Comments on Preliminary Bioaccumulation

Assessment of Medium Chain Chlorinated Paraffins

(MCCPs)

Prepared for The MCCP REACH Consortium

Final Report

Prepared by:

Jon Arnot, Ph.D.

April 30, 2013
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Executive Summary

This report evaluates bioaccumulation information presented in the draft medium chain chlorinated 

paraffin (MCCP) REACH substance evaluation (SEV) document developed by the United Kingdom and 

associated 4 April 2013 draft decision letter from the European Chemicals Agency (ECHA) and provides 

an assessment of the MCCP bioaccumulation conclusions and testing recommendations. Key technical 

information regarding the interpretation of bioaccumulation testing data and the bioaccumulation 

assessment of hydrophobic organic chemicals is first presented. This includes illustrations of the critical 

relationships between the chemical bioavailability in the water (i.e., the dissolved chemical fraction

available for uptake into aquatic organisms), growth dilution rates and metabolic biotransformation 

rates on bioaccumulation metrics; namely the bioconcentration factor (BCF). These illustrations show 

the relative importance of the primary biotransformation rate constant (kM) on the level of 

bioaccumulation of hydrophobic organic chemicals in fish.

Robust study summaries for two bioconcentration tests and three dietary bioaccumulation 

(biomagnification) tests for fish are provided in this report. A review of a field study including 

bioaccumulation factor (BAF), biomagnification factor (BMF) and trophic magnification factor (TMF) data 

is also included. Novel kM calculations for expected MCCP constituents and related chemicals less likely 

to be found in commercial products (i.e., medium chain polychlorinated alkanes, MC-PCAs) are 

presented. The kMs are similar in magnitude for expected MCCP constituents and MC-PCAs that have 

been tested. Growth corrected BCFs based on dissolved water concentrations calculated here are 

generally consistent with the BCFs calculated in the SEV with respect to the ECHA bioaccumulation 

assessment. Based on this analysis it is unlikely that further testing will provide new information for the 

bioaccumulation assessment of MCCPs using the BCF assessment metric and current BCF criteria. There 

is some uncertainty in some of the existing testing data related to the use of total radioactivity; 

therefore, any further testing that may be considered should be done with parent chemical specific 

analysis to address these uncertainties. While expected MCCP constituents show calculated growth 

corrected BCFs that exceed regulatory criteria, the current weight of evidence indicates that expected 

MCCP constituents are not likely to biomagnify in fish and in aquatic food webs based on laboratory and 

field BMFs and field TMFs, respectively.
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Table of Contents

Executive Summary .................................................................................................................................2

Table of Contents....................................................................................................................................3

Introduction ............................................................................................................................................4

Scope of Review ..................................................................................................................................4

Bioaccumulation – Background Terminology and Key Relevant Concepts.............................................4

Chemical bioavailability in the water................................................................................................6

Growth dilution ...............................................................................................................................7

Biotransformation rates...................................................................................................................9

Measured Bioaccumulation Data Review............................................................................................... 10

Measured Laboratory Bioconcentration Data – BCFs ......................................................................... 10

Robust Study Summary: Fish BCF Test #1 (Thompson et al. 2000) .................................................. 10

Robust Study Summary: Fish BCF Test #2 (Hurd and Vaughan, 2010) ............................................. 11

Measured Laboratory Dietary Bioaccumulation Data – BMFs............................................................. 13

Robust Study Summary: Fish BMF Test #1 (Fisk et al. 1998) ........................................................... 13

Robust Study Summary: Fish BMF Test #2 (Fisk et al. 1996) ........................................................... 14

Robust Study Summary: Fish BMF Test #3 (Fisk et al. 2000) ........................................................... 14

Measured Field Bioaccumulation Data – BAFs, BMFs and TMFs ......................................................... 16

Robust Study Summary: Fish BAF and BMF field data (Houde et al. 2008)...................................... 16

Biotransformation Rate Constants (in vivo, in silico) - kM.................................................................... 17

Incorporating kM,N for Bioaccumulation Assessment - BCFs ................................................................ 20

General Comments on SEV Document................................................................................................... 21

Comment on “SEV Report section 8.1.4 Summary and overall conclusions on PBT and vPvB 

properties” ......................................................................................... 21

References............................................................................................................................................ 22
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Introduction

Scope of Review

This document provides general technical knowledge and commentary regarding bioconcentration 

and bioaccumulation in biota (primarily fish), measured data, mass balance models and quantitative 

structure-activity relationships (QSARs). The document provides a review and assessment of some of the 

bioaccumulation information presented in the draft medium chain chlorinated paraffin (MCCP) REACH 

substance evaluation (SEV) document developed by the United Kingdom and associated 4 April 2013 

draft decision letter from the European Chemicals Agency (ECHA). Given the time frame for preparing 

the present document, the objective here is not to provide a thoroughly documented review for all of 

the available measured (laboratory and field) and modelled bioaccumulation data for MCCPs and 

medium chain length polychlorinated alkanes (MC-PCAs) presented in the SEV document and elsewhere. 

If desirable, future work could expand upon the current document by examining data not presented in 

the SEV and for other chain length PCAs and CPs. The focus here is (i) to critically examine the 

acceptable (usable) quality measured bioaccumulation data to better understand the bioaccumulation 

of MCCPs in aquatic organisms and food webs and based on this analysis (ii) determine if further 

requested dietary based bioaccumulation testing of MCCP constituents is warranted. Through the 

course of this analysis key technical information relating to the bioaccumulation process of neutral 

organic chemicals in aquatic organisms is briefly presented with detailed published studies cited for 

brevity. If desirable, more analysis and documentation can be provided in future work.

MCCPs are a mixture of chemicals, considered a UVCB substance (i.e. a substance of unknown or 

variable composition, complex reaction products or biological materials), registered for evaluation under 

a single CAS No (85535-85-9). MCCPs are linear alkanes comprised of 14 to 17 carbons with a range of 

chlorine substitution. Theoretically, there could be approximately 120,000 different constituents in the 

MCCP mixture. The range of properties for constituents is therefore also large with predictions for the 

octanol-water partition coefficient (KOW) spanning a few orders of magnitude, e.g., log KOWs from ~7 to 

>9.0 [1]. While it is recognized that all of the different MCCP constituents and congeners have different 

bioaccumulative properties, the general focus of this review is on the available bioaccumulation data 

and the interpretation of the data included in the SEV document. 

A brief overview of relevant information for assessing the bioaccumulation potential of organic 

chemicals is first provided, including some key terminology and concepts. Bioaccumulation data 

included in the SEV document deemed most relevant and usable (“acceptable quality”) are briefly 

summarized. Modelled bioaccumulation data not included in the SEV document are developed here to 

seek insights into differences in chlorine substitution patterns and chain length on the 

biotransformation rate and bioaccumulation assessment of MCCPs. Finally some general comments and 

responses to “initial charge questions” are provided.

