Statistical Post-Processing Method for Evaluating Bioaccumulation in Fish Due to Dietary Exposure in Japan.

In 2018, the dietary exposure bioaccumulation fish test of the Organization for Economic Co-operation and Development Test Guideline No. 305 was introduced into Japan's Chemical Substances Control Law. The Japanese government has adopted a single definitive testing criterion for the absence of high bioaccumulation: the growth-corrected kinetic dietary magnification factor (BMFKg) must be less than 0.007. The aim of this study was to decrease regulatory restrictions in order to increase newly developed chemical substances and their subsequent approval of their manufacture and import, i.e., the present study was motivated by concerns over the criterion being too restrictive, rather than scientific concerns, such as uncertainty in criterion. We used statistical post-processing to assess the possibility of expanding the criteria for not being highly bioaccumulative. Based on our results, we proposed the criterion that the test substance should be considered not highly bioaccumulative if the following two conditions are met: (1) The ratio of the maximum to the minimum measured 5% lipid-standardized biomagnification factor at the end of the uptake phase (BMF5%, n = 5) for the test substance and reference substance should be less than 3.0, and (2) For the measured BMF5% of the test substance (n = 5), the probability that the next (the sixth) BMF5% is below 0.0334 should exceed 95% based on statistical post-processing. It is worth noting that the BMF5% values should only be applied for non-ionizable lipid soluble compounds. Application of our suggested approach to Japan implies that the criterion for chemical substances that are not highly bioaccumulative in the dietary exposure bioaccumulation fish test would be increased from 0.007 to 0.0149.

Comparison of laboratory-derived biomagnification factors for hexachlorobenzene in common carp conducted under 9 test conditions.

Nine dietary exposure bioaccumulation fish tests with hexachlorobenzene (HCB) were conducted with common carp to explore how differences in test conditions (different test foods and feeding rates) influenced the lipid-corrected, growth-corrected kinetic biomagnification factor (BMFkgL ) value (BMFkgL = BMFkg × lipid content of test food/lipid content of test fish). The BMFkgL values for HCB differed by approximately a factor of 5 among the tests. The average, median, 95% confidence interval, and coefficient of variation of the BMFkgL values were 0.925, 0.998, 0.578 to 1.27, and 49%, respectively. The BMFkgL value differed markedly between tests conducted using test food with lipid contents of approximately 5 and 15%. Different feeding rates (2 or 3% of body weight/d) had comparatively little effect on the BMFkgL of HCB. The present study revealed that the lipid content of test fish was correlated with the growth-corrected kinetic BMF (BMFkg ) value of HCB but the lipid content of test food was poorly correlated with BMFkg . This lack of correlation might explain the large variations of the BMFkgL values observed. The value of the BMFkg normalized to a fish with a 5% lipid content (defined as the 5% lipid-normalized BMFkg in the present study) did not differ markedly between tests conducted using test food with different lipid contents (5-15%). It is therefore useful to report the 5% lipid-normalized BMFkg as well as the BMFkgL when dietary exposure tests are conducted. Environ Toxicol Chem 2018;37:1032-1039. © 2017 SETAC.

Prediction of the bioconcentration factor in common carp (Cyprinus carpio L.) using data from the dietary exposure bioaccumulation fish test.

An approach to predicting the bioconcentration factor (BCFpre ) from the predicted uptake rate constant (k1 pre ) and the depuration rate constant measured in the dietary exposure bioaccumulation fish test (k2 dietary ) [BCFpre = k1 pre /k2 dietary ] is proposed in test guideline 305 of the Organization for Economic Cooperation and Development Guidelines for Testing of Chemicals. Data were collected on the BCFs of 197 test chemicals from Japan's Chemical Substances Control Law database. To demonstrate how the BCFpre compares with experimentally derived BCF under optimum conditions, 48 of 197 test chemicals, including a number of studies that could be considered problematic, were excluded from the analysis. The k1 pre was calculated by using 22 published prediction methods: the correlations between experimental uptake rate constants (k1 aqueous ) and k1 pre for all prediction methods were very low and were statistically nonsignificant (p > 0.05). Three prediction methods were also selected that gave relatively good values for the geometric mean of k1 pre /k1 aqueous and calculated values of BCFpre for 12 test chemicals. Linear relationships (p < 0.05) are presented between logarithm of experimental and predicted BCF. The correlation coefficients of growth-corrected experimental and predicted BCF tended to be higher than values that were not growth corrected. For some test chemicals, use of predicted BCF led to a bioaccumulation classification different from that of existing regulatory criteria.

