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Bioaccumulation is broadly defined as a process by which the concentration of a chemical in an organism exceeds that in the respiratory medium (e.g. water for fish, air for mammals), in the diet, or both (Gobas et al, 2009). Highly bioaccumulative chemicals are perceived as problematic because harmful (toxic) concentrations may be achieved in organisms, including humans, at the top end of food chains, even though the ‘source’ (e.g. water) concentrations are not directly toxic. Although the various regulatory schemes attempt to set both quantitative and qualitative criteria to define the level of bioaccumulation deemed hazardous, interpretation of these criteria in relation to the different types of scientific data that are, or are not, available for a particular chemical is often difficult. In response to this difficulty, Gobas et al (2009), as part of SETAC Pellston Workshop in 2008 considering all factors relevant to the identification of PBTs and POPs, reviewed the state of the science and proposed a framework for the evaluation of bioaccumulation in relation to the regulatory situation.


In the case of medium-chain chlorinated paraffin (MCCP), alkanes, C14-17, chloro, there are a variety of data available to assess the bioaccumulation potential, including:

·       Octanol-water partition coefficient (KOW), both estimated and measured;

·       Bioconcentration factor (BCF) as measured in laboratory tests;

·       Bioaccumulation factor (BAF) as measured in laboratory tests and field studies;

·       Biota-Sediment Accumulation Factor (BSAF) as measured in laboratory tests;

·       Biomagnification factor (BMF) as measured in laboratory tests and field studies; and

·       Trophic magnification factor (TMF) as measured in field studies.


By definition KOW, BCF, BAF, BSAF, BMF and TMF are steady-state metrics, i.e., there are no significant changes in chemical concentrations over time. KOWis used as a surrogate for lipid-water equilibrium partitioning and has recognized limitations for B assessment, primarily because it is only a chemical property and ignores biological processes such as biomagnification and biotransformation (Arnot 2008, Nichols 2007, Arnot 2013). The BCF is the ratio of the chemical concentration in a fish to the chemical concentration in the water following chemical exposure from the water only. The BCF is measured under controlled laboratory conditions; there is no dietary exposure. The BAF is the ratio of the chemical concentration in a fish to the chemical concentration in the water as a result of all routes of exposure (i.e. water and food). The BMF is the ratio of the chemical concentration in an organism to the chemical concentration in its diet. The BMF can be determined through laboratory (model) testingorfield measurements. Field BMFs include all routes of exposure, whereas laboratory BMFs only include dietary exposures under controlled conditions in which there is no exposure to chemical in the water. The TMF is the average factor by which the chemical concentration in biota of a food web increases per trophic level and is determined from environmental monitoring data, i.e., organisms are exposed to chemical from the environment and diet. Obtaining accurate BCFs and BAFs for very hydrophobic chemicals like MCCP constituents is challenging because of technical difficulties and a general lack of extensive scientific knowledge on the actual dissolved (bioavailable) chemical concentration in the water for such hydrophobic (“water-hating”) chemicals. Rationales for lipid correction (normalizing) for neutral organic chemicals and growth correction of the measured data are provided elsewhere (Gobas 2009, Burkhard 2012, Arnot 2013, Connolly 1988, Mackay 1982, OECD. 2012).


Growth Correction of Bioaccumulation Results


The issue of growth correction for BCF values has been raised by reviewers in the context of the bioaccumulation assessment of MCCP given the potential concern for growth dilution in laboratory studies. Historically growth correction has not been applied in B assessments (i.e., pre-2012) and it is unclear how these corrections are to be evaluated against historical BCF standards which themselves are based on non-growth corrected data. Laboratory bioaccumulation studies are typically started with young fish so limiting growth is almost an impossibility. In the interest of being responsive and transparent on this issue, growth corrected values have been provided on key studies where the data permit (e.g. Thompson 2000 and Hurd and Vaughan 2010), though caution should be applied in their interpretation. This is perhaps yet another reason to view the broad array of data available to assess the bioaccumulation potential of MCCP as opposed to any one study or specific type of data.


Octanol-Water Partition Coefficient Data


Measurements and predictions for the octanol-water partition coefficient (KOW) for MCCP constituents span a few orders of magnitude (log KOWs from ~5.5 to >9.0). These very high KOWs reflect the fact that MCCP constituents are very hydrophobic (“water-hating”) and hence readily partition from water to other phases.


