3-4).
NIP and HCB in both diet and fish tissue were measured using the same GC-MS conditions as described for the aqueous exposure test. Ion monitoring for HCB was at m/z = 283. BMF was calculated according to Anonymous (2004a) as follows:
alpha () = (C0,fish × Ktotal)/(I × Cdiet)/(1 – exp(–Ktotal × t)) (2)
BMF = I × /Kelim (3)
BMFlipid = BMF/F (4)
where alpha () is calculated uptake efficiency of test chemical from diet, C0,fish is concentration in fish at the beginning of depuration derived from the regression line intercept, Ktotal is overall elimination rate constants, I is feeding rate, Cdiet is concentrations of test substance in the test diet, t is duration of uptake phase, BMF is biomagnification factor, Kelim is growth-corrected elimination rate constants, BMFlipid
is lipid-corrected BMF and F is lipid corrected factor: fishlipid/dietlipid.
There was no mortality in any exposed or control fish. The overall growth rates constants (during entire study) of fish in the aqueous exposure test were 0.0186 g d–1 (high level), 0.0186 g d–1 (low level) and 0.0188 g d–1 (control). The overall growth rates constants in the dietary exposure test were 0.0278 g d–1 (high level), 0.0291 g d–1 (low level) and 0.0265 g d–1 (control). There were no significant differences between growth rates at either exposure level for both tests (t test, P > 0.05).
The measured concentrations of NIP in the water of the aqueous exposure tests were 4.68 ± 0.32 µg L–1 (high level) and 0.466 ± 0.025 µg L–1 (low level). The BCFk was 5100 (high level) and 5100 (low level), BCFss was 5400 (high level) and 4700 (low level). The 5%-lipid normalised BCFk (BCFk, lipid) was 5200 (high level) and 6100 (low level). The concentration of NIP in fish tissues (µg [g wet wt]–1) from the aqueous exposure test was highest in viscera, followed by head, hepatopancreas, digestive tract and dermis, with the lowest concentration in muscle (Table 3-4).
Concentrations of NIP in the test diet (Cdiet) were 1000 ± 21 µg g–1 (high level) and 104 ± 6 µg g–1 (low level). The concentrations of HCB in the test diet were 96.7 ± 3.2 µg g–1 (high level) and 93.6 ± 2.4 µg g–1 (low level). The BMFlipid was 0.137 (high level) and 0.137 (low level). The concentration of NIP in tissues of fish from the dietary exposure test was highest in viscera, followed by hepatopancreas, head, digestive tract and dermis, with the lowest concentration in muscle (Table 3-4).
Test fish in the dietary exposure test were exposed to a complex diet containing both NIP and HCB, whereas fish in the aqueous exposure test were only exposed to NIP. The use of a reference substance provides a check on the assimilation efficiency resulting from the method used to spike the diet, but the test substance and reference substance should not interact with each another (Anonymous 2004b). In this
test, the author observed high assimilation efficiencies (α) for HCB (0.717–0.733), which indicates a sufficient bioavailability of the spiked diet. HCB is generally not metabolized by fish and so should not affect the metabolic capacity of the fish. In fact, BMF parameters for HCB did not change under the different dosing concentrations of NIP in this test. Therefore the author concludes that the use of HCB did not affect the accumulation potential or the comparison of NIP distribution in tissue in this study.
Growth-corrected elimination half lives (t1/2) were similar values between both exposure tests; 2.1–3.0 d via aqueous exposure and 2.7–2.9 d via dietary exposure.
To compare the distributions of NIP and lipid content in tissues, the author measured both the lipid and NIP content in the same group of fish. The comparison of percentage distributions on a whole-body basis is shown in Figure 3-6. Despite the difference in uptake routes, the percentage of whole-body NIP found in muscle was almost the same for the two routes—27% via aqueous exposure and 26% via dietary exposure.
Furthermore, the distribution patterns of NIP and the lipid content of tissues on a percentage basis were similar—highest in the head, followed by muscle, viscera, dermis, digestive tract and hepatopancreas. There was no specific accumulation in the dermis or digestive tract, which were in direct contact with the NIP. The author concludes that there is a strong relationship between tissue distribution of NIP and lipid content, independent of the uptake route.
4. Conclusion
In this study, the author demonstrate that the comparison of NIP uptake via water and food and its distribution in Carp. The BCF of NIP was 5100, and the
lipid-corrected biomagnification factor was 0.137. Growth-corrected elimination half lives were similar; 2.1–3.0 d (aqueous exposure) and 2.7–2.9 d (dietary exposure).
From either uptake route, the accumulation potential of NIP in fish tissues depends on the lipid content. These results provide laboratory data that are useful for environmental and human risk assessments of chemical substances.
