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

𝑩𝑪𝑭 =𝑪𝒇𝒊𝒔𝒉

𝑪𝒘𝒂𝒕𝒆𝒓=𝒌𝟏𝒌𝑻

Variability?

Water-based exposure?

Mixtures?1Arnot & Gobas 20042Arnot & Gobas 2006

kD

k1

k2 kE

kG

kM

𝑑𝐶F𝑑𝑡

= 𝑘1𝐶W + 𝑘𝐷𝐶𝐷 − 𝑘2 + 𝑘𝐸 + 𝑘𝑀 + 𝑘𝐺 𝐶F

Arnot & Gobas, QSAR, 2003，Chen et. al. ES&T 2018, OECD 305 2012 Revisions

Using dietary exposure

𝑩𝑪𝑭 =𝒌𝟏𝒌𝑻

measured

estimated

What is benchmarking and howcan it help?

Benchmarking with a conservative substance

𝑑𝐶

𝑑𝑡⇒𝑑𝐶/𝑑𝐶𝐵𝑀

𝑑𝑡 DaysLn(C

/ n

gg-1

ww

)

No benchmark

Days

Benchmarkd with HCB

3,4

3Xiao et al. 20134Chen et al. 2018

Can we measure the BCF of mixtures?

Benefits

• More representative of the chemical product

• Reducing the number of test animals

Evaluation

• 16 chemicals with published tested and

predicted BCF data for individual substances

• Reported BCFs from < 100 to ~17000

BM-BCF

BCF-literature

BM

-BC

FBCF-literature

Not statistically different

(t-test, p = 0.33)

Threshold benchmarking5

Is the depuration rate faster or slowerthan the benchmark?

5Zou et al. 2015

Ln(C

/ n

gg-1

ww

)

No benchmark

Days

Days

Benchmarkd with PeCB

Very Bioaccumulative

𝑩𝑪𝑭 =𝒌𝟏𝒌𝑻

Mixtures?

Uncertainty of k1 estimate

𝑩𝑪𝑭𝑩𝑴 =𝒌𝟏𝒌𝑻𝑮

Water-based exposure?

Variability?

BCF of Essential oils

Pine oil

• > 60 components• Analytical standards for compounds

> 1% contribution (n = 9)• > 88% of the mixture

Cedarwood oil (virginian)

• ~ 60 components• Analytical standards for compounds > 1%

contribution (n = 7, detected n = 6)• 75% - 80% of the mixture

(depending on batch)

Image from wikipedia.org

Image from wikipedia.org

β-pinene (BPN) Carene (CAN)

Terpinolene (TPN) Borneol (BNL) Bornyl Acetate (BAC) β-caryophyllene (BCP)

Measured pine oil constituents

α-pinene (APN) Camphene (CAM) Limonene (LIM)

Results: Pine oil

Log BCFBM = 0.97 x Log KOW - 0.88

R² = 0.95

B limit

vB limitMedian BCF for BCP meets B criterion

No constituents are B at the 95% confidence level

Thujopsene (Thu)

Cuparene (Cup) Cedrol (CDL) α-Funebrene (aFun)

Measured Cedarwood oil constituents

α-Cedrene (aCed) β-Cedrene (bCed)

Biotransformation of Cedarwood oil?

Log BCFBM = 0.70 x Log KOW + 0.16

R² = 0.94

B limit

vB limit

Biotransformation of Cedarwood oil constituents exceeds biotransformation of the reference substances

BUT: Four out of six constituents are B or vB at the 95% confidence level

Comparison with literature data

Benchmark substancesanalysed in theCedarwood oil study

Benchmark substancesanalysed in thePine oil study

Dichlorobenzene (DiCB)Hexachlorobenzene (HCB)Musk xylene (MX)PCB52Pentachlorobenzene (PeCB)Trichlorobenzene (TrCB)

What about P and T of Cedarwood oil?