Bioconcentration Factor Data


There are 2 existing GLP guideline laboratory bioaccumulation studies on MCCP related test materials in fish (Thompson 2000 and Hurd and Vaughan 2010) that reported bioconcentration factor (BCF) results. Only Thompson (2000) was conducted on a full range MCCP test material at approximately 51% Cl by weight and this study also employed a radiolabelled pentadecane (C15) at approximately 51% Cl by weight. Several published dietary fish studies are also available on chlorinated C14 and C16 test materials (Fisk 1996, 1999, 2000) and one in earthworms using a C16 chlorinated test material (Fisk 1998). An additional earthworm toxicity study (Thompson 2001) on a full-range MCCP product at 52% chlorination by weight also provided some estimates of bioaccumulation, though these results have limitations including high test material concentrations. A new dietary bioaccumulation study in fish according to the OECD guideline 305 on a chlorinated tetradecane (C14) at 50% chlorine by weight is in progress and should be completed in 2018. This study will be the first to include analysis of individual chloroalkane congener groups. 


Thompson (2000) is a GLP OECD guideline 305 study in rainbow trout (Oncorhynchus mykiss) using full range MCCP and a radiolabelled n-pentadecane-8-14C (both at 51% chlorine by weight). The bioconcentration factor (BCF) based on a kinetic estimate was 1087 and 349 at a nominal exposure concentration of 1 and 5 µg/L, respectively, at day 35. The kinetic estimates of time for tissue concentrations to reach 95% steady state levels were 67 and 74 days at 1 and 5 µg/L nominal, respectively, suggesting that steady-state conditions were not achieved. The uptake rate constants (k1) were 48.7 and 14.2 /d and the depuration half-lives were 15 and 17 daysfor the low and high exposure treatments, respectively. Total elimination rate constants (“k2”) were 0.0448 and 0.0407 /d for the low and high exposure treatments, respectively. A growth rate constant (“kG”) of 0.021 /d can be calculated from the test data.As the analytical method used did not distinguish between parent compound and metabolites, these values represent the realistic upper limit for the true BCF of the substance and its metabolites. The fish BCF value of 1087 from this study is considered the most reliable. This value is supported by other experimental studies which derived fish BCF values of about 600 for MCCPs tested at around the water solubility, and is also in line with an alternative predictive method (SRC BCF WIN program) which derived a BCF of 1549, as described in the RAR (EU, 2005).There are key technical issues related to data quality, uncertainty and interpretation with study including a lack of correction of mass balance for parent test chemical (i.e., total radioactivity for both parent and possible metabolites was quantified) the position of the radiolabel on the test substance is not reported, and, as previously mentioned, steady-state conditions were not approached during the test (i.e. time to reach ~95% of steady-state = 4xt1/2~60 d). Finally, growth corrected BCF values, not reported in the original study, of 2164 and 686 were determined for the low and high test concentrations, respectively. 


Hurd and Vaughan (2010) is a GLP study conducted according to OECD guideline 305 in rainbow trout using a 14C-labelled n-tetradecane (45% chlorinated) at a single exposure concentration of 0.5 µg/L (nominal) in a flow-through system. The study had 35-day accumulation phase and a 42-day depuration phase. During the accumulation phase, tissue levels of test material, measured by radiochemical analysis, increased to 2265 µg/kg bw (at day 35) at a mean measured exposure concentrations of 0.34 µg/L. During the depuration phase, tissue levels declined to 438 µg/kg bw. The whole body BCF at the end of the exposure phase was 6660 (3230 normalised to 5% lipid content; mean lipid content of the fish was 10.3%). Based on the whole body concentration of test substance equivalents at day 35, the extent of depuration of test substance after 42 days was 81%. Using a kinetic approach, the uptake rate constant (k1) was determined to be 395 /day and the depuration rate constant (k2) 0.0432 /day, giving a kinetic BCF of 9140 (4440 normalised to 5% lipid content). The growth corrected BCF values, not reported in the original study, were determined to be 29924 (fish lipid content =10.3%) and 14526, when normalized for 5% lipid content. The kinetic data also allowed estimates to be made for the depuration half-life (16 days) and the time to 95% of steady state (69 days). Since all these calculations are based on radioactivity measurements, and therefore do not distinguish between parent compound and possible metabolites, these values represent the worst-case values. In conclusion, the whole body BCF for chlorinated n-tetradecane (45% chlorinated) in rainbow trout (Oncorhynchus mykiss) exposed to a nominal concentration of 0.5 µg/l for 35 days was 6660 and the kinetic BCF, based on the calculated uptake and depuration rate constants, was 9140. Normalised to 5% lipid content, these values would be 3230 and 4440, respectively. The BCF of 6660 appears to be unusually high compared with the other BCF values obtained to date, and the method employed may overestimate the BCF with decreasing chlorination content. Samples from this study were subjected to further analysis to determine the proportions of radioactivity present as parent compound or as polar or bound metabolites (Leonards and van Beuzekom, 2010). Only samples of fish at the end of the depuration phase were available. The results showed that no significant extractable metabolites of chlorinated tetradecane were present, but non-extractable tissue metabolites were found, which accounted on average for 21% of the total 14C activity in the fish. It is considered likely that the proportion of metabolites present at the end of the uptake phase would have been different, and potentially higher, than those measured at the end of the depuration phase. 