Bioaccumulation – Background Terminology and Key Relevant Concepts

Full definitions of key terminology for bioaccumulation and bioaccumulation hazard assessment are 

available elsewhere [2-5]. Briefly, bioaccumulation is the net result of competing rates of chemical 

uptake and elimination in an organism under a defined set of exposure conditions. Fish are commonly 

used as a model organism for assessing bioaccumulation hazard. Key routes of chemical uptake in fish 

include uptake at the gill surface and through chemical exposure in the diet and key routes of chemical 

elimination include gill elimination, fecal egestion and metabolic biotransformation. Growth dilution is a 
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process in which the chemical concentration in the organism can be reduced; however, the parent 

chemical is not actually eliminated from the body. Growth rate “constants” are often included in mass 

balance models predicting chemical concentrations and bioaccumulation. A typical steady-state mass 

balance model for calculating chemical concentrations in fish (CF) is [5-7]:

CF = [(k1×CWD) + (kD×CD)] / (kR + kE + kG + kM)

where k1 is the gill uptake rate constant of chemical from water, CWD is the dissolved (bioavailable) 

chemical concentration in the water, kD is the dietary uptake rate constant of chemical from the diet, CD

is the chemical concentration in the diet and kR, kE, kG and kM are respectively the first-order rate 

constants for gill elimination, fecal egestion, growth dilution and metabolic biotransformation. The 

numerator reflects chemical uptake processes and the denominator reflects chemical elimination 

processes. The sum of the elimination rate constants is often referred to as the total elimination rate 

constant (i.e. kT; e.g., units d-1

) or depuration rate constant with various nomenclatures (e.g., kd or k2) 

and corresponds to the whole body total elimination half-life t1/2 (e.g., units d) as ln2/kT.

Typical bioaccumulation hazard assessment metrics include KOW, the bioconcentration factor (BCF; 

L/kg), the bioaccumulation factor (BAF; L/kg), and the biomagnification factor (BMF; kg-lipid/kg-lipid) [3, 

8]. Other metrics, such as the trophic magnification factor (TMF) and the biota-sediment accumulation 

factor (BSAF; kg-OC/kg-lipid) are also considered useful for bioaccumulation assessment [3, 8]. KOW is 

used as a surrogate for lipid-water partitioning and has recognized limitations for bioaccumulation 

assessment, primarily because it ignores key biological processes such as uptake and elimination, most 

notably biotransformation [9, 10]. Therefore, KOW is no longer typically used alone to assess 

bioaccumulation. KOW is still an important input parameter for the majority of bioaccumulation models. 

The BCF is the steady-state ratio of the chemical concentration in a fish to the chemical concentration in 

the water following chemical exposure from the water only (as occurs under controlled laboratory 

conditions), i.e., BCFss = Cf/Cw. The BCF can also be determined from the kinetic (rate constant) 

information as BCFk = k1/kT. The BAF is the steady-state 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, food, and 

potentially sediment), i.e., BAFss = Cf/Cw. The BAF typically represents environmental exposures and is 

derived under field conditions. The BCF and BAF can be calculated using wet weight or lipid normalized 

tissue concentrations and total or dissolved water concentrations. For most neutral organic chemicals 

the lipid normalized / dissolved water concentration BCF and BAF metrics provide information on the 

thermodynamic status for the chemical distribution between the organism and the water. The BMF is 

the steady-state ratio of the lipid normalized chemical concentration in an organism to the lipid 

normalized chemical concentration in its diet [3, 8]. The BMF can be determined through laboratory 

testing or field measurements. It is noted that all of these metrics used to evaluate bioaccumulation 

hazard are normalized to an exposure source (i.e., the concentration in the surrounding environment or 

the concentration in the diet). While monitoring data are useful for exposure and risk assessment, they 

are not useful for bioaccumulation hazard assessment without some quantification of the actual 

exposures.

Bioaccumulation hazard assessment seeks to evaluate and compare the inherent properties of 

chemicals in a consistent manner. Several parameters other than the “inherent chemical properties” can 

influence chemical bioaccumulation and thus the interpretation of bioaccumulation test results. Some 

examples of key parameters other than the “inherent chemical properties” that can influence

bioaccumulation include organism properties (lipid content) and exposure conditions (diet lipid content, 

organic carbon content in the water). Standardized testing methods and guidance have been developed 
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in efforts to assess the inherent properties of the chemicals under defined conditions [11], in essence 

these guidelines seek to provide “a testing model” for evaluating bioaccumulation in fish. The

recommended testing methods and technical guidance continue to evolve with the science.

For screening-level bioaccumulation assessments, lipid normalization, growth correction and 

dissolved water concentration based bioaccumulation metrics provide a consistent means for chemical 

comparisons. Benchmarking with a positive control or standard chemical [12, 13] is an additional 

approach that may address variability and uncertainty and result in improved and more consistent data 

interpretation for chemical evaluations. The rationale of lipid normalization for neutral organic 

chemicals is well established [3, 14, 15], although still not consistently adapted in all chemical 

evaluations. The confounding influences of bioavailability (i.e. the actual dissolved water concentration) 

and growth dilution have not been commonly and consistently recognized and addressed in most 

historical bioaccumulation assessments; therefore, the influence of these two parameters on fish 

bioaccumulation data interpretation are highlighted here. The rate at which a hydrophobic chemical is 

biotransformed is a critical determinant of the overall extent of bioaccumulation for hydrophobic 

chemicals [9, 10, 16]; therefore, highlighting the influence of kM is also a focus of this document.

Chemical bioavailability in the water

Understanding the concept of chemical bioavailability in the water is critical for interpreting and 

applying aquatic-based bioaccumulation metrics such as the BCF and the BAF because the denominator 

of these steady-state calculations includes an estimate of the water concentration, i.e., BCFss = Cf/Cw. 

Only the dissolved chemical in the water is available for uptake through the gills [17]. Organic carbon 

(OC) in the water can influence the bioavailable (dissolved) solute fraction (BSF) for hydrophobic 

chemicals. The fraction dissolved (or BSF) can be estimated as [6, 18]:

BSF = 1/(1 + [POC]×0.35×KOW + [DOC]×0.08×KOW)

where [POC] is the particulate organic carbon concentration, [DOC] is the dissolved organic carbon 

concentration (operationally defined as OC passing through a 0.45 μm filter), and the values 0.35 [19]

and 0.08 [20] are proportionality constants relating KOW to the types of organic carbon. Figure 1 

illustrates the relationship between BSF, KOW, DOC and POC [18]. Note that even seemingly small 

quantities of OC can have significant effects on the actual fraction of chemical that is available for 

uptake into an aquatic organism when KOW gets large (e.g. >107

) due to the high level of hydrophobicity 

associated with these chemicals.

The total water concentration (CWT) for hydrophobic neutral organic chemicals is typically measured 

in bioaccumulation and toxicity testing and field studies using solvent extraction and therefore includes 

both the dissolved fraction and the fraction sorbed to OC (not bioavailable). The dissolved water 

concentration (CWD) is therefore CWD = CWT×BSF. As shown in Figure 1, BSF = 1 (i.e., CWD ≈ CWT) for lower

KOW chemicals even when POC and DOC concentrations are relatively high; however, for higher KOW

chemicals BSF < 1 (i.e., CWD < CWT) even when POC and DOC concentrations are seemingly low (i.e., less 

than recommended “acceptable values” in OECD 305 BCF testing [11].