Table 3-1 Chemical structures of test and reference substances for the study Nitrofen (NIP; 2,4-dichloro-1-(4-nitrophenoxy) benzene)
CAS number 1836-75-5
Hexachlorobenzene (HCB) CAS number 118-74-1
Cl
Cl Cl
Cl
Cl Cl
O Cl
Cl
NO2
Table 3-2 Parameters used for calculation of bioconcentration factors (BCFs) for Nitrofen (NIP) under high- and low-exposure conditions
NIP exposure level Parameter
High Low fishlipid (%)
Kuptake (d–1)
4.97 1800
4.21 1270 Ktotal (d–1)
Kgrowth (d–1) Kelim (d–1) t1/2 (d) BCFk BCFss
BCFklipid
0.351 0.0186 0.332 2.1 5100 5400 5200
0.248 0.0186 0.230 3.0 5100 4700 6100
fishlipid; fish lipid content based on wet weight, Kuptake; uptake rate constants, Ktotal; overall elimination rate constants, Kgrowth; overall growth rate constants for fish, Kelim; growth-corrected elimination rate constants, t1/2; growth-corrected elimination half-life, BCFk; kinetic bioconcentration factor, BCFss; BCF at steady state, BCFk lipid; 5%
lipid-normalised BCF.
Table 3-3 Parameters used for calculation of biomagnification factors (BMFs) for Nitrofen (NIP) and hexachlorobenzene (HCB) under high- and low-exposure conditions
NIP HCB Parameter
High Low High Low
dietlipid (%) fishlipid (%) C0,fish (µg g–1) Ktotal (d–1) Kgrowth (d–1) Kelim (d–1) t1/2 (d) alpha () BMF
Lipid factor (F) BMFlipid
15.5 5.34 40.1
0.286 0.0278 0.258 2.7 0.406 0.0471 0.344 0.137
15.4 5.34 4.11 0.269 0.0291 0.240 2.9 0.380 0.0476 0.347 0.137
15.5 5.34 13.8
0.0930 0.0278 0.0652 10.6
0.733 0.337 0.344 0.982
15.4 5.34 13.6
0.0846 0.0291 0.0555 12.5
0.717 0.388 0.347 1.12
dietlipid; diet lipid content based on wet weight, fishlipid; fish lipid content based on wet weight, C0,fish; concentration in fish at the beginning of depuration derived from the regression line intercept, Ktotal; overall elimination rate constants, Kgrowth; overall growth rate constants for fish, Kelim; growth-corrected elimination rate constants, t1/2; growth-corrected elimination half-life, alpha (); calculated uptake efficiency of test chemical from diet, BMF; biomagnification factor, Lipid factor (F); lipid corrected factor: fishlipid/dietlipid, BMFlipid; lipid- corrected BMF.
Table 3-4 Tissue concentrations and quantity of Nitrofen (NIP) and hexachlorobenzene (HCB)
a Value in parentheses shows percentage distribution of test substances on a whole-body basis.
Aqueous exposure test Dietary exposure test
NIP NIP HCB
Part of fish
Wet weight
(g)
Concentration (µg g–1)
Quantity a (µg)
Wet weight
(g) Concentration (µg g–1)
Quantity a (µg)
Concentration (µg g–1)
Quantity a (µg) Dermis
Head
Digestive tract Hepatopancreas Viscera
Muscle
6.31 16.7
2.39 0.881 2.34 25.1
1.59 3.74 2.03 2.99 7.83 1.45
10.0 ( 7) 62.5 (46) 4.85 ( 4) 2.63 ( 2) 18.3 (14) 36.4 (27)
2.36 6.14 1.05 0.362 0.780 8.85
3.44 5.80 3.70 5.91 17.8
2.55
8.12 ( 9) 35.6 (41) 3.89 ( 5) 2.14 ( 2) 13.9 (16) 22.6 (26)
9.11 13.1 10.0 22.0 53.4 7.08
21.5 (10) 80.4 (36) 10.5 ( 5) 7.96 ( 4) 41.7 (19) 62.7 (28) Total 53.7 2.51 135 (100) 19.5 4.41 86.2(100) 11.5 225 (100)
Conditions of column chromatograph Sep-Pak Plus C18
Conditionings Acetonitrile approx. 10mL
Ultra pure water approx. 10 mL Loading Whole volume of the solution was loaded.
Elution Eluent Acetonitrile 6 mL
Figure 3-1 Pretreatment for analysis of Nitrofen (NIP) in the test water (aqueous exposure).
Sample for GC-MS analysis Test water
20 mL (high level), 200 mL (low level)
Eluate
←Water for recovery test 180 mL (graduated cylinder) (only high level)
・Column chromatography
・Evaporation to dryness
(rotary evaporator, about 40 ºC, nitrogen purge)
←n-Hexane 1 mL (measuring pipette)
・Ultrasonic irradiation (approx. 30 sec.)
・Filling up to 2 mL (n-hexane, volumetric flask)
Figure 3-2 Pretreatment for analysis of Nitrofen (NIP) in the test fish (aqueous exposure).
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 3-5 g (analytical balance)
←Acetonitrile 15 mL (graduated cylinder)
・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)
・Evaporation to dryness
(rotary evaporator, approx 40 ºC, nitrogen purge)
←n-Hexane 5 mL (measuring pipette)
・Ultrasonic irradiation (approx. 30 sec.)
