Conclusions

FAs (0.0092). This can be attributed to the characteristic properties of fluorine atoms (F, atomic weight 19), which are the smallest after hydrogen atoms (H, atomic weight 1) in three dimensions (H = 0.120 nm, F = 0.135 nm, on the basis of the van der Waals radius). In addition, the ability of a fluorine atom to bind to a carbon atom (C–F bond, 116 kcal mol^-1) is stronger than that of a hydrogen atom (C–H, 99.5 kcal mol^-1) or the other halogen atoms (e.g., C–Cl, 78 kcal mol^-1). The rigid molecular structure of compounds like PFAAs, with linear perfluoroalkyl chains (Deff = 0.61–0.96 nm in C = 8–18 PFAAs) may enable them to pass through biological membranes more easily (Anliker et al. 1988) and lead to higher bioaccumulation (Dimitrov et al. 2002). These specific physicochemical properties of PFCAs would result in an underestimation of the steric bulk effect of molecule size (e.g., Dmax) when evaluated on the basis of MW, and the higher bioaccumulation potential of PFCAs even with their relatively large MWs.

BCF = 320–430).

This study also provides evidence to explain the persistent residual of PFAAs with long perfluoroalkyl chains (e.g., PFTA and PFUnA) in aquatic organism; more reliable hazard information for PFAAs is required in the future.

Table 4-1 Chemical structures of perfluoroalkyl acids (PFAAs)

Perfluoroalkyl carboxylic acid (PFCA)

Perfluoroalkyl sulfonic acid (PFSA)

Perfluoroalkyl phosphonic acid (PFPA) CF₃(CF₂)_n S

O O CF₃(CF₂)_n S O O O

O CF₃(CF₂)_n C

O CF₃(CF₂)_n C O O

O

CF₃(CF₂)_n P O

O O CF₃(CF₂)_n P O

O O

Table 4-2 Bioconcentration test conditions for test substances

Nominal test concentration^a

Measured test concentration^a,b Test substance

(acronym)

Chemical structure

High Low High Low

Exposure period

(d)

Depuration Period

(d) Perfluorooctanoic acid

(PFOA) CF3(CF2)6COOH 50 5 47.6 (1.5) 4.71 (0.10) 28 -^c

Perfluoroundecanoic acid

(PFUnA) CF3(CF2)9COOH 1 0.1 0.946 (0.030) 0.0911 (0.0034) 60 66 or 23^d Perfluorododecanoic acid

(PFDoA) CF3(CF2)10COOH 1 0.1 0.978 (0.031) 0.0978 (0.0043) 60 28 or 47^e Perfluorotetradecanoic acid

(PFTA) CF3(CF2)12COOH 1 0.1 0.890 (0.068) 0.0893 (0.0083) 60 29

Perfluorohexadecanoic acid

(PFHxDA) CF3(CF2)14COOH 1 0.1 0.998 (0.020) 0.100 (0.002) 60 77 Perfluorooctadecanoic acid

(PFODA) CF3(CF2)16COOH 1 0.1 0.967 (0.020) 0.0974 (0.0032) 60 24 Perfluorooctane sulfonic acid,

potassium salt (PFOS) CF3(CF2)7SO3K 20 2 16.0 (1.1) 1.88 (0.09) 58 37

a Test concentrations are in µg L^–1.

b Value in parentheses is the standard deviation of the measured test concentration.

c There was no depuration phase for the PFOA test.

d Depuration period: 66 d for high level, 23 d for low level.

e Depuration period: 28 d for high level, 47 d for low level.

Table 4-3 Analytical conditions for test substances

Test substance Analytical method^a Mobile phase^b (vol:vol) Detection PFOA LC-MS Acetonitrile/water (60:40) m/z = 413 PFUnA LC-MS Methanol/water (80:20) m/z = 563 PFDoA LC-MS/MS Methanol/water (90:10) m/z = 613 > 569 PFTA LC-MS Methanol/water (90:10) m/z = 713 PFHxDA LC-MS/MS Methanol/water (95:5) m/z = 813 > 769 PFODA LC-MS/MS Methanol/water (95:5) m/z = 913 > 869 PFOS LC-MS Acetonitrile/water (60:40) m/z = 499

a LC-MS, liquid chromatography–mass spectrometry; LC-MS/MS, liquid chromatography–tandem mass spectrometry.

b Also contained 5 mmol L^–1 ammonium acetate for PFUnA or 5 mmol L^–1. di-n-butylammonium acetate for the other test substances.

