAR, 2003 ， Chen et. al. ES&T 2018, OECD 305 2012 Revisions

What is benchmarking and how can it help?

Benchmarking with a conservative substance Ln(C / ng g -1 ww) 3,4 𝑒𝐷 𝑒𝑢 ⇒ 𝑒𝐷/𝑒𝐷 𝐶𝑁 No benchmark 𝑒𝑢 Days Benchmarkd with HCB Days 3 Xiao et al. 2013 4 Chen et al. 2018

Can we measure the BCF of mixtures? Benefits • More representative of the chemical product BM-BCF • Reducing the number of test animals BCF-literature Evaluation BM-BCF • 16 chemicals with published tested and Not statistically different predicted BCF data for individual substances (t-test, p = 0.33) • Reported BCFs from < 100 to ~17000 BCF-literature

Threshold benchmarking 5 Ln(C / ng g -1 ww) Very Bioaccumulative Is the depuration rate faster or slower No benchmark than the benchmark? Days Benchmarkd with PeCB Days 5 Zou et al. 2015

Water-based exposure? Uncertainty of k 1 estimate 𝑪𝑫𝑮 = 𝒍 𝟐 𝑪𝑫𝑮 𝑪𝑵 = 𝒍 𝟐 𝒍 𝑼 𝒍 𝑼𝑯 Variability? Mixtures?

BCF of Essential oils Pine oil • > 60 components • Analytical standards for compounds > 1% contribution (n = 9) • > 88% of the mixture Cedarwood oil (virginian) • Image from wikipedia.org ~ 60 components • Analytical standards for compounds > 1% contribution (n = 7, detected n = 6) • 75% - 80% of the mixture Image from wikipedia.org (depending on batch)

Measured pine oil constituents α -pinene (APN) β -pinene (BPN) Camphene (CAM) Carene (CAN) Limonene (LIM) β -caryophyllene (BCP) Terpinolene (TPN) Borneol (BNL) Bornyl Acetate (BAC)

Results: Pine oil Log BCF BM = 0.97 x Log K OW - 0.88 R² = 0.95 Median BCF for BCP meets B criterion vB limit No constituents are B at the 95% confidence level B limit

Measured Cedarwood oil constituents α -Cedrene (aCed) β -Cedrene (bCed) Thujopsene (Thu) α -Funebrene (aFun) Cuparene (Cup) Cedrol (CDL)

Biotransformation of Cedarwood oil? Log BCF BM = 0.70 x Log K OW + 0.16 R² = 0.94 Biotransformation of Cedarwood oil constituents exceeds biotransformation of the reference substances vB limit BUT: Four out of six constituents are B or vB at the 95% confidence level B limit

Comparison with literature data Dichlorobenzene (DiCB) Hexachlorobenzene (HCB) Musk xylene (MX) PCB52 Pentachlorobenzene (PeCB) Trichlorobenzene (TrCB) Benchmark substances Benchmark substances analysed in the analysed in the Cedarwood oil study Pine oil study

What about P and T of Cedarwood oil? Screening T criterion: EC50 < 0.01 mg/L (potential T), < 0.01 mg/L (T) • α -Cedrene: not P, vB, potential T • Cedarwood oil has applications as natural biocide/insecticide 6 . • Cedrol: not P, not B, unknown acute T, publications indicating potential • α -Cedrene EC50: 0.044 mg/L (Daphnia pulex) 7 genotox, ED • Cedrol: indications for endocrine disruption 8 and gentoxicity 9 • Thujopsene: not readily but significant 56% degradation, B, unknown T • No T results for the remaining Cedarwood oil constituents • No experimental results for most of the analysed constituents Readily biodegradable: ≥ 60% degradation in a ready biodegradation test • α -Cedrene and Cedrol: > 75% biodeg in 28 days (OECD 301 test) 10 • Thujopsene: 36 % biodeg in 28 days test, 56 % in 60 days test (OECD 301) 10 • No biodeg test results for the remaining Cedarwood oil constituents

Take home messages • Dietary exposure with internal benchmarking provides a robust method for depuration rate measurements of complex mixtures in fish • Results for reference substances match published measurements for individual components • Number of test animals can be reduced • Higher biotransformation of Pine oil and Cedarwood oil compared to reference substances • Pine oil likely contains at least one B constituent but most constituents are not B • Most measure constituents of Cedarwood oil are B or vB • More information on NCSs and constituents is needed Thank you for your attention!

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Ultrasound assisted extraction - Purge & trap – GC-MS ACN hexane MQ water Hexane Elute with Hex phase

BCF BM-BCF Target 5th Median 95th 5th Median 95th α -Cedrene 4600 138000 infinite 6900 11000 23000 β -Cedrene 4400 69000 infinite 6000 9200 20000 Thujopsene 3000 8600 infinite 3600 4800 6900 Cuparene 1900 2900 6900 1700 2100 2600 Cedrol 570 710 940 520 600 720 α -Funebrene 4100 23000 infinite 4400 6900 15000 BMs TrCB 610 800 1200 650 750 870 PeCB 3500 12000 infinite 3800 5500 9900 HCB 12000 -10615 infinite n.a. n.a. n.a. PCB3 2800 6900 infinite 2800 4100 7700

Threshold benchmarking Name k T k TG α -Cedrene 0.001 ± 0.029 -0.012 ± 0.01 β -Cedrene 0.002 ± 0.029 -0.01 ± 0.009 Thujopsene 0.016 ± 0.03 0.004 ± 0.011 Cuparene 0.047 ± 0.027 0.044 ± 0.012 Cedrol 0.195 ± 0.049 0.203 ± 0.036 α -Funebrene 0.006 ± 0.028 -0.004 ± 0.009 BMs TrCB 0.173 ± 0.053 0.15 ± 0.024 PeCB 0.012 ± 0.027 n/a HCB -0.013 ± 0.024 -0.025 ± 0.011 PCB3 0.02 ± 0.03 0.01 ± 0.014

Composition [%]