Uptake and biological effects of synthetic glucocorticoids in common carp (Cyprinus carpio).

Uptake and biological effects of synthetic glucocorticoids (GCs) were analyzed using common carp (Cyprinus carpio). Fish were exposed to clobetasol propionate (CP) or clobetasone butyrate (CB) individually or in mixture at 1 µg L(-1) for 21 days. Bioconcentration factor (BCF) of CB was calculated as 100, and BCF of CP was less than 16. No effects were found in fish erythrocyte and leukocyte numbers and serum glucose levels after exposure to the selected GCs. On the other hand, serum concentrations of free amino acids significantly increased in GC-exposed groups. Thus, exposures to synthetic GCs at relatively low concentrations seemed to cause enhancement of protein degradation and subsequent increase of serum free amino acids without a corresponding increase in serum glucose levels, an effect which might be related to partial induction of gluconeogenesis by GC.

Resampling the bioconcentration factors data from Japan's chemical substances control law database to simulate and evaluate the bioconcentration factors derived from minimized aqueous exposure tests.

Existing standard bioconcentration tests (e.g., the Organization for Economic Cooperation and Development [OECD] test guideline 305) require large numbers of test animals and resources. The minimized aqueous exposure test is a new approach based on the standard bioconcentration test but allows estimation of bioconcentration factor (BCF) by minimized sampling of the test fish. The authors collected BCF data (298 curves from 155 chemicals, using common carp as test species) from Japan's Chemical Substances Control Law database and resampled the data to simulate the calculation of BCF that would be obtained if studies had been designed to obtain kinetic BCF derived from minimized aqueous exposure tests (BCF(km)). The correlation was high (r(2) = 0.967) between BCF derived from standard bioconcentration tests (BCF(full)) and BCF(km). The average value of the BCF(full) to BCF(km) ratio (BCF(full):BCF(km)) was 1.04 and ranged from 0.54 to 1.93, the 5th and 95th percentiles being 0.74 and 1.45, respectively. The results based on the 5th and 95th percentiles of the BCF(full):BCF(km) ratio suggest that BCF(full) 2,000 corresponds to BCF(km) 1,400 to 2,700, whereas BCF(full) 5,000 corresponds to BCF(km) 3,400 to 6,800. The authors also emphasize that the standard bioconcentration test should be performed when the resulting BCF(km) is in the region of regulatory concern.

Comparison of bioconcentration and biomagnification factors for poorly water-soluble chemicals using common carp (Cyprinus carpio L.).

Existing regulatory criteria for bioaccumulation assessment of chemicals are mainly based on a bioconcentration factors (BCF) not a biomagnification factors (BMF). We performed dietary exposure tests for nine poorly water-soluble chemicals and developed a linear regression between the 5 % lipid normalized BCF (BCF(L)) and the lipid-corrected BMF (BMF(L)). The BMF(L) of substances with BCF(L) = 5,000 was 0.31 (95 % CI 0.11-0.87), whereas the BCF(L) of substances with BMF(L) = 1 was 13,000 (95 % CI 5,600-30,000). Five substances can be considered very bioaccumulative (vB) according to the BCF end point (BCF > 5,000), but only two substances were recognized to biomagnify according to the BMF end point (BMF ≥ 1). Although our results are highly suggestive of a relationship between BCF and BMF, additional BMF and trophic magnification factor data for chemicals are required to support this relationship, and new techniques (e.g., fugacity approach) may help in resolving the apparent contradiction in hazard categorization.

Enhanced response of T lymphocytes from Pgap3 knockout mouse: Insight into roles of fatty acid remodeling of GPI anchored proteins.