Table 4-4 Steady-state bioconcentration factors (BCFss) and physicochemical properties of test substances BCFss Depuration half-life (d)

Test

substance High level Low level High level Low level

Water solubility^a

(mg L^–1) log Powb log Kowc Clog P^d MW^e,f Deffe,g

(nm)

Dmaxe,h

(nm)

PFOA 3.1 <5.1–9.4 -ⁱ - 3300 2.8 6.30 3.23 414.1 0.61 1.36

PFUnA 2300 3700 29 15 0.597 4.0 9.20 3.94 564.1 0.79 1.55

PFDoA 16,000 10,000 9 8 0.520 - 10.2 4.17 614.1 0.82 1.72

PFTA 16,000 17,000 13 18 0.296 5.1 12.1 4.64 714.1 0.63 2.18

PFHxDA 4800 4700 48 54 0.153 - 14.0 5.11 814.1 0.96 2.16

PFODA 430 320 12 11 0.00470 - 16.0 5.58 914.1 0.92 2.48

PFOS 720 1300^j 45 52 910 - 4.13 3.09 538.2 0.63 1.55

a Measured by following the OECD TG 105 (OECD 1995).

b Measured by following the OECD TG 117 (OECD 2004).

c Calculated by using Kowwin v. 1.67 (US Environmental Protection Agency).

d Calculated by using Clog P v. 4.0 (Biobyte Corp., Claremont, CA, USA).

e Calculated by OASIS Software–Database Manager ver.1.4 (Laboratory of Mathematical Chemistry, Bourgas, Bulgaria).

f MW, molecular weight.

g Deff, effective cross-sectional diameter.

h Dmax, maximum diameter.

i '-', Not measured.

j Average BCF value on the final day of exposure phase.

Table 4-5 Concentrations of test substances and other type of chemicals, and lipid content in each fish tissue and ratio of the concentrations to that in the head

Test concentration Integument^a Head Viscera Remaining parts

Test substance

µg L^–1 ng g^–1 (ratio)

PFUnA 0.1 229 (0.80) 287 (1) 402 (1.40) 73.2 (0.26)

PFDoA 0.1 1110 (0.89) 1240 (1) 1500 (1.21) 386 (0.31)

PFTA 0.1 3020 (1.19) 2540 (1) 3460 (1.36) 724 (0.29)

PFHxDA 0.1 464 (0.97) 480 (1) 1030 (2.12) 229 (0.48)

PFOS 2 5120 (0.99) 5170 (1) 8550 (1.66) 1600 (0.31)

Fluorinated ether^b 0.1 680 (0.65) 1040 (1) 1260 (1.21) 410 (0.39)

Fluorinated alcohol^c 1 2830 (0.79) 3560 (1) 6750 (1.90) 1560 (0.44)

HCB^d 0.005 86.8 (0.65) 134 (1) 190 (1.41) 56.1 (0.42)

Lipid content (µg g^–1)^e - 88.4 (0.97) 91.0 (1) 198 (2.18) 50.9 (0.56)

a Including alimentary canal and gills.

b 2,2-Bis[p-(1,1,2,2-tetrafluoroethoxy)phenyl]-4-methylpentane (Yakata et al. 2003).

c 1H,1H,11H-Eicosafluoro-1-undecanol: H(CF2)10CH2OH.

[National Institute of Technology and Evaluation, Japan. CHEmical Collaboration Knowledge database (J-CHECK) (1994)].

d Hexachlorobenzene (Ministry of Economy, Trade and Industry unpublished data).

e Data from Inoue et al. (2011).

Cl

Cl Cl

Cl

Cl Cl

F₂HCF₂CO C CH₃ CH₂

OCF₂CHF₂ CH

H₃C CH₃

Table 4-6 Physicochemical properties of fatty acids

Substance Chemical structure MW^{a, b} Dmaxa, c

Octanoic acid CH3(CH2)6COOH 144.21 13.02 Undecanoic acid CH3(CH2)9COOH 186.29 17.04 Dodecanoic acid CH3(CH2)10COOH 200.32 18.51 Tetradecanoic acid CH3(CH2)12COOH 228.37 20.74 Hexadecanoic acid CH3(CH2)14COOH 256.42 23.27 Octadecanoic acid CH3(CH2)16COOH 284.48 26.10

a Calculated by OASIS Software–Database Manager ver.1.4 (Laboratory of Mathematical Chemistry, Bourgas, Bulgaria).

b MW, molecular weight.

c Dmax, maximum diameter, unit is nm.