For bioaccumulation assessments consistent comparisons of “inherent chemical properties” using

the BCF and BAF should therefore be based on CWD since this concentration is independent of [POC] and 

[DOC]. In other words, BCFs and BAFs based on CWD are most appropriate for bioaccumulation hazard 

categorization assessments so that chemicals can be compared against a consistent set of conditions 
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(i.e. the concentration of dissolved chemical in the exposure medium). For exposure and risk 

assessments, chemical partitioning to OC in the water column must be quantified to obtain reliable 

estimates of exposure based on the total (bulk) water concentrations.

It is recognized that estimating the truly dissolved chemical concentrations of very hydrophobic 

chemicals in water is technically challenging and thus measurements and model calculations are 

uncertain. Furthermore, the vast majority (more than 95%) of existing measured bioaccumulation data 

for fish (i.e. BCFs and BAFs) are based on total water concentrations. Thus, most screening-level 

bioaccumulation models (i.e., regression-based BCF models using laboratory BCF data from total water 

concentrations) make predictions based on the total water concentration under “typical” or assumed 

laboratory exposure conditions (i.e., relatively lower POC and DOC than the environment, but still some

OC). In the MCCP SEV document, the issue of bioavailability does not seem to be well understood.

Figure 1. Illustration of the effect of chemical hydrophobicity (KOW) and Particulate Organic Carbon (POC) 

and Dissolved Organic Carbon (DOC) concentrations on the dissolved (bioavailable) fraction of the total 

chemical concentration in the exposure water. This figure is part of Figure 2 in [18]. The fraction 

dissolved is estimated here as 1/(1 + [POC]×0.35×KOW + [DOC]×0.08×KOW) [1] where the values 0.35 [19]

and 0.08 [20] are proportionality constants relating KOW to types of organic carbon.

Growth dilution

As illustrated below, the organism growth rate can also be a confounding factor for certain 

hydrophobic chemicals and thus this parameter requires consideration for the consistent interpretation 

of bioaccumulation metrics. When key chemical elimination processes are slow (i.e., gill elimination, 

biotransformation, fecal egestion), the growth rate can confound the true depuration or elimination 

rate of the chemical from the test organism, i.e., growth rate constant becomes a principal factor 

resulting in decreased chemical concentrations in the organism over time, not actual chemical 

elimination from the body. In this regard, growth rates are expected to be most relevant for relatively 

hydrophobic, slowly biotransformed chemicals (i.e., key elimination rates are relatively slow, growth 

dilution becomes more dominant “rate process” in the overall total elimination). Thus there is a 

rationale for considering growth correction for consistent chemical hazard assessments that seek to 
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evaluate and compare the “inherent properties” of the chemicals independent of the exposure or 

organism conditions (i.e., differences in growth rates). Growth correction is essentially kT - kG and is 

sometimes referred to as k2g.

Figure 2 uses the mechanistic Arnot and Gobas screening-level mass balance model included in EPI 

Suite to illustrate the influence of growth rates and bioavailability on the BCF [1, 21]. A spreadsheet 

version of the model is available that allows for these parameter manipulations and illustrations

(www.arnotresearch.com). The model is parameterized here for a small fish (~10 g) with a 5% whole 

body lipid content (i.e., consistent with the suggested lipid content for bioaccumulation assessment

against “standardized criteria”). The water temperature is set to 15 °C similar to BCF and BMF testing 

conditions for cold-water species (i.e., rainbow trout). For Figure 2 the biotransformation rate constant

(kM) is assumed to be zero to highlight the relative roles of growth and bioavailability (dissolved chemical 

fraction) on the BCF. An assumed “laboratory” growth rate constant (kG) of 0.03 /d is initially used to 

parameterize the model and a second set of simulations assumes kG = 0 /d, consistent with an approach 

for growth correction, i.e., “growth normalization”. These first two simulations include the default 

model parameters for POC and DOC concentrations in the water, i.e., 0.5 mg/L for both. The model 

shows that growth rate and bioavailability have negligible influence on the BCF for lower KOW chemicals, 

i.e., log KOW < ~4.5. Growth normalization begins to influence the BCF as KOW increases, particularly when 

log KOW > ~6. The differences between “measured total water concentration BCFs” (including growth, 

blue diamonds) and “growth corrected total water concentration BCFs” (blue squares) can be very large 

for hydrophobic chemicals, when biotransformation is assumed negligible. It is emphasized that when 

log KOW > 5, kM ~ 0 and BCFs are corrected for growth (kG = 0), all of the wet weight BCFs are greater than 

5,000 L/kg. Under these conditions, growth corrected BCFs calculated from total water concentrations 

“plateau” for higher KOW chemicals because all of the other rates of chemical elimination are low (or 0)

and the steady-state BCF is essentially controlled by the fecal egestion rate constant. When the BCF is 

calculated based on the dissolved water concentrations (i.e., BSF = 1), the BCF continues to increase 

with increasing KOW. This is explained by inspection of the BCF and BSF calculations and Figure 1. 

Notably, when log KOW > ~5, kG = 0 and kM ~ 0, the BCFs are > 5,000 whether or not the BCF is calculated 

based on the total or dissolved water concentrations. 
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Figure 2. Illustration of the effect of growth rate (KG / d) and bioavailable solute fraction (BSF, see Figure 

1) on the bioconcentration factor (BCF) as a function of hydrophobicity (KOW).

Growth rates can vary considerably based on energy density of the diet, digestibility of the diet, 

organism activity, life-stage, etc. In general terms, organisms grow when the uptake of energy exceeds 

the expenditure of energy. Organisms are not expected to grow (or live very long) if they are not 

feeding. Fish in the environment feed and grow at various rates throughout the life-cycle and often for 

some species at different rates depending on the season. While growth correction of bioaccumulation 

metrics is relatively straightforward if measured properly during a laboratory test, i.e., for lab BCFs and 

BMFs, the issue is more complicated in the environment.

Biotransformation rates

Building from the illustrations in Figure 2, the role of the biotransformation rate constant (kM) is 

illustrated next in Figure 3. In a series of BCF calculations kM is increased from 0 to 0.03 to 0.15 to 0.3 /d. 

Figure 3 shows that when growth corrected BCFs for an assumed 5% lipid content fish are calculated 

based on the dissolved water concentration (i.e., normalized exposure conditions), the kM needs to be 

about 0.15 /d for BCF < 5,000 and about 0.3 /d for BCF < 2,000 regardless of the chemical KOW. Given the 

uncertainty in the actual KOW of MCCP constituents (or other organic chemicals), these illustrations help 

put into context the importance of the biotransformation rate constant in determining the overall 

bioaccumulation of hydrophobic organic chemicals. Thus the “inherent bioaccumulation properties” of 

hydrophobic chemicals (i.e., log KOW > 6) in aquatic organisms is largely dependent on reliable estimates 

of the biotransformation rate constant (kM). For these reasons an effort was made here to estimate kM
10

from the “acceptable quality” bioaccumulation test data for MCCPs. Further examples of the role of 

biotransformation rates on bioaccumulation and exposure assessment are available elsewhere [9, 10, 

16, 22, 23].

Figure 3. The role of the primary biotransformation rate constant (KM /d) on the growth corrected (i.e., 

KG = 0 /d) bioconcentration factor (BCF) calculated from the dissolved (bioavailable) water 

concentration, i.e., BSF = 1.

