For the assessment of bioaccumulation, the commonly used test design corresponds to the OECD 305 guideline “Bioconcentration: Flow-through Fish Test” (OECD 1996). During this test, fish are maintained in aquaria or basins while being continuously exposed to an aqueous solution of a test substance. Usually, concentrations are kept constant by using flow-through conditions. The 2 parameters to be experimentally determined during the test are the concentration of test substance in the exposure medium and in fish. The uptake of the test substance derives from fish respiration with a contaminated surrounding medium, and occurs primarily through the gills. The test result, therefore, is defined as a BCF. The application of this test design is appropriate for substances with moderate hydrophobicity (log octanol–water partitioning coefficient [KOW] = 1.5 to 6), and exposure time normally lasts 28 days. It may be prolonged to a maximum of 56 days if the steady state between uptake and elimination can not be reached within the normal time frame. This reflects a compromise between the need to reach a steady state and the biological and technical limits constraining the test system's capability to provide reproducible results.
Highly hydrophobic organic substances
Within the scope of application from log KOW = 1.5 to 6, the OECD 305 fish test has been demonstrated to generate reproducible results. In the interval between log KOW = 3 to 6, a nearly linear correlation of log KOW and log BCF has been found (Neely et al. 1974; Veith et al. 1979). However, when very hydrophobic organic compounds (VHOCs) are tested with log KOW > 6, the reliability of experimentally determined BCF data is questionable. A frequent observation is that the linear correlation between log KOW and measured log BCF no longer applies to VHOCs, with log BCF values either leveling off or declining. This observation is referred to as a hydrophobicity cutoff, and possible reasons for this behavior are still being debated. In this context, it needs to be pointed out that log KOW values reported for some VHOCs are often just as uncertain, differing due to inappropriate measuring methods or erroneous data compilation (Linkov et al. 2005). Apart from uncertainties in a given log KOW, one explanation for the declining log BCF is based on a true mechanistic background. It has been proposed that excessive molecular dimensions of VHOCs cause gradually diminished gill membrane permeation ability (Opperhuizen et al. 1985). Furthermore, a reduced bioavailability of VHOCs due to the very low aqueous solubility and the strong binding propensity to sediment and dissolved organic carbon (DOC) has also been suggested as a basis for the argument for the existence of a hydrophobicity cutoff (Arnot et al. 2006).
In contrast, experimental evidence for a continuing linear correlation up to log KOW = 7 to 8 has also been reported (Kraij et al. 2003; van der Wal et al. 2004). Some authors attribute the observed hydrophobicity cutoff to experimental artifacts, e.g., nonequilibrium conditions and third-phase effects (Jonker and van der Heijden 2007). Nonequilibrium conditions are mainly caused by 2 kinds of effects. On one hand, diminished membrane diffusion constants, determined by increasing molecular dimensions, may retard gill permeation. On the other hand, the very low aqueous solubility and low concentration of VHOCs also cause a deceleration in the diffusion rate (Kelly et al. 2004). In both cases, a reduced uptake rate will result. Therefore, in certain situations, a steady state may not be attained during the exposure period, and the steady-state BCF (BCFSS) will be underestimated. However, decelerated diffusion processes through membranes will affect both uptake and depuration rate in the same way. Consequently, the determination of the kinetic BCF (BCFKIN) will be influenced to a lesser extent compared to BCFSS.
Oversaturation of test solutions may constitute another reason for declining BCF observed beyond a log KOW of 6. Commonly, only the freely dissolved portion of a substance is considered to be bioavailable, whereas the aggregate or bound fraction is not accessible for uptake via gill permeation (Barron 1990; Voutsas et al. 2002). In older test protocols, solubilizers such as acetone and methanol have been used frequently to facilitate the preparation of stock solutions of VHOCs. However, when these stock solutions are diluted, oversaturated aqueous test solutions with higher aggregate structures may result. In this case, only a fraction of the initial amount of test substance is freely dissolved and bioavailable, and accordingly, the BCF is underestimated.
A similar underestimation of BCFSS may occur when liquid–liquid extraction is applied to quantify the amount of test substance in the exposure medium. This method will include both the detection of freely dissolved substance as well as aggregate and DOC-bound substance (Mayer et al. 2000). Therefore, this extraction method is likely to overestimate the concentration of freely dissolved substance, and thus, the BCF is underestimated. Feasible test procedure modifications to avoid these experimental artifacts have been described by Jonker and van der Heijden (2007). Nonequilibrium conditions can be avoided by prolonging the exposure time, provided that the organism's life span allows for such an extension. A further alternative to disregard the nonbioavailable portion is the use of passive sampling devices such as solid-phase microextraction (SPME) and semipermeable membrane devices. These sampling devices differentiate between the freely dissolved amount of a substance and its bound portion. SPME has already been used as an analytical method in a bioaccumulation study on fish (Tolls and van Dijk 2002).