Figure 4-1-1 Pretreatment for analysis of perfluorooctanoic acid (PFOA) in the test water.

・Filling up to 10 mL (water for recovery test, volumetric flask) (only Level 1)

・Taking out 2.5 mL (transfer pipette) (only Level 1)

・Filling up to 5 mL (ultra pure water, volumetric Sample for LC-MS analysis

Test water

1 mL (high level), 2.5 mL (low level)

Conditions of column chromatograph Sep-Pak Plus C8

Conditionings Methanol approx. 10mL

Ultra pure water approx. 10 mL Loading Whole volume of the solution was loaded.

Elution Eluent Methanol 8 mL

Figure 4-1-2 Pretreatment for analysis of perfluoroundecanoic acid (PFUnA) in the test water.

Sample for LC-MS analysis Test water

50 mL (high level), 500 mL (low level)

Eluate

←Water for recovery test 450 mL (graduated cylinder) (only high level)

←Acetic acid 1 mL (transfer pipette)

・Column chromatography

・Filling up to 10 mL (ultra pure water, volumetric flask)

Conditions of column chromatograph Sep-Pak Plus C8

Conditionings Methanol approx. 10mL

Ultra pure water approx. 10 mL Loading Whole volume of the solution was loaded.

Elution Eluent Methanol 9 mL

Figure 4-1-3 Pretreatment for analysis of perfluorododecanoic acid (PFDoA) in the test water.

Sample for LC-MS/MS analysis Test water

50 mL (high level), 500 mL (low level)

Eluate

←Water for recovery test 450 mL (graduated cylinder) (only high level)

←Formic acid 1 mL (transfer pipette)

・Column chromatography

・Filling up to 10 mL (ultra pure water, volumetric flask)

Conditions of column chromatograph Sep-Pak Plus C18

Conditionings Methanol approx. 10mL

Ultra pure water approx. 10 mL Loading Whole volume of the solution was loaded.

Elution Eluent Methanol containing 0.1% formic acid 10 mL

Figure 4-1-4 Pretreatment for analysis of perfluorotetradecanoic acid (PFTA) in the test water.

Sample for LC-MS analysis Test water

50 mL (high level), 500 mL (low level)

Eluate

←Water for recovery test 450 mL (graduated cylinder) (only high level)

←Formic acid 1 mL (transfer pipette)

・Column chromatography

・Concentration approx. 3mL (rotary evaporator, approx. 40 ºC)

←Ultra pure water 5 mL (transfer pipette)

・Filling up to 10 mL (methanol, volumetric flask)

Conditions of column chromatograph Sep-Pak Plus C8

Conditionings Methanol approx. 10mL

Ultra pure water approx. 10 mL Loading Whole volume of the solution was loaded.

Elution Eluent Methanol 10 mL

Figure 4-1-5 Pretreatment for analysis of perfluorohexadecanoic acid (PFHxDA) in the test water.

Sample for LC-MS/MS analysis Test water

40 mL (high level), 400 mL (low level)

Eluate

←Water for recovery test 360 mL (graduated cylinder) (only high level)

←Formic acid 2 mL (measuring pipette)

・Column chromatography

・Concentration approx. 1mL (rotary evaporator, approx. 40 ºC)

←Methanol/ultra pure water (95/5 V/V) approx. 1 mL (measuring pipette)

・Filling up to 10 mL (methanol, volumetric flask)

・Ultrasonic irradiation (approx. 1 min.)

・Filling up to 2 mL (methanol/ultra pure water (95/5 V/V), volumetric flask)

Conditions of column chromatograph Sep-Pak Plus C18

Conditionings Methanol approx. 10mL

Ultra pure water approx. 10 mL Loading Whole volume of the solution was loaded.

Elution Eluent Methanol 10 mL

Figure 4-1-6 Pretreatment for analysis of perfluorooctadecanoic acid (PFODA) in the test water.

Sample for LC-MS/MS analysis Test water

40 mL (high level), 400 mL (low level)

Eluate

←Water for recovery test 360 mL (graduated cylinder) (only high level)

←Formic acid 1 mL (transfer pipette)

・Column chromatography

・Concentration approx. 0.5 mL (rotary evaporator, approx. 40 ºC)

←Methanol/ultra pure water (95/5 V/V) approx. 1 mL (measuring pipette)

・Ultrasonic irradiation (approx. 1 min.)

・Filling up to 2 mL (methanol/ultra pure water (95/5 V/V), volumetric flask)

Figure 4-1-7 Pretreatment for analysis of perfluorooctane sulfonic acid, potassium salt (PFOS) in the test water.