Screening T criterion: EC50 < 0.01 mg/L (potential T), < 0.01 mg/L (T)

• Cedarwood oil has applications as natural biocide/insecticide6.

• α-Cedrene EC50: 0.044 mg/L (Daphnia pulex)7

• Cedrol: indications for endocrine disruption8 and gentoxicity9

• No T results for the remaining Cedarwood oil 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

• α-Cedrene: not P, vB, potential T• Cedrol: not P, not B, unknown acute T, publications indicating potential

genotox, ED• Thujopsene: not readily but significant 56% degradation, B, unknown T

• No experimental results for most of the analysed constituents

Thank you for your attention!

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

References1. Arnot JA, Gobas FAPC. 2004. A food web bioaccumulation model for organic chemicals in aquatic ecosystems. Environ. Toxicol. Chem. 23 (10): 2343−2355.

2. Arnot JA, Gobas FAPC. 2006. A review of bioconcentration factor (BCF) and bioaccumulation factor (BAF) assessments for organic chemicals in aquatic organisms. Environ.

Rev. 14: 254–297.

3. Xiao RY, Adolfsson-Erici M, Akerman G, McLachlan MS, MacLeod M. 2013. A Benchmarking Method to Measure Dietary Absorption Efficiency of Chemicals by Fish. Environ.

Toxicol. Chem. 32 (12): 2695−2700.

4. Chen CL, Löfstrand K, Adolfsson-Erici M, McLachlan MS, MacLeod M. 2018. Deriving in Vivo Bioconcentration Factors of a Mixture of Fragrance Ingredients Using a Single

Dietary Exposure and Internal Benchmarking. Environ. Sci. Technol. 2018, 52, 5227−5235.

5. Zou H, Radke M, Kierkegaard A, MacLeod M, McLachlan MS. 2015. Using Chemical Benchmarking to Determine the Persistence of Chemicals in a Swedish Lake. Environ. Sci.

Technol. 2015, 49, 1646−1653.

6. Kramer A, Guggenbichler P, Heldt P, Juergen K, Ladwig A, Thierbach H, Weber U, Daeschlein G. 2006. Hygenic Relevance and Risk Assessment of Antimicrobial-Impregnated

Textiles. In: Hipler UC & Elsner P. Biofunctional Textiles and the Skin. Current Problems in Dermatology, Vol. 33. ISBN: 3-8055-8121-1. page 94.

7. Passino-Reader DR, Hickey JP, Ogilvie LM. 1997. Toxicity to Daphnia pulex and QSAR Predictions for Polycyclic Hydrocarbons Representative of Great Lakes Contaminants.

Bull. Environ. Contam. Toxicol. 59:834-840.

8. Simon C, Onghena M, Covaci A, VanHoeck E, Van Loco J, Vandermarken T, Van Langenhove K, Demaegdt H, Mertens B, Vandermeiren K, Scippo ML, Elskens M. 2016.

Screening of endocrine activity of compounds migrating from plastic baby bottles using a multi-receptor panel of in vitro bioassays. Toxicology in Vitro 37. 121–133.

9. Mertens B, Simon C, Van Bossuyt M, Onghena M, Vandermarken T, Van Langenhove K, Demaegt H, VanHoeck E, Van Loco J, Vandermeiren K, Covaci A, Scippo ML, Elskens

M, Verschaeve L. 2016. Investigation of the genotoxicity of substances migrating from polycarbonate replacement baby bottles to identify chemicals of high concern. Food

and Chemical Toxicology 89. 126-137.

10. Jenner KJ, Kreutzer G, Racine P. 2011. Persistency assessment and aerobic biodegradation of selected cyclic sesquiterpenes present in essential oils. Environ Toxicol

Chem30:1096 – 1108.

Hexane phase

ACN

Elute with Hex

Ultrasound assisted extraction - Purge & trap – GC-MS

MQ water

hexane

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

Name kT kTG

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

Threshold benchmarking

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