Measured Bioaccumulation Data Review 

Measured Laboratory Bioconcentration Data – BCFs

Robust Study Summary: Fish BCF Test #1 (Thompson et al. 2000)

Test substance: radiolabelled n-pentadecane-8-

14C (C15H26Cl6, ~51% chlorinated w/w)

Test organism: rainbow trout (Oncorhynchus mykiss) ~2 g initial wet weight; lipid content not measured.

The test followed OECD 305 guidelines (1996) [24] and good laboratory practise [25]. Uptake and 

elimination kinetics were calculated based on 35 d exposure and 42 d depuration phases. Kinetic BCF 

estimates of 1,087 L/kg-ww and 349 L/kg-ww for exposure concentration treatments of 1 and 5 μg/L

(nominal), respectively, at about 15 °C. Uptake rate constants (k1) were 48.7 and 14.2 /d for the low and 
11

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 confirming the estimate used in the SEV document. There are key 

technical issues related to data quality, uncertainty and interpretation:

• There was no correction of mass balance for parent test chemical [2, 18], i.e., total radioactivity 

for both parent and possible metabolites was quantified. Given the flow through conditions of 

the test, the tissue concentrations and ensuing BCFs represent likely upper bound estimates of 

the BCF based on total water concentrations. The position of the radiolabel on the test 

substance is not reported. This issue is a potential source of uncertainty with the reported BCF 

and kinetic information.

• Depuration half-lives were 15 and 17 days for nominal exposure concentration treatments of 1 

and 5 μg/L, respectively. Steady-state was not approached during the test, i.e. time to reach 

~95% of steady-state = 4xt1/2 ~60 d, suggesting the BCF calculated as Cf/Cw is an underestimate 

of the steady-state value supporting the rationale for using the kinetic BCF estimates. 

• The exposure concentrations are near the estimated water solubility limits for the test chemical. 

Based on w/w% Cl, an assumed representative SMILES notation was generated 

[CC(Cl)CC(Cl)CC(Cl)CC(Cl)CCC(Cl)CC(Cl)CC] and predicted KOW and water solubility (Sw) estimates 

are log Kow ~8.8, Sw ~0.1 μg/L [1] and log Kow ~6.8, Sw ~43 μg/L (ACD Labs). It is possible that 

the measured exposure concentration of 5 μg/L exceeds the water solubility limit for the test 

chemical. It is recognized that there are uncertainties in measured and predicted KOW and water 

solubility; however, the relatively lower BCF for the higher exposure concentration is consistent 

with a pattern of observations of underestimated BCFs for the same chemical when water 

solubility is exceeded [2]. When chemical exposure concentrations are above the water 

solubility limit, the measured total water concentration is an overestimate of the actual 

bioavailable water concentration resulting in underestimates of the BCF. This issue is a key 

source of uncertainty with the reported BCFs, particularly for the higher exposure level.

• Total water concentrations were measured throughout the exposure; however, non-purgeable 

organic carbon (NPOC) concentrations during the test ranged from 0.69 to 2.4 mg/L (some 

higher values also reported). Although the values and nature of the OC are uncertain, the BCFs 

based on the dissolved (bioavailable) water concentration are higher than the BCFs based on 

measurements of the total water concentration. For example, based on common model 

estimates for the dissolved fraction and log KOW ~8 and [DOC] ~1 mg/L, the dissolved fraction 

may be about 10% of the total measured concentration (see Figure 1). This issue is a key source 

of uncertainty with the reported BCFs.

• Solvent was used, may be a minor factor; however, there are conflicting studies on possible cosolvent effects and it is probably a relatively minor source of uncertainty in this case.

Robust Study Summary: Fish BCF Test #2 (Hurd and Vaughan, 2010)

Test substance: 1-

14C-labelled n-tetradecane (C14H25.5Cl4.5, ~42-48% chlorinated w/w)

Test organism: rainbow trout (Oncorhynchus mykiss) ~2.9 g initial wet weight; mean lipid content 10.3%

The test followed OECD 305 guidelines [24] and good laboratory practise [25]. Uptake and 

elimination kinetics were calculated based on 35 d exposure and 42 d depuration phases. Kinetic BCF 

estimates of 9,140 L/kg-ww (4,440 L/kg-ww when normalized to 5% lipid content) for nominal exposure 
12

concentration of 0.5 μg/L - mean measured total water concentration of 0.34 μg/L, at about 15 °C. 

Uptake rate constant (k1) of 395 /d and total elimination rate constant (“k2”) of 0.0432/d. A growth rate 

constant of 0.030 /d is reported. Generally a good quality study; however, there are key technical issues 

related to data uncertainty and interpretation:

• There was no correction of mass balance for parent test chemical [2, 18], i.e., total radioactivity 

for both parent and possible metabolites was quantified. Given the flow through conditions of 

the test, the tissue concentrations and ensuing BCFs represent likely upper bound estimates of 

the BCF based on total water concentrations. The position of the radiolabel on the test 

substance is reported to be on a terminal carbon atom. 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). Samples of fish at the end 

of the depuration phase showed that no significant extractable metabolites of chlorinated 

tetradecane were present, but non-extractable (presumably polar) tissue metabolites were 

found, which accounted on average for 21% of the total 14C activity in the fish. This issue is a 

potential source of uncertainty with the reported BCF and kinetic information.

• Depuration half-life was 16 days, i.e. time to ~95% of steady state is 64 days, indicating the BCF 

calculated as Cf/Cw is an underestimate of the steady-state value supporting the rationale for 

using the kinetic BCF estimate. 

• It is likely that the measured exposure concentration of 0.34 μg/L is below the water solubility 

limit for the test chemical. Based on w/w% Cl, an assumed representative SMILES notation was 

generated [CC(Cl)CCCC(Cl)CC(Cl)CCCC(Cl)CC] and predicted KOW and water solubility (Sw) 

estimates are log Kow ~7.9, Sw ~1 μg/L [1] and log Kow ~6.8, Sw ~86 μg/L (ACD Labs). Note 

these predictions are assumed for 4 Cls, not 5. It is recognized that there are uncertainties in 

measured and predicted KOW and water solubility.

• Total water concentrations were measured throughout the exposure and total organic carbon 

concentrations are reported as <3.0 mg/L. DOC of 0.45 mg/L was measured at one point during 

the test. BCFs based on the dissolved (bioavailable) water concentration are expected to be 

higher than the BCFs based on measurements of the total water concentration, e.g., based on 

log KOW ~7.5 and [DOC] ~0.5 mg/L, the dissolved fraction may be about 40% of the total 

measured concentration. This issue is a potential source of uncertainty with the reported BCF.

• Solvent was used, may be a minor factor; however, there are conflicting studies on possible cosolvent effects and it is probably a relatively minor source of uncertainty in this case.

The potential error in k2 estimation is a result of uncertainties associated with chemical specific 

analysis (i.e., both parent and metabolites quantified). There may be ~20% error in k2 estimation in this 

case; however, it is difficult to say with certainty from the available data; however, as outlined below in 

the estimation of biotransformation rate constants assuming a 20% faster value for k2, this level of 

uncertainty may be relatively inconsequential in terms of the overall bioaccumulation assessment.