・Filling up to 10 mL (water for recovery test, volumetric flask) (only Level 1)

・Taking out 2.5 mL (transfer pipette)

・Filling up to 5 mL (methanol, volumetric flask) Sample for LC-MS analysis

Test water

1 mL (high level), 10 mL (low level)

Figure 4-2-1 Pretreatment for analysis of perfluorooctanoic acid (PFOA) in the test fish.

・Taking out 1 - 5 g (analytical balance) Sample for storage

Fine sample Test fish

Supernatant Residue

・Measurement of weight and body length

・Chopping into pieces (scissors)

・Refinement (Polytron, 2 min. or more, on ice water)

・Taking out 5 g (analytical balance)

←Acetonitrile 20 mL (graduated cylinder)

・Homogenization (Polytron, approx. 1 min.)

・Washing (acetonitrile 7 mL)

・Centrifugation (7100  g, 5 min.)

・Filtration (absorbent cotton)

・Filling up to 50 mL (acetonitrile, volumetric flask)

・Taking out 1 mL (transfer pipette)

・Filling up to 25 mL (ultra pure water, volumetric flask) Sample for LC-MS analysis

Figure 4-2-2 Pretreatment for analysis of perfluoroundecanoic acid (PFUnA) in the test fish.

・Taking out 2 - 5 g (analytical balance) Sample for storage

Fine sample Test fish

Supernatant Residue

・Measurement of weight and body length

・Chopping into pieces (scissors)

・Refinement (Polytron, 2 min. or more, on ice water)

・Taking out 5 g (analytical balance)

←Acetonitrile 15 mL (graduated cylinder)

←Acetic acid 0.5 mL (measuring pipette)

・Homogenization (Polytron, approx. 1 min.)

・Washing (acetonitrile 4 mL)

・Centrifugation (7000  g, 5 min.)

・Filtration (absorbent cotton)

・Filling up to 50 mL (acetonitrile, volumetric flask)

・Taking out 1 mL (transfer pipette)

・Filling up to 20 mL (methanol/ultra pure water (8/2 V/V), volumetric flask)

Sample for LC-MS analysis

Figure 4-2-3 Pretreatment for analysis of perfluorododecanoic acid (PFDoA) in the test fish.

・Taking out 1 - 5 g (analytical balance) Sample for storage

Fine sample Test fish

Supernatant Residue

・Measurement of weight and body length

・Chopping into pieces (scissors)

・Refinement (Polytron, 2 min. or more, on ice water)

・Taking out 4 - 5 g (analytical balance)

←Acetonitrile 15 mL (graduated cylinder)

←Formic acid 0.5 mL (measuring pipette)

・Homogenization (Polytron, approx. 1 min.)

・Washing (acetonitrile 3 mL)

・Centrifugation (7000  g, 5 min.)

・Filtration (absorbent cotton)

・Filling up to 25 mL (acetonitrile, volumetric flask)

・Taking out 1 mL (transfer pipette)

・Filling up to 20 mL (methanol/ultra pure water (9/1 V/V), volumetric flask)

Sample for LC-MS/MS analysis

Figure 4-2-4 Pretreatment for analysis of perfluorotetradecanoic acid (PFTA) in the test fish.

・Taking out 2 - 5 g (analytical balance) Sample for storage

Fine sample Test fish

Supernatant Residue

・Measurement of weight and body length

・Chopping into pieces (scissors)

・Refinement (Polytron, 2 min. or more, on ice water)

・Taking out 5 g (analytical balance)

←Acetonitrile 15 mL (graduated cylinder)

←Formic acid 0.5 mL (measuring pipette)

・Homogenization (Polytron, approx. 1 min.)

・Washing (acetonitrile 5 mL)

・Centrifugation (7000  g, 5 min.)

・Filtration (absorbent cotton)

・Filling up to 25 mL (acetonitrile, volumetric flask)

・Taking out 1 mL (transfer pipette)

・Filling up to 40 mL (methanol/ultra pure water (1/1 V/V), volumetric flask)

・Taking out 2.5 mL (transfer pipette)

・Filling up to 5 mL (methanol/ultra pure water (1/1 V/V), volumetric flask)

Sample for LC-MS analysis

Figure 4-2-5 Pretreatment for analysis of perfluorohexadecanoic acid (PFHxDA) in the test fish.