Thompson et al. (2001c) determined the uptake of a radiolabelled C15 chlorinated paraffin (n-pentadecane-8-14C; 51% chlorinated) by earthworms (Eisenia fetida) from soil as part of an earthworm toxicity study. The concentration of MCCPs in adult worms at day 28 was 169, 802 and 732 mg/kg wet wt at measured soil concentrations of 70, 800 and 8200 mg/kg wet wt respectively, resulting in BSAF values of 2.4, 1.0 and 0.089 respectively. For juvenile worms at day 56, the mean tissue concentrations were 140 and 1011 mg/kg wet wt from soil concentrations over 28-56 days of 61 and 748 mg/kg wet wt, resulting in BSAF values of 2.3 and 1.4 (no juvenile worms were produced from adults exposed to the highest soil concentration). In an expert evaluation of the study data (EU, 2005), it was noted that the maximum BCF value in this study of 2.4 was derived from soil containing 4.7% organic carbon. Using a default value of 2% for organic carbon content the maximum BSAF value for MCCPs in earthworms would be estimated to be 5.6. The potential for uptake by worms from soil and sediment appears to reduce with increasing chlorine content of the MCCP. There are some limitations to the use of this study for bioaccumulation testing in the fact that the primary purpose of this study was to evaluate the toxicity of the test material to the earthworm, not bioaccumulation, and the concentrations are much higher than what would have likely been used in a separate B study. Further, it is not recommended to calculate B metrics using total radioactivity, as was done in this study, because it quantifies the parent and metabolite concentration in the tissues.


Bioaccumulation Factor, Biomagnification Factor and Trophic Magnification Factor Data


There are a range of BAF, BMF and TMF data available on MCCP. These data have been extensively assessed in the reviews by Thompson and Vaughan (2014), published, and Arnot (2014), unpublished (attached in Section 13 of dossier). Both expert assessments concluded that vast majority of these BAF, BMF and TMF data show that MCCP is not bioaccumulating in the food chain, especially when considering samples from the natural environment.


Houde et al. (2008) reported lipid-normalised log BAFs results in the range of 6.5 to 7.3 for MCCP as analyzed based on their field study of various invertebrates and fish in the Great Lakes region of Canada and USA. Lipid-normalised values are generally employed for field data. Houde (2008) also determined a range of field-derived BMFs, which range from 0.11 to 0.94 for total MCCP for various invertebrate to fish in Lake Michigan and Lake Ontario. It should be noted that Houde (2008) also reported a BMF value for sculpin-Diporeia above one in Lake Michigan, though this result was not considered reliable as they are based on a single Diporeia sample as discussed in Houde (2008) and Arnot (2014). For comparison, the sculpin-Diporeia BMF for Lake Ontario was 0.88 for total MCCP in Houde (2008).


The accumulation and depuration of two radiolabelled C16 chlorinated paraffins ([1-14C] n-hexadecane; 34% chlorinated, and [U-14C] n-hexadecane; 69% chlorinated) by oligochaete worms (Lumbriculus variegatus) from spiked sediment has been investigated. Worms were exposed to the 34% chlorinated material at two concentrations (47.1 and 135 µg/g dry weight sediment) and to the 69% chlorinated material at 264 µg/g for 21 days. After transfer to un-spiked sediment, organisms were kept for a further 42 days. Rates of uptake and depuration were determined and kinetic biota-sediment bioaccumulation factors (BSAF) calculated. All determinations were based on measurements of total radioactivity. Recovery of oligochaetes at the end of the study was at least 97%. The rate of uptake of radioactivity from sediment was 0.076 to 0.093 g/g/d for the 34% C16 chlorinated paraffin and 0.013 g/g/d for the 69% material, with equilibrium being reached by 21 days. The BSAF,lipid, organic carbon and growth corrected, was 4.4 for the low chlorination material and 0.6 for the high-chlorination material (Fisk et al. 1998).