Alternative test designs for highly hydrophobic substances
The use of passive sampling devices such as SPME may improve the reliability of test results and the quality of BCF values for substances with log KOW > 6. However, the test design by itself implies a major drawback. Under environmental conditions, VHOCs show an increasing tendency to bind or adsorb to dissolved and particulate matter such as humic acids, DOC, particulate organic carbon, and sediment (Haitzer et al. 1998; Voutsas et al. 2002; Parkerton et al. 2008). Consequently, aqueous exposure via gill uptake is reduced and no longer accounts for the prevailing exposure pathway. Instead, exposure by ingestion of sediment or food gains in importance. To overcome this shortcoming, 2 alternative test protocols have been suggested. The foremost of these is the OECD 315 test “Bioaccumulation in Sediment-dwelling Benthic Oligochaetes” (OECD 2008), followed by the “Fish Dietary Bioaccumulation Study” (EMBSI 2004).
The bioaccumulation test using sediment-dwelling oligochaetes was established in order to assess the bioaccumulation potential of sediment-associated substances and their impact on benthic ecosystems. Benthic organisms, in particular oligochaetes as the most abundant species, play an important ecological role. They contribute to the bioturbation of sediment and the degradation of dead organic material. In addition, they serve as prey for benthic-feeding fish. By ingesting sediment or burrowing in sediment, these organisms are exposed to sediment-bound substances via multiple uptake routes. This may lead to a possible mobilization of sediment-associated substances and a subsequent entry into food chains. According to the OECD 315 test protocol, oligochaetes are exposed to contaminated sediment for 28 days, and uptake as well as depuration is monitored. The scope of application of this test covers all stable organic substances that partition to sediments, having log KOW values from 3 to 6, as well as VHOCs. Exposure occurs via multiple uptake routes (ingestion of sediment, direct dermal contact with sediment or via porewater), therefore the test endpoint has to be considered as a bioaccumulation factor (BAF) or a biota-to-sediment accumulation factor (BSAF).
In general, the calculation of a BAF is based on the test substance's sediment concentration, but the analytically accessible sediment concentration does not necessarily reflect the bioavailable portion. The bioavailability is strongly affected by the organic carbon fraction fOC of the sediment as well as the history of the spiked sediment. Field-contaminated sediments with a longer aging period often show, in particular, a different behavior compared to laboratory-contaminated sediments. When deriving BSAFs from BAFs, the sediment concentration is already amended for the organic carbon fraction fOC. An additional possibility to estimate the bioavailable amount of test substance in sediment is to measure the porewater concentration by using passive sampling methods. Specifically, SPME has already been used in sediment tests, and the authors even reported the endpoint as a BCF (van der Waal et al. 2004; Jonker and van der Heijden 2007). Regardless of the method used for sediment analysis, an exact description of the analytical procedure and calculation method must be stated to allow for a comparable BAF. According to Annex XIII of the REACH regulation, the ultimate decisive bioaccumulation criterion is the BCF measured using aquatic organisms. In the current REACH guideline R.11 (ECHA 2008a), the BAF for benthic organisms is implemented only as additional indicator to support proposed bioaccumulation. Furthermore, due to the lack of a commonly agreed method to deduce BCFs from BAFs, the value of the OECD 315 test has been limited in regulatory aspects up to now. Therefore, it is suggested that BAF be included as an additional indicator for bioaccumulation when Annex XIII is updated.
The fish dietary study was designed especially to test VHOCs with log KOW > 6. Here, fish are exposed to the test compound via contaminated food for 7 to 14 d, followed by a consecutive depuration phase of 28 d. During the depuration phase, the depletion of the substance in fish is monitored, and the depuration rate constant is calculated analogously to the OECD 305 test. Based on the dietary exposure pathway, the result of the fish-feeding study is referred to as a biomagnification factor (BMF) rather than a BCF. However, unlike the OECD 315 sediment test, an equation to estimate a BCFKIN from depuration kinetics has been proposed (Weisbrod, Woodburn, et al. 2009). In this approach, the depuration rate constant or half-life time, respectively, is experimentally determined, whereas the uptake rate constant is estimated from fish weight, log KOW, and a default value for the concentration of DOC. Although this approach is a rather rough estimate for a BCF, it may facilitate the application of the fish-feeding study in a regulatory context. The major advantage of oral administration via food is the feasibility to apply higher concentrations of VHOC. The troublesome solubilization of VHOCs during the preparation procedure of aqueous test solutions can also be circumvented. Moreover, because contact time of spiked food and water is normally short, and the food is consumed by fish instantaneously, substances also known to be unstable in aqueous solutions can be administered in this manner.
Compared to the OECD 315 sediment test, the fish dietary test provides certain benefits. In principle, the same fish species as recommended in the OECD 305 test can be used. This allows for a comparison of BMF and BCF values for substances within the scope of application of the OECD 305 test (log KOW < 6). However, uptake via ingestion is a major difference relative to uptake by gill permeation. A high concentration of digestive enzymes is present in the gastrointestinal tract of fish, e.g., hydrolases, esterases, and proteases. Hence, certain substances may already be converted prior to traversing the intestinal epithelium. In addition, there are great differences in the constitution of digestive systems (length of intestine, retention time in gut, expressed enzyme profile) among fish species. Therefore, BMFs derived from different species may vary widely. Apart from these differences, exposure via ingestion mirrors an environmentally realistic pathway of substance uptake, including possible mitigating effects, such as enzymatic cleavage in the gastrointestinal tract.