・Taking out 1 - 5 g (analytical balance) Sample for storage

Fine sample Test fish

Supernatant Residue

・Measurement of weight and body length

・Chopping into pieces (scissors)

・Refinement (Polytron, 2 min. or more, on ice water)

・Taking out 4 - 5 g (analytical balance)

←Acetonitrile 15 mL (graduated cylinder)

←Formic acid 2 mL (measuring pipette)

・Homogenization (Polytron, approx. 1 min.)

・Washing (acetonitrile 3 mL)

・Centrifugation (7000  g, 5 min.)

・Filtration (absorbent cotton)

・Filling up to 25 mL (acetonitrile, volumetric flask)

・Taking out 1 mL (transfer pipette)

←Ultra pure water 2.5 mL (measuring pipette)

・Filling up to 5 mL (acetonitrile, volumetric flask) Sample for LC-MS/MS analysis

Figure 4-2-6 Pretreatment for analysis of perfluorooctadecanoic acid (PFODA) in the test fish.

・Taking out 1 - 5 g (analytical balance) Sample for storage

Fine sample Test fish

Supernatant Residue

・Measurement of weight and body length

・Chopping into pieces (scissors)

・Refinement (Polytron, 2 min. or more, on ice water)

・Taking out 4 - 5 g (analytical balance)

←Acetonitrile 15 mL (graduated cylinder)

←Formic acid 0.5 mL (measuring pipette)

・Homogenization (Polytron, approx. 1 min.)

・Washing (acetonitrile 3 mL)

・Centrifugation (7000  g, 5 min.)

・Filtration (absorbent cotton)

・Filling up to 25 mL (acetonitrile, volumetric flask)

・Taking out 2.5 mL (transfer pipette)

・Filling up to 5 mL (ultra pure water, volumetric flask) Sample for LC-MS/MS analysis

Figure 4-2-7 Pretreatment for analysis of perfluorooctane sulfonic acid, potassium salt (PFOS) in the test fish.

・Taking out 1 - 5 g (analytical balance) Sample for storage

Fine sample Test fish

Supernatant Residue

・Measurement of weight and body length

・Chopping into pieces (scissors)

・Refinement (Polytron, 2 min. or more, on ice water)

・Taking out 3 g (analytical balance)

←Methanol 15 mL (graduated cylinder)

・Homogenization (Polytron, approx. 1 min.)

・Washing (methanol 5 mL)

・Centrifugation (5000  g, 5 min.)

・Filtration (absorbent cotton)

・Filling up to 25 mL (methanol, volumetric flask)

・Taking out 0.5 mL (transfer pipette)

・Filling up to 20 mL (methanol/ultra pure water (1/1 V/V), volumetric flask)

Sample for LC-MS analysis

Figure 4-3 Relationship between BCF and exposure/depuration periods.

PFOA; perfluorooctanoic acid, PFUnA; perfluoroundecanoic acid, PFDoA; per- fluorododecanoic acid, PFTA; perfluorotetradecanoic acid, PFHxDA; perfluoro- hexadecanoic acid, PFODA; perfluorooctadecanoic acid, PFOS; perfluorooctane sulfonic acid, potassium salt

Solid line shows BCFs during exposure phase and the dashed line shows BCFs during depuration phase.

1 10 100 1000 10000 100000

0 20 40 60 80 100 120 140 160

Test period (d)

BCF

PFOA PFUnA PFDoA PFTA PFHxDA PFODA PFOS

High exposure level

1 10 100 1000 10000 100000

0 20 40 60 80 100 120 140 160

Test period (d)

BCF

PFOA PFUnA PFDoA PFTA PFHxDA PFODA PFOS

Low exposure level

Figure 4-4 Relationships between log BCF and number of carbon atoms of test substances.

Solid line (log BCF = –0.104[number of carbons]² + 2.87[number of carbons] – 15.5;

r² = 0.984) shows the curvilinear regression from PFCA data for the relationship between log BCF and the number of carbons. PFOS data are not included in the regression line. The symbols representing PFCA and PFOS data are as in Figure 4-3.

0 1 2 3 4 5

5 8 11 14 17 20

Number of carbon atoms

log BCF

0 1 2 3 4 5

5 8 11 14 17 20

Number of carbon atoms

log BCF

Figure 4-5 Relationships between log BCF and physicochemical properties of test substances (log BCF versus log Kow).

Solid line (log BCF = –0.111[log Kow]² + 2.65[log Kow] – 11.5; r² = 0.984) shows the curvilinear regression derived from log BCF and log Kow data for PFCAs. PFOS data are not included in the regression line. Symbols identifying PFCA and PFOS data are as in Figure 4-3. Filled and hollow circles show the measured log Pow of PFOA, PFUnA and PFTA under high and low exposure conditions, respectively. These data were not included in the regression analysis.