・Filling up to 10 mL (n-hexane, volumetric flask) Sample for GC-MS analysis
・Taking out 1 - 5 g (analytical balance) Sample for storage
Figure 3-3 Pretreatment for analysis of Nitrofen (NIP) and hexachlorobenzene (HCB) in the test diet (dietary exposure).
Test diet
Supernatant Residue
・Taking out 1 g (analytical balance)
←n-Hexane 15 mL (graduated cylinder)
・Homogenization (Polytron, approx. 1 min.)
・Washing (n-hexane 5 mL)
・Centrifugation (7000 g, 5 min.)
・Filtration (absorbent cotton)
・Filling up to 25 mL (n-hexane, volumetric flask)
・Taking out 1 mL (transfer pipette)
・Filling up to 10 mL (n-hexane, volumetric flask) Sample for GC-MS analysis
Figure 3-4 Pretreatment for analysis of Nitrofen (NIP) and hexachlorobenzene (HCB) in the test fish (dietary exposure).
・Taking out 1 - 5 g (analytical balance)
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)
←Acetone 15 mL (graduated cylinder)
・Homogenization (Polytron, approx. 1 min.)
・Washing (acetone 5 mL)
・Centrifugation (7000 g, 5 min.)
・Filtration (absorbent cotton)
・Filling up to 50 mL (acetone, volumetric flask) Sample for storage
Sample for GC-MS analysis (HCB)
Sample for GC-MS analysis (NIP)
・Taking out 1 mL (transfer pipette)
・Filling up to 30 mL
(acetone, volumetric flask)
Figure 3-5 Concentration of Nitrofen (NIP) and hexachlorobenzene (HCB) concentration in test fish during test period.
Blue (NIP) and red (HCB) diamond shows measured concentration in test fish.
Solid line shows the calculated concentration in test fish using following equation.
Cfish = Cdiet × I×α × (1-e-ktotal × t) / ktotal 0 < t <10 Cfish = Cdiet × I × α × (e-ktotal × (t-10) - e-ktotal × t) / ktotal t >10
Uptake phase Depuration phase
0 10 20
0 5 10 15 20 25 30
Test period (d) Concentration in test fish (µg g-1) a
0 10 20
0 5 10 15 20 25 30
Test period (d) Concentration in test fish (µg g-1) a
Figure 3-6 Distribution of lipids and test substances in fish tissues on a whole-body basis.
Value in column shows percentage distribution of test substances on a whole-body basis.
10 9 7
13
36 41
46 34
5 5
4 5
4 2
2 3
19 16
14 14
28 26 27 31
0% 25% 50% 75% 100%
Dietary exposure (HCB) Dietary exposure
(NIP) Aqueous exposure
(NIP) Lipid content
Dermis Head Digestive tract Hepatopancreas Viscera Muscle
Chapter 4
Evaluation of bioconcentration for perfluoroalkyl acids based on their physicochemical properties using common carp (Cyprinus carpio)
1. Introduction
Perfluoroalkyl acids (PFAAs) are a family of anionic fluorinated surfactants that consist of a carbon chain, typically 4–14 carbon atoms in length, and a charged functional moiety (primarily perfluoroalkyl phosphonic acid [PFPA], perfluoroalkyl carboxylic acid [PFCA] and perfluoroalkyl sulfonic acid [PFSA]) (Lau et al. 2007) (Table 4-1). Despite a strict regulation for bioaccumulative substances, there is enormous numbers of reports that PFAAs, especially perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) have been detected in environmental media, wildlife, and human tissues in many different geographic locations throughout the world (Kannan 2011).
The development of quantitative structure–activity relationships (QSARs) has allowed the prediction of bioconcentration factors (BCFs) from the physicochemical properties of chemical substances, but these relationships are not well studied for PFAAs and few data are available for the physicochemical properties of PFAAs (Lei et al. 2004, Rayne and Forest 2009, Kannan 2011). Therefore, in Chapter 4, the author clarifies BCFs of PFAAs using common carp (Cyprinus carpio) and discusses the bioaccumulation potential of PFAAs based on their unique physicochemical properties.
2. Materials and Methods Test substances
Perfulorooctanoic acid (PFOA, 98%) was purchased from Daikin Industries, Ltd. (Osaka, Japan). Perfluoroundecanoic acid (PFUnA, 99.0%), perfluoro- tetradecanoic acid (PFTA, 97.3%) and perfluorooctadecanoic acid (PFODA, 98.7%)
were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Perfluorododecanoic acid (PFDoA, 98.5%) was purchased from Wako Chemical, Ltd.
(Kanagawa, Japan). Perfluorohexadecanoic acid (PFHxDA, 100.67%) was purchased from Lancaster Synthesis Ltd. (Lancashire, UK). Perfluorooctane sulfonic acid (PFOS [potassium salt], 100.3%) was purchased from Kishida Chemical Co., Ltd. (Osaka, Japan).
3. Measurement of bioconcentration factors