Summary comments of relevant BCF data

BCF Test #2 is considered to be of better quality than BCF Test #1 because the exposure 

concentration in Test #2 is “further from the estimated water solubility limits” than Test #1, i.e., 

greater confidence that exposure concentrations are below the water solubility of the test chemical

and potential errors related to the dissolved (bioavailable) fraction of chemical in the water are 

expected to be lower. It is noted that the elimination rate constants and half-lives are comparable
13

(i.e., independent of potential exposure errors), but the estimated uptake rate constants (k1) were 

lower in Test #1, possibly an artefact of the true bioavailability of the chemical (total, not dissolved,

water concentrations used in the regression equations) and water solubility uncertainties (errors).

Measured Laboratory Dietary Bioaccumulation Data – BMFs

There are three published studies by Fisk and colleagues (1996, 1998, and 2000), differing mostly by 

the formulations of the test chemicals and the concentrations in the diet and whether or not the test 

material was radiolabelled or not. The formulation of the mixtures (i.e., actual structures being tested) 

could be a critical consideration when interpreting the bioaccumulation data (i.e., chain length and 

chlorine substitution patterns). Note that the bioaccumulation data by Fisk and colleagues are

reported in the publications as lipid normalized and growth corrected.

Robust Study Summary: Fish BMF Test #1 (Fisk et al. 1998)

Test substance: C14H26Cl4 (42% Cl w/w), C14H25Cl5 (48% Cl w/w), C14H24Cl6 (53% Cl w/w): SCCPs (C10 and 

C11) also tested. No radiolabelled chemicals used in this test; however, terminal Cl-substitution not 

considered representative of MCCP constituents.

Test organism: rainbow trout (Oncorhynchus mykiss) ~4.5 g initial wet weight; mean lipid content 2.8%

Fish were exposed to chemical in their diet for 40 days, followed by an 80 day depuration period. A 

growth corrected depuration rate constant (“k2g”) and associated half-life were calculated. From these 

kinetic data the assimilation efficiency (α) and BMF are calculated assuming constant feeding rates and 

chemical concentrations in the food. The test chemicals were derived from alkenes with double bonds 

on both of the terminal carbons resulting in terminal and adjacent chlorination, i.e. 4 chlorine atoms -

two at both ends. After those double bonds are saturated, the location of the remaining chlorine 

substitutions is believed to be random for C14H25Cl5 and C14H24Cl6; therefore, these test chemicals are not 

specifically defined with respect to structure. Fisk et al. [26] point out that “the PCAs used in these 

experiments are not likely to be found at high proportions in commercial CP mixtures because they 

contain Cl on terminal and adjacent carbons, which is chemically and energetically unfavourable under 

free radical reaction conditions used in the production of commercial CP products”. With respect to 

primary biotransformation, precisely where the non-terminal Cls are on the alkane chain may be less 

significant relative to the known substitution of the two Cls at both ends of the alkane chain. This is 

examined in more detail below.

The bioaccumulation data from Fisk et al. 1998 [26] using the terminal Cl-substituted PCAs include 

several data points in which the calculated chemical assimilation efficiencies (α) are > 100%. 

Bioaccumulation kinetic data with chemical assimilation efficiencies >100% should generally be 

considered to have some source of critical error since the only plausible explanation for a “true 

assimilation efficiency” > 100% is the formation of the chemical in the organism (or perhaps the GIT) 

during the testing and analysis. Note this has been hypothesized for certain polybrominated diphenyl 

ethers, but it is not considered a likely event for the chlorinated alkane data here. The assimilation 

efficiency (α) and BMF are calculated from the depuration rate constants and assumed “totally 

consistent” feeding rates and chemical concentrations in the food. When assimilation efficiency is > 

100% this suggests some source of error, perhaps in one of these parameters used in the calculation. In 

particular, the bioaccumulation data for C14H24Cl6a reported in Table 26 of the SEV should not be used for 
14

the bioaccumulation assessment since α > 100%. The other test chemicals with assimilation efficiency 

estimates > 100% reported in Fisk et al. [26], and elsewhere, should also be considered highly uncertain

and not used for bioaccumulation assessment. Also note in Table 26 of the SEV document, the BMF for 

C14H25Cl5b is not correct - it is not 29; the reported value by Fisk et al. [26] is 2.9.

A general finding in Fisk et al. 1998 [26] is that half-lives (t1/2s) of PCAs increase with increasing 

chlorination, chain length, and KOW. This is consistent with other studies cited in their paper; however, 

the precise location of the Cl substitution could have some influence on biotransformation rates and 

hence overall half-life. Excluding the uncertain data point for C14H24Cl6a the whole body half-lives for MCPCAs range from 39 to 53 days, with a median value of 44.5 days. These growth corrected half-lives are 

from juvenile rainbow trout that weighed about 4.5 g at the start of the 120 day test. Based on the 

reported growth rate of about 0.016 /d [26], the estimated average fish mass at 80 days (i.e., mid-point 

of depuration) is about 30 g. Note the kinetic, growth corrected lipid normalized BMFs are > 1 for these 

test chemicals.

Robust Study Summary: Fish BMF Test #2 (Fisk et al. 1996)

Test substance: 14C16H31Cl3 (32% Cl w/w) and 14C16H21Cl13 (68% Cl w/w), other CPs also evaluated. 

Radiolabelled test chemicals used in this test.

Test organism: rainbow trout (Oncorhynchus mykiss) ~4.5 g initial wet weight; mean lipid content ~5.7%

Fisk et al. 1996 [27] tested CPs that were more similar to the commercial industrial mixture than 

the Fisk et al. 1998 tests that were synthesized from dienes, i.e., terminal Cls. Fish were exposed to 

chemical in their diet for 40 days, followed by a 160-173 day depuration period. A growth corrected 

depuration rate constant (“k2g”) and associated half-life were calculated. From these kinetic data the 

assimilation efficiency (α) and BMF are calculated from the assuming constant feeding rates and 

chemical concentrations in the food. Whole-body t1/2s based on total radioactive residue (TRR), i.e., 

parent and metabolites, ranged from 37 to 77 d for MCCPs (median) and from 39 to 87 d for SCCPs. 

These t1/2s are from juvenile rainbow trout that weighed about 4.5 g at the start of the ~200 day test. 

Based on the reported growth rate of about 0.012 /d [27], the estimated average fish mass at 120 days 

(i.e., mid-point of depuration) is about 20 g. Kinetic, growth corrected BMFs for MCCPs based on total 

radioactive residue (TRR), i.e., parent and metabolites, ranged from 0.4 to 1.1 with a median of 0.72. 

Assimilation efficiencies range from 9.4% to 35.1% for the MCCPs, note assimilation efficiency estimates 

<50%. Depuration rate constants are based on TRR; and all other kinetic parameters use these data for 

calculation and therefore because this does not constitute chemical specific analysis there are 

uncertainties in these data [2, 18].

Robust Study Summary: Fish BMF Test #3 (Fisk et al. 2000)

Test substance: 14C14H21.3Cl6.7 (~55% Cl w/w), SCCP and LCCPs also evaluated. Radiolabelled test 

chemicals used in this test.

Test organism: rainbow trout (Oncorhynchus mykiss) ~3 g initial wet weight; mean lipid content 5-11%

In 2000, Fisk and colleagues [28] published another fish bioaccumulation study for CPs using two 

different dietary exposure concentrations for each group of CP. In this case they used CPs that are 

generally more consistent with the structures expected in the industrial mixture, i.e., no apparent 
15

terminal Cls. Fish were exposed to chemical in their diet for 40 days, followed by a 160 day depuration 

period. A growth corrected depuration rate constant (“k2g”) and associated half-life were calculated. 