0 1 2 3 4 5

0 5 10 15 20

log K_owor log P_ow

log BCF

0 1 2 3 4 5

0 5 10 15 20

log K_owor log P_ow

log BCF

Figure 4-6 Relationships between log BCF and physicochemical properties of test substances (log BCF versus Clog P).

Solid line (log BCF = –1.88[Clog P]² + 17.3[Clog P] – 35.4; r² = 0.984) shows the curvilinear regression derived from log BCF and Clog P data for PFCAs. Symbols identifying PFCA and PFOS data are as in Figure 4-3. Filled and hollow circles show the measured log Pow of PFOA, PFUnA and PFTA under high and low exposure conditions, respectively. These data were not included in the regression analysis.

0 1 2 3 4 5

0 5 10 15 20

Clog P or log P_ow

log BCF

0 1 2 3 4 5

0 5 10 15 20

Clog P or log P_ow

log BCF

Figure 4-7 Relationships between log BCF and physicochemical properties of test substances (log BCF versus MW).

Solid line (log BCF = –4.15 × 10^–05[MW]² + 0.0585[MW] – 16.3; r² = 0.984) shows the curvilinear regression derived from log BCF and MW data for PFCAs. PFOS data are not included in the regression line. Symbols identifying PFCA and PFOS data are as in Figure 4-3. Dashed line shows the threshold MW cutoff criteria for bioaccumulation potential (EC 2003).

MW >700

less likely to bioaccumulate (EC 2003)

0 1 2 3 4 5

300 500 700 900 1100

MW

log BCF

MW >700

less likely to bioaccumulate (EC 2003)

0 1 2 3 4 5

300 500 700 900 1100

MW

log BCF

Figure 4-8 Relationships between log BCF and physicochemical properties of test substances (log BCF versus Dmax).

Solid line (log BCF = –8.80[Dmax]² + 34.8[Dmax] – 29.9; r² = 0.887) shows the curvilinear regression derived from log BCF and Dmax data for PFCAs. PFOS data are not included in the regression line. Symbols identifying PFCA and PFOS data are as in Figure 4-3. Dashed line shows the threshold Dmax cutoff criteria for bioaccumulation potential (Dimitrov et al. 2003).

Dmax >1.47 nm

BCFs <5000 (Dimitrov et al. 2003) 0

1 2 3 4 5

1.0 1.5 2.0 2.5 3.0

D_max(nm)

log BCF

Dmax >1.47 nm

BCFs <5000 (Dimitrov et al. 2003) 0

1 2 3 4 5

1.0 1.5 2.0 2.5 3.0

D_max(nm)

log BCF

Figure 4-9 Relationship between Dmax and MW of PFCAs (number of carbons; 8–18) and fatty acids (FAs, number of carbons; 8–18).

Solid line (Dmax-PFCA = 0.00233[MWPFCA] + 0.338; r² = 0.943) shows the linear regression derived from Dmax and MW data for PFCAs. Symbols identifying PFCA data are as in Figure 4-3. Dashed line shows the linear regression (Dmax-FA = 0.00919[MWFA] – 0.0134; r² = 0.999) derived from Dmax and MW data (×) for FAs.

×

×

×

×

×

× PFCAs (C = 8-18)

FAs (C = 8-18)

1.0 1.5 2.0 2.5 3.0

0 200 400 600 800 1000

MW Dmax(nm)

×

×

×

×

×

× PFCAs (C = 8-18)

FAs (C = 8-18)

1.0 1.5 2.0 2.5 3.0

0 200 400 600 800 1000

MW Dmax(nm)

Chapter 5 General discussion

Bioaccumulation assessment is a key part of chemical regulatory programs.

In the most existing regulatory programs, aqueous exposure based bioconcentration factor (BCF) is used as the bioaccumulation criteria, but some technical challenges and limitations remain when using BCF for assessing chemical substances with low water solubility. The Organization for Economic Co-operation and Development (OECD) is currently considering a revision of Test Guideline 305 to incorporate a dietary exposure test. This test guideline is recommended for chemical substances that cannot be tested using an aqueous exposure method (i.e., a bioconcentration test), including poorly water-soluble chemical substances, highly lipophilic chemical substances, and very adsorptive chemical substances. For these types of chemical substances, dietary testing is considered to be more relevant, because dietary exposure is the most important exposure route in aquatic environments.