The mean bioaccumulation factor (BAF) for a radiolabelled C15 chlorinated paraffin (n-pentadecane-8-14C; 52% chlorinated) from soil by the roots of carrot (Daucus carota) was calculated to be 0.045, based on the relative concentration of radioactivity in soil and carrot root on days 50 to 70 (Thompson et al. 2005).


Overall Bioaccumulation Assessment


Figure 1 is from Arnot 2014 (full report attached in the dossier) and illustrates the application of a bioaccumulation (B) assessment framework proposed in Burkhard et al. (2012). The measured B data for MCCP constituents are from various aquatic species (plankton, invertebrates, fish) from laboratory testing (BCF, BMF) and environmental monitoring (BMF, BAF, TMF). A total of 97 measured data points are compared against the B assessment criterion of 1 (red horizontal line) proposed by Burkhard et al. (2012). Data derived from field studies, and in particular TMF values, are considered to be the ultimate indicator of a compound’s potential to bioaccumulate in the natural environment (Gobas 2009). A total of 93% of the data in Figure 1 are from environmental (field) studies and are thus considered highly relevant (“real world”) B assessment data. Of these 97 measured data points, 7 (7.2%) met or exceeded the threshold criterion and 93 (92.8%) were lower than the threshold criterion. The median value (central tendency) is 0.27 (black dashed line). The SETAC POP/PBT expert workshop experts considered that a TMF >1 represented the most conclusive evidence of the bioaccumulative nature of a chemical (Gobas 2009). Figure 1 shows that all of the TMFs for the MCCP constituents < 1. The current weight of evidence indicates that MCCP constituents are not likely to biomagnify in fish and in aquatic food webs.


Figure 1. Fugacity ratios calculated using the recommended methods (Burkhard et al., 2012) for available relevant and reliable bioaccumulation data for MCCP constituents. Values > 1 (red line) indicate biomagnification (bioaccumulation) hazard. 93% of the data points are < 1 and the median value = 0.27.


Additional References

Arnot JA, Mackay D, Bonnell M. 2008. Estimating metabolic biotransformation rates in fish from laboratory data.Environ Toxicol Chem27:341-351.

Arnot, J. 2014. Bioaccumulation Assessment of Medium-chain chlorinated paraffins (MCCPs). April 28, 2014. Unpublished reported provided in Section 13 of the REACH dossier. ARC Arnot Research & Consulting Inc., Toronto, ON.

Arnot J. 2013. Comments on Preliminary Bioaccumulation Assessment of Medium Chain Chlorinated Paraffins (MCCPs). ARC Arnot Research & Consulting Inc., Toronto, ON.

Burkhard LP, Arnot JA, Embry MR, Farley KJ, Hoke RA, Kitano M, Leslie HA, Lotufo GR, Parkerton TF, Sappington KG, Tomy GT, Woodburn KB. 2012. Comparing laboratory and field measured bioaccumulation endpoints.Integr Environ Assess Manage8:17–31.

Connolly JP, Pedersen CG. 1988. A Thermodynamic-Based Evaluation of Organic Chemical Accumulation in Aquatic Organisms. Environ Sci Technol 22:99-103.

Gobas, FAPC, de Wolf W, Burkhard L, Verbruggen E, Plotzke K. (2009) Revisiting Bioaccumulation Criteria for POPs and PBT Assessments.Integr Environ Assess Manage5(4): 624–637.

Mackay D. 1982. Correlation of Bioconcentration Factors.Environ Sci Technol16:274-278.

Nichols JW, Fitzsimmons PN, Burkhard LP. 2007.In vitro-in vivo extrapolation of quantitative hepatic biotransformation data for fish. II. Modeled effects on chemical bioaccumulation.Environ Toxicol Chem26:1304-1319.

OECD. 2012. OECD Guidelines for Testing Chemicals. Test No. 305: Bioaccumulation in Fish: Aqueous and Dietary Exposure. Organization for Economic Co-operation and Development, Paris.

Thompson R, Vaughn M. 2014. Medium-chain chlorinated paraffins (MCCPs): A review of bioaccumulation potential in the aquatic environment.Integr Environ Assess Manage10(1): 78–86.

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