From these kinetic data the assimilation efficiency (α) and BMF are calculated assuming constant 

feeding rates and chemical concentrations in the food. Based on the reported growth rate of about 

0.014 /d, the estimated average fish mass at 120 days (i.e., mid-point of depuration) is about 16 g. The 

reported half-lives for the two MCCP treatments were 58 and 41 d for the low and high dose, 

respectively. The corresponding assimilation efficiencies and kinetic, growth corrected BMFs for MCCPs 

based on total radioactive residue (TRR), i.e., parent and metabolites, were 10% and 11% and 0.43 and 

0.27, respectively. Note the kinetic, growth corrected lipid normalized BMFs are < 1 for all MCCPs for 

these test chemicals.

Summary comments of relevant BMF data

Reduced chemical dietary uptake (assimilation) efficiency (α, or sometimes referred to as ED) has 

been observed for very hydrophobic organics in a range of species (see, e.g., [6, 7, 21, 29, 30]. 

Empirical models (i.e., regressions based on measured data) for α for organic chemicals in fish show 

median values for α of about 50% for chemicals with log KOW from ~5 to 6.5 and decreasing values for 

α when log KOW > ~6.5 [6, 30]. Therefore, the rationale in Fisk et al. 2000 [28] and elsewhere to assume 

assimilation efficiencies of 50% and 90% to calculate some of the BMFs for CPs and PCAs instead of α

values derived from the test data is questionable and seemingly unjustified. The BMFs calculated with 

the measured α are considered more reliable and applicable for bioaccumulation hazard assessment. 

There are merits and limitations to all of the existing laboratory BMF test data. BMF Test #1 

included chemical specific analysis (albeit, perhaps unconfirmed congener analysis), whereas Tests #2 

and #3 are based on total radioactivity generating some uncertainty in the derived kinetics and 

bioaccumulation data. BMF Test #1 uses chemical formulations that are less likely commercial MCCP 

products (i.e., terminal Cl-substitution). Chain lengths for the MCCPs and MC-PCAs studied in dietary 

bioaccumulation testing range from 14 to 16 carbons and chlorine substitution ranges from 32 to 68% 

w/w. Estimated log KOWs range from about 7 to greater than 9. The kinetic, growth corrected lipid 

normalized BMFs for the expected MCCP constituents from these three studies are < 1, except for 1 of 

the 11 reliable quality measurements (i.e., 1.07 for low dose C16H31Cl3). The BMFs for the MC-PCAs (i.e. 

terminal Cl-substitution) show BMFs >1. A potential difference between the BMF results for terminal 

Cl-substituted MC-PCAs and the expected MCCP constituents may be the slightly higher α generally 

observed for the MC-PCAs. Assuming that all of the data are reasonably accurate and that TRR use in 

the MCCP tests (i.e., #2 and #3) is an insignificant variable, this apparent difference could be due to 

some slow, but relevant, degradation rate of MCCPs in the gastro-intestinal tract (GIT) by 

microorganisms and/or some first-pass effects in the GIT tissues and liver that do not occur, or do not 

occur as quickly, for the terminal Cl-substituted MC-PCAs.

The reliable quality whole body growth corrected half-lives for these MCCPs and MC-PCAs from 

the dietary studies range from 37 to 77 days. The median t1/2 of 50 days from the dietary testing is 

greater than the median t1/2 from the BCF testing (16 days); however, it is recognized that t1/2s are a 

function of organism properties (e.g., body size, lipid content).
16

Measured Field Bioaccumulation Data – BAFs, BMFs and TMFs

Robust Study Summary: Fish BAF and BMF field data (Houde et al. 2008)

BAFs

Houde et al. [31] report MCCP concentrations in biota and water samples from Lake Ontario and 

Lake Michigan. Lipid weight BAFs are calculated between water and four species of fish and plankton 

from Lake Ontario. Whole body wet weight BAFs for “total MCCP” range from about 24,000 (Lake trout) 

to about 163,000 (Alewife). As noted in Houde et al. [31] and by Thompson (2010) these BAFs are based 

on water samples including some different locations and years (mostly 2002-2004) to the biota samples

(mostly 2001). The measured water concentration of ΣMCCP varied from 47 pg/l in 2002 to <0.5 pg/l in 

2004 at the same sample location. The former value is considered an “outlier” by the authors [31]. The 

biota were sampled from the western, industrialised end of Lake Ontario, whereas the water samples 

were taken in the western, central and eastern areas of Lake Ontario. Furthermore, as also noted by 

Thompson (2010), sample sizes were small and several water samples were below the detection limit

(for BAF calculations 0.5DLs assumed). It is unclear if the BAFs are based on ratios of the same MCCP 

constituents. For these reasons general confidence in the reported BAFs is low. Due to the water 

measurement uncertainties and the spatial and temporal discrepancies between fish and water, the 

BAFs are not considered to be usable for definitive quantification; however, as outlined in the present 

study it is likely that many of the MCCP BAFs based on dissolved water concentrations are > 5,000 L/kgww.

BMFs

As noted by Gobas et al. [3] and Burkhard et al. [8] assessing whether or not a chemical 

biomagnifies in the environment is a critical consideration for bioaccumulation hazard assessment. The 

best use of the Houde et al. [31] data is for the BMF calculations (water and spatial/temporal issues less 

problematic in this regard; however, sample sizes are still rather small). Trout BMF data are considered 

more reliable than the Sculpin-Diporeia BMF data since n=1 for the latter. BMF data should be lipid 

normalized for a thermodynamic analysis, i.e., is the chemical fugacity increasing from diet to consumer 

[8].

From Lake Ontario all lipid normalized fish BMFs for trout/alewife, trout/sculpin, trout/smelt

(n=36) for MCCP isomers analyzed were < 1 (ranging from 0.01 for C16Cl7 to 0.86 for C14Cl7). From Lake 

Michigan all lipid normalized fish BMFs for trout/alewife (n=8) were < 1 (ranging from 0.14 to 0.40 for 

C14Cl5 up to C15Cl6). In Lake Michigan some lipid normalized fish BMFs for trout/sculpin (n=3) MCCPs 

isomers were > 1 and some (n=5) were < 1 (ranging from 0.61 to 1.2 for C14Cl5 up to C15Cl6). For MCCPs 

with molar mass greater than C15Cl6, BMFs could not be calculated in Lake Michigan and Lake Ontario 

because the isomer concentrations were below detection limits or not analyzed. A weight of evidence

from the field BMF data (i.e. 94% of the data) indicate that these MCCPs do not biomagnify in fish.

TMFs

TMFs were calculated for the C14, C15 and C16 isomer groups and for total MCCP from the Lake 

Ontario samples. TMF values for Lake Ontario ranged from 0.14 (C16) to 0.29 (C14), with a value of 0.22 

for total MCCP. These TMF data also indicate that these MCCP constituents do not biomagnify in 

aquatic food webs in the environment.
17

Other measured data – Other BCFs, BSAFs

Given the time frame of the current project, the other bioaccumulation studies listed in the various 

documents were not examined and reviewed here. Such data could be evaluated and could possibly to 

add to the analysis and weight of evidence presented herein.