In Chapter 2, the author investigated the relationship between BCFs and water solubility for poorly water-soluble chemicals. The difference in BCFs for chemical substances tested with different exposure concentrations was the largest for 2-(2-hydroxy-3,5-di-tert-butyl phenyl)-5-chlorobenzotriazole (CBT). For CBT, the BCF of 5700 was obtained when the test level was 1.0 × 10^-5 mg L^-1, and this was almost 6000 times greater than the BCF of 1.0 determined when the test level was 4.0 × 10^-1mg L^-1. This huge variability was caused by low water solubility of CBT (3.3 × 10^-5 mg L^-1). This indicates that the concentration dependence of the BCF (i.e., the underestimation of the actual BCF due to the reduced bioavailability of chemical substances in water) is closely related to its water solubility, as only freely dissolved chemical substances will accumulate in fish. This also indicated that, using CBT as example, CBT is considered to be a very bioaccumulative (vB) substance (BCF

≥5000) based on various regulatory criteria (e.g., the Chemical Substances Control Law [CSCL] and the Registration, Evaluation, and Authorisation of Chemicals [REACH]), but if tested at aqueous concentrations above it’s water solubility, CBT would not be considered to be a vB substance.

Due to the concentration dependence of the BCF, the CSCL (2003) now recommends performing the bioconcentration test at two different concentration levels which are both below the water solubility of the test substance. In addition, when the BCF is suspected to be concentration-dependent after testing at two concentration levels, a third test should be performed at an even lower concentration to obtain an actual BCF.

The author performed dietary exposure tests for nine poorly water-soluble chemicals (Binox M, 4,4'-methylenebis (2,6-di-tert -butylphenol); PeCB, pentachlorobenzene; NIP, 2,4-dichloro-1-(4-nitrophenoxy) benzene; Solvent Blue 36, 1,4-bis(isopropylamino) anthraquinone; DNPD, N,N'-di-2-naphthyl-p- phenylene- diamine; Musk-xylene, 1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenzene;

TP, o-terphenyl; MXC, methoxychlor) and clarified the linear relationship between BCF and BMF. Five chemical substances (Binox M, PeCB, Solvent Blue 36 Musk-xylene and HCB) have BCFs > 5000, and are classified as very bioaccumulative based on regulatory criteria. Only two of those substances (Binox M and HCB) have lipid-corrected BMFs > 1, indicating they are likely to biomagnify via a food chain. Thus, three (PeCB, Solvent Blue 36 and Musk-xylene) of the nine test substances would not be considered bioaccumulative based on their BMF value but would be very bioaccumulative based on their BCFs. This contradiction in hazard categorization between different the existing bioaccumulation

assessment criteria requires further analysis; additional dataset of BCF and BMF of the chemical substances is required to resolve this issue.

The equation to calculate the 5% lipid normalized BCF (BCFL) from the lipid-corrected biomagnification factor (BMFL) showed that the BMFL for substances with BCFL =5000 ranged from 0.11 to 0.87 (95% confidence interval), indicating that these substances may not biomagnify through successive trophic levels in a food chain. However, substances with BMFL = 1 had BCFL values ranging from 5600 to 30,000, indicating that these substances may bioaccumulate in aquatic organisms via water and dietary exposure, i.e., bioconcentration and biomagnification. A good relationship was found between the BCF and BMF of poorly water-soluble chemicals and the author explored the effectiveness of these bioaccumulation endpoints. In the future, we need to clarify the correlation between BCF and BMF for regulatory bioaccumulation assessment criteria (e.g., CSCL and REACH), and more BCF and BMF datasets for chemical substances are required to support this relationship.

In Chapter 3, common carp (Cyprinus carpio) were exposed to Nitrofen (NIP) by different uptake routes (water or food) to compare bioaccumulation parameters and tissue distribution. The BCF for NIP was 5100, the BMFL was 0.137, and the growth-corrected elimination half-lives were 2.1–3.0 d via aqueous exposure and 2.7–2.9 d via dietary exposure. From either uptake route, the distribution of NIP was highest in the head, followed by the muscle, viscera, dermis, digestive tract and hepatopancreas, which were highly correlated with the tissue lipid content. The author concluded that the uptake route has no influence on the tissue distribution of NIP and that the accumulation potential of different tissues depends on their lipid

content. These results suggest that both BMF and BCF are effective bioaccumulation endpoints for poorly water-soluble chemicals.