Glycosylphosphatidylinositol (GPI) is a complex glycolipid that serves as a membrane anchor for many cell-surface proteins, such as Thy-1 and CD48. GPI-anchored proteins (GPI-APs) play important roles in many biological processes, such as signal transduction and cell-cell interaction, through their association with lipid rafts. Fatty acid remodeling of GPI-APs in the Golgi apparatus is required for their efficient association with lipid rafts, i.e., the unsaturated fatty acid at the sn-2 position of the PI moiety is exchanged for the saturated fatty acid by PGAP2 and PGAP3. To investigate the immunological role of the fatty acid remodeling of GPI-APs, we generated a Pgap3 knockout mouse. In this mouse, GPI-APs are expressed on the cell surface without fatty acid remodeling, and fail to associate with lipid rafts. Male Pgap3 knockout mice were born alive at a ratio lower than expected from Mendel's law, whereas the number of female mice followed Mendel's law. All mice exhibited growth retardation and abnormal reflexes such as limb grasping. We focused T cell function in these mice and found that T cell development in the absence of Pgap3 was normal. However, the response of T cells was enhanced in Pgap3 knockout mice in both in vitro and in vivo studies, including alloreactive response, antigen-specific immune response, and experimental autoimmune encephalomyelitis. Cross-linking of Thy-1 in wild-type cells inhibited the signal transduced by the T cell receptor (TCR), whereas cross-linking of Thy-1 in Pgap3 knockout cells enhanced the TCR signal. These results suggest that GPI-APs localized in lipid rafts may modulate signaling through the TCR.

Unique physicochemical properties of perfluorinated compounds and their bioconcentration in common carp Cyprinus carpio L.

Carp (Cyprinus carpio L.) was exposed to perfluorinated compounds (PFCs)-perfluoroalkyl carboxylic acids (number of carbon atoms, C = 8, 11, 12, 14, 16, and 18) and perfluorooctane sulfonate (PFOS)-in bioconcentration tests to compare the bioconcentration factors (BCFs) and physicochemical properties of each specific compound. Despite having the same number of carbon atoms (C = 8), the BCFs of perfulorooctanoic acid (PFOA) and PFOS differed by more than two orders of magnitude (PFOA BCF = < 5.1 to 9.4; PFOS BCF = 720 to 1300). The highest BCFs were obtained from perfluorododecanoic acid (BCF = 10,000 to 16,000) and perfluorotetradecanoic acid (BCF = 16,000 to 17,000). The longest observed depuration half-lives were for perfluorohexadecanoic acid (48 to 54 days) and PFOS (45 to 52 days). The concentrations of PFCs were highest in the viscera, followed by the head, integument, and remaining parts of the test fish. PFCs concentrations in the integument, which was in direct contact with the test substances, were relatively greater than that of other lipophilic substance (hexachlorobenzene). It is likely that Clog P would be a better parameter than log K (ow) for the prediction of BCFs for PFCs. Threshold values for PFCs bioaccumulation potential (molecular weight = 700, maximum diameter = 2 nm) seemed to deviate from those generally reported because of the specific steric bulk effect of molecule size.

Effects of 4-tert-pentylphenol on the gene expression of P450 11beta-hydroxylase in the gonad of medaka (Oryzias latipes).

Alkylphenols including 4-tert-pentylphenol (4-PP) have been shown to alter sexual differentiation in fish due to their estrogenic properties. Medaka (Oryzias latipes) is so sensitive to these substances because morphological sex reversal and testis-ova induction are well developed in the exposed males. However, little work has been done to characterize the molecular effects of estrogenic substances on the morphological and gonadal feminization in male fish. Cytochrome P450 11beta-hydroxylase (P450(11beta)) is a key steroidogenic enzyme in production of 11-ketotestosterone which is the predominant androgen in male fish. In this study, we cloned a cDNA encoding medaka testicular P450(11beta), and then investigated the gene expression of P450(11beta) in the testes of genetically male medaka exposed to 4-PP. The cDNA contains 1740 nucleotides that encode a protein of 543 amino acids, which shares 68.9% and 73.4% homology with testicular P450(11beta)s from Japanese eel (Anguilla japonica) and rainbow trout (Oncorhynchus mykiss), respectively. HeLa cells transfected with an expression vector containing the open reading frame of medaka P450(11beta) cDNA showed 11beta-hydroxylating activity in the presence of exogenous testosterone. Analysis of tissue distribution by RT-PCR showed great abundance of P450(11beta) mRNA in testis. In the partial life-cycle exposure with 4-PP, morphologically sex-reversal was observed in XY medaka exposed to 4-PP concentrations of > or =238 microg/L. Furthermore, exposure to 4-PP completely inhibited P450(11beta) mRNA expression in the gonads of sex-reversed XY fish at 60-day posthatch. These results suggest that xeno-estrogen 4-PP may have inhibitory effects on the synthesis of testicular 11-oxygenated androgens through downregulation of P450(11beta) expression in the genetically male fish.