Biotransformation Rate Constants (in vivo, in silico) - kM

As highlighted in Figure 3 and noted in the literature, the biotransformation rate constant (kM) is a 

critical parameter determining the overall bioconcentration and bioaccumulation for hydrophobic 

organic chemicals in aquatic and terrestrial organisms. Based on in vitro and in vivo biotransformation 

studies for fish, birds and mammals, the primary biotransformation pathway for n-linear alkanes in fish 

is through hydroxylation at the end of the chain into corresponding alcohols, which are then 

transformed into the fatty acid, which are then subject to β-oxidation [32, 33]. Chain length can 

influence which enzymes are involved in the biotransformation reaction. Variation in the chlorine 

substitution pattern, primarily at the ends of the alkane chain, may therefore also be key determinants 

of the kinetics for the first transformation reaction. There is clear evidence that MCCP congeners and 

MC-PCAs are biotransformed from the in vivo dietary fish bioaccumulation studies. The objective here is 

to estimate the rates at which these chemicals are biotransformed from the in vivo laboratory test data 

and compare these values in terms of chain length, chlorine substitution and exposure route (Water-BCF

vs. Diet-BMF). 

A mass balance method for estimating in vivo biotransformation rate constants (kM /d) from 

laboratory data (i.e., BCF and BMFs tests) was used here [9]. Because the method accounts for testspecific information (i.e., fish body size, lipid content, temperature, feeding rate, growth rate, etc) and 

these factors are known to influence bioaccumulation kinetics (e.g. [34]), the mass balance estimation

method is superior to the kM estimates provided by Fisk et al. [35] (see also [9, 36]). The mass balance 

method used here also includes a screening-level uncertainty analysis to characterize uncertainty in 

calculated kMs based on the uncertainty in the measured data (i.e., rate constants, body size, lipid 

content), key model parameters and chemical properties used to parameterized the model (i.e., KOW). 

The method was parameterized here using median values from the laboratory tests. For example, from 

the data reported in BCF Test #2 for C14H25.5Cl4.5 and a log KOW of 8 a median kM estimate is 0.013 /d. A 

QSAR model for estimating kM from chemical structure was also used to compare with the in vivo 

estimates [1, 37]. 

Table 1 summarizes in vivo and QSAR estimates for kM,N (note “normalized for body mass”, i.e., 10 g) 

for the chemicals that have been tested and reviewed to be of usable quality in the present document. 

The uncertainty factors for these in vivo kM,N estimates are about 2, i.e., 95% probability that the kM,N

value lies between a factor of 2 of the listed median. A general rule of thumb for the QSAR predictions is 

about a factor of 5 [37]. There is notable overlap between the in vivo and QSAR kM,N estimates for a 

selected constituent thus providing some confidence in the plausible range of values. It is recognized 

that the measured k2g values derived without chemical-specific analyses are more uncertain than k2g

derived using parent chemical specific analysis. The uncertainties related to this issue are not captured 

in the uncertainty assigned to the measured k2g data for these kM estimates. However, as a case study 

using the BCF Test #2 data (Hurd and Vaughan, 2010) and noted in Table 1 as C14H25.5Cl4.5b, the measured 

k2 value (before growth correction) was assumed to be 20% faster to approximate a potential difference 

in the rates based on in the percentage of metabolites estimated at the end of the test. Note that this 
18

“20% faster assumption” is a first approximation based only on the percentage of metabolites 

determined at the end of the test; the approximation is assumed and unconfirmed.

The kM,N estimates are all within the same “order of magnitude”. The in vivo values range from 0.010 

to 0.022 /d and the median value is 0.016 /d (i.e., < factor of 3 for the range of values) regardless of 

exposure route (i.e., BCF-water or BMF-diet) and fish lipid content and body size. There are no easily 

recognizable differences in kM,N estimates for these chemicals based on differences in carbon chain 

length and chlorine substitution; however, given the relatively small differences for the chemicals 

studied here compared to the range of structural differences for all PCAs and CPs, there may be 

relationships between biotransformation rates and structure when greater chemical diversity is 

considered. There were no clear differences in the Cl-terminal substituted chemical kM,N estimates

compared to the non Cl-terminal substituted chemicals (i.e., expected MCCP constituents). In general, 

the degree of chlorination based on %w/w may be misleading with respect to the potential differences 

in biotransformation rates. The in silico values range from 0.003 to 0.022 with a median value of 0.016 

/d. Given the general uncertainties in biological data such as biotransformation rate constants and 

predicting such data from structure, the strong similarities between the in vivo and QSAR rates shown 

here are notable. The QSAR predictions reflect a relationship of slower biotransformation rates for 

larger, more chlorinated CPs; however, the pattern is not as recognizable in the in vivo estimates for 

these MCCPs. The potential reason for this trend in the QSAR predictions is because the QSAR includes 

KOW as a predictor and KOW predictions by KOWWIN are automatically included in the screening-level EPI 

Suite kM,N QSAR predictions.

Decreasing biotransformation rates of PCAs has been associated with increasing Cl content and 

carbon chain length in birds and fish [27]. This observation and inference may be more apparent by 

considering a broader range of CPs (i.e., including SCCPs and LCCPs); however, based on the current 

analysis for MC-PCAs and MCCPs in fish the differences in the biotransformation rate constant appear to 

be relatively small. Further analysis of the hypothesis could be examined using the laboratory data for 

SCCPs, etc.

It is recognized that it is not feasible to measure and test all MCCP constituents. There is no 

notable trend in differences in kM for the range of carbon length and chlorine substitution included in 

the available MC-PCA and MCCP test data. This suggests that additional requested testing may not 

provide any “new” information, although a test with non Cl-terminal substitution MCCP constituents 

conducted with chemical-specific analysis could perhaps provide more definitive estimates (less 

uncertainty) of key bioaccumulation parameters such as BCF, BMF, BAF, half-life and kM.

The upper bound kM,N estimate is less than the value suggested by the model calculations in Figure 

3 to lower the dissolved water concentration BCF below 5,000. 
Table 1. Summary of in vivo and in silico (QSAR) kM estimates.

in vivo in vivo in silico in silico

Test Reference MolFormula k2g kM,N kM,N log Kow Representative structure

BCF Test #2 Hurd and Vaughan, 2010 C14H25.5Cl4.5a 0.013 0.014 0.022 7.37 CC(Cl)CCCC(Cl)CC(Cl)CCCC(Cl)CC

BCF Test #2a Hurd and Vaughan, 2010 C14H25.5Cl4.5b 0.022 0.024 0.022 7.37 CC(Cl)CCCC(Cl)CC(Cl)CCCC(Cl)CC

BMF Test #1 Fisk et al. 1998 C14H26Cl4 0.018 0.019 0.018 7.74 C(Cl)C(Cl)CCCCCCCCCCC(Cl)C(Cl)

BMF Test #1 Fisk et al. 1998 C14H25Cl5a 0.013 0.014 0.017 7.69 C(Cl)C(Cl)CCCCC(Cl)CCCCCC(Cl)C(Cl)

BMF Test #1 Fisk et al. 1998 C14H25Cl5b 0.015 0.016 0.017 7.69 C(Cl)C(Cl)CCCCCCCCC(Cl)CC(Cl)C(Cl)