The uptake of substances by fish generally occurs by penetration through a biomembrane. These mechanisms are known as diffusional transport (simple diffusion across a lipid bilayer), solvent drag, energy-dependent transport, and cytosis (Hayton et al. 1990, Isaia 1982, Nakai et al. 2005). The uptake of lipophilic chemical substances is mainly controlled by diffusional transport and solvent drag (McKim et al. 1985). There is a strong linear relationship between the BCF and log Kow values up to a log Kow value of approximately 6 for non-polar organic compounds, which are poorly metabolised (EC 2003). The distribution of such non-polar organic compounds in fish tissue may be similar to NIP, but there are only a few experimental data comparing the tissue distribution of other types of chemical substances (e.g., metals and surfactants) taken up by different uptake routes.

Stapleton et al. (2004) showed that after the dietary administration of decabromodiphenylether (deca-BDE), which has a large molecular weight (MW = 959) and is minimally bioavailable (<1%), persistent and bioaccumulative metabolites (penta-BDE and hexa-BDE) are formed. Therefore, the tissue distribution of chemical substances and their metabolites from different uptake routes, even those with relatively large molecular sizes, also need to be clarified.

In Chapter 4, common carp (Cyprinus carpio) were exposed to perfluoroalkyl carboxylic acids (PFCAs; C = 8, 11, 12, 14, 16 and 18) and perfluorooctane sulfonate (PFOS) in bioconcentration tests to compare the BCFs and physicochemical properties of each compound. Despite having the same number of carbon atoms (C = 8), the BCFs of perfluorooctanoic acid (PFOA) and PFOS

differed by more than two orders of magnitude (PFOA, BCF <5.1 to 9.4; PFOS, BCF = 720–1300). The highest BCFs were measured with perfluorododecanoic acid (BCF = 10,000–16,000) and perfluoro- tetradecanoic acid (PFTA, BCF = 16,000–17,000). The longest duration half-lives were observed for perfluorohexadecanoic acid (PFHxDA, 48–54 d) and PFOS (45–52 d). The concentrations of perfluoroalkyl acids (PFAAs) were highest in the viscera, followed by the head, integument and remaining parts of the test fish. The PFAA concentrations in the integument, which was in direct contact with the test substances, were somewhat higher than the concentrations of other lipophilic substances (e.g., HCB). The author suggested that Clog P, which is a model for calculating the log P from chemical structure based on fragmental method (Hansch and Leo 1995) would be a better parameter than log Kow for predicting the BCFs of PFCAs. The molecular size threshold values for the PFCA bioaccumulation potential (MW = 700, maximum diameter = 2 nm) seemed to deviate from the generally reported values because of the specific steric bulk effect of the PFCA molecular sizes.

The Japanese regulatory authority uses molecular weight as the criteria for identifying chemical substances with low bioconcentration potential. In the CSCL (2003), chemical substances with a MW ≥800 are excused from the need for a bioconcentration test, because of their limited ability to penetrate biomembranes, resulting in non-bioaccmulative chemical substances. However, the molecular weight cutoff criteria for the bioaccumulation potential of chemical substances with more than two halogen atoms has been revised to MW ≥1000 following this study (PFTA [MW = 714]: BCF = 16,000–17,000; PFHxDA [MW = 814]: BCF =

4700–4800; and PFODA [MW = 914]: BCF = 320–430).

The author found that the physicochemical properties of PFAAs led to unique bioconcentration potentials in fish. PFAA pollution in the aquatic environment is important, because fish is a primary source of protein for human consumption and is therefore an important pathway for human exposure to PFAAs (Jin et al. 2009, Yeung et al. 2009, Yoo et al. 2009, Zushi and Masunaga 2009a, b, Tsuda et al. 2010). However, there are many other pathways for human exposure to PFAAs, including the atmosphere, drinking water, food, plants and soils (So et al.

2006, Stahl et al. 2009, Lau 2010, Murakami et al. 2011, Xiao et al. 2011). We also need to consider the fate and effects of PFAAs via multiple human exposure routes and complete an integrated risk assessment for PFAAs.

In summary, the author found a highly suggestive relationship between the BCF and BMF values of poorly water-soluble chemicals, and explored the effectiveness of BMF derived from a dietary exposure test as a bioaccumulation endpoint. The author also identified different cutoff criteria (MW ≥1000) for the bioaccumulation potential of chemical substances with more than two halogen atoms.

This study provides laboratory data that are useful for environmental and human risk assessments of chemical substances. I hope that this study will advance knowledge about the relationships between humans, chemical substances and the environment.

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