BMF Test #1 Fisk et al. 1998 C14H24Cl6b 0.016 0.017 0.015 7.64 C(Cl)C(Cl)CCCC(Cl)CCCCC(Cl)CC(Cl)C(Cl)

BMF Test #3 Fisk et al. 2000 low dose C14H23.3Cl6.7 0.012 0.013 0.015 7.64 CC(Cl)CC(Cl)CC(Cl)CCCCC(Cl)CC(Cl)C(Cl)

BMF Test #3 Fisk et al. 2000 high dose C14H23.3Cl6.7 0.017 0.020 0.015 7.64 CC(Cl)CC(Cl)CC(Cl)CCCCC(Cl)CC(Cl)C(Cl)

BMF Test #2 Fisk et al. 1996 low dose C16H31Cl3 0.014 0.016 0.013 8.41 CC(Cl)CCCC(Cl)CCCCCCCC(Cl)CC

BMF Test #2 Fisk et al. 1996 high dose C16H31Cl3 0.019 0.022 0.013 8.41 CC(Cl)CCCC(Cl)CCCCCCCC(Cl)CC

BMF Test #2 Fisk et al. 1996 low dose C16H21Cl13 0.012 0.014 0.003 8.70 CC(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)CC

BMF Test #2 Fisk et al. 1996 med dose C16H21Cl13 0.011 0.013 0.003 8.70 CC(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)CC

BMF Test #2 Fisk et al. 1996 high dose C16H21Cl13 0.009 0.010 0.003 8.70 CC(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)C(Cl)CC

aNote these data are for an assumed and unconfirmed faster rate of elimination based on measured 20% metabolites at the end of the test, 

thus they are considered “exploratory”
Incorporating kM,N for Bioaccumulation Assessment - BCFs 

Although the BMF and TMF are purported to provide the most appropriate answers (insights) to the 

question as to whether or not a chemical biomagnifies in organisms and food webs, there is seemingly 

endless interest in the BCF for bioaccumulation hazard assessment. Thus some BCF estimates for the 

chemicals in Table 1 are provided in Table 2 using a BCF model and the in vivo kM estimates derived 

above. For the BCFs predicted based on total water concentrations, a value of 1 mg/L DOC was assumed 

based on values inferred from BCF Test #2. The BCFs are for a fish of 10 g, 5% lipid content at 15 °C

generated using the spreadsheet version of the lower trophic level fish in the BCFBAF model [1, 21]

(www.arnotresearch.com). Note that the column on the right shows growth corrected BCFs based on 

the dissolved water concentrations, i.e., BCF data most appropriate for comparing the “inherent 

chemical properties”. Also note that the most relevant BCF model calculation for comparison with the 

measured BCF data from Test #2 (best quality BCF available: 4,440) is the value of 4,500 L-total water / 

kg-wet weight.

The BCF findings here are generally consistent with the recommendations in the SEV report with 

respect to growth corrected BCF calculations and REACH BCF bioaccumulation criteria; however, these 

bioaccumulation hazard categorizations seemingly contradict the measured observations for potential 

chemical biomagnification in the field (i.e., BMFs and TMFs < 1). There is uncertainty in all measured and 

modelled data, including those data compiled, evaluated and applied in the SEV document and 

discussed here. Burkhard and colleagues [8] developed a weight of evidence approach for assessing 

whether or not a chemical is expected to biomagnify in the environment and such an approach could be 

considered for MCCPs for further data analysis. 

Table 2. Summary of some BCF calculations using in vivo kM estimates from various studies.

BCF (L-total/kg-ww) BCF (L-total/kg-ww) BCF (L-diss/kg-ww)

Test MolFormula Including growth1 Growth-corrected Growth-corrected

BCF Test #2 C14H25.5Cl4.5a 4500 17400 33700

BCF Test #2 C14H25.5Cl4.5b 3800 11600 22500

BMF Test #1 C14H26Cl4 2200 8900 28300

BMF Test #1 C14H25Cl5a 2700 12500 37100

BMF Test #1 C14H25Cl5b 2600 11000 32600

BMF Test #1 C14H24Cl6b 2800 11200 30400

BMF Test #3 C14H23.3Cl6.7 3100 14100 38300

BMF Test #3 C14H23.3Cl6.7 2700 9900 27000

BMF Test #2 C16H31Cl3 600 3200 36600

BMF Test #2 C16H31Cl3 500 2400 27000

BMF Test #2 C16H21Cl13 300 2100 43300

BMF Test #2 C16H21Cl13 400 2300 47400

BMF Test #2 C16H21Cl13 400 2700 57700 1 assuming kG = 0.03 /d, simulating BCF Test #2
21

General Comments on SEV Document

The REACH SEV document presents a review and analysis on bioaccumulation assessment for 

MCCPs. The analysis includes REACH methods and criteria and shows creative efforts to characterize the 

bioaccumulation of different untested expected MCCP congeners based on different measured 

endpoints. There are some apparent misunderstandings related to BCF calculations (i.e. total vs. 

dissolved water concentrations) for BCF data and current model interpretation. The SEV document 

includes an “overabundance” of k1 models for calculating BCFs from dietary test data (i.e., Tables 27 and 

28); a single model or an average of k1 models would have been sufficient in the proper context. 

Extrapolation methods applied to “scale BCFs” are questionable (pages 67-71 and pages 178-186), since 

the bioaccumulation conditions and chemicals are not consistent; thus the approximated scaling 

approach is limited and confusing in the current context. Mechanistic interpretations of the measured 

data would be more appropriate (e.g., as illustrated here). The half-life comparison is good, but could be 

improved as outlined here to address differences body composition, mass and temperature. 

There is uncertainty in the actual MCCP constituent / congener structure (i.e., chlorine substitution); 

however, this is an issue for all interpretations of the data and is not only an issue in the SEV document. 

The approach to examine different KOW estimates in the SEV document is a reasonable first approach to 

address uncertainty in constituents and KOW; however, as shown here the uncertainty in the actual 

structure of MCCP constituents and KOW may be relatively inconsequential for the bioaccumulation 

assessment when using BCF and BAF endpoints and criteria.

Comment on “SEV Report section 8.1.4 Summary and overall conclusions on PBT and 

vPvB properties”

“The PBT assessment is complicated because both the biodegradability and bioconcentration 

factor of MCCP constituents depend on chlorine content and chain length, and the trends in these are 

opposite (i.e. persistence appears to increase with increasing chlorine content whilst the 

bioconcentration factor appears to decrease).”

The analysis provided in the present document explains some of the “apparent contradictions” 

mentioned in the SEV report when interpreting measured and modeled BCF data. In particular, the 

present analysis illustrates why some measured BCFs “go up” while some predicted Arnot and Gobas 

BCFs “go down”, i.e., the Arnot and Gobas BCF and BAF models in BCFBAF use default POC and DOC 

values typical for North American surface water, i.e., 0.5 mg/L and 0.5 mg/L, respectively. POC and DOC 

values in filtered tap water will be lower than surface water, thus increasing the bioavailable solute 

fraction (and hence measured BCFs) for hydrophobic organics under most laboratory conditions. This 

apparent contradiction does not exist when the fish bioconcentration data are considered in terms of 

the estimated bioavailable (dissolved) concentrations in the water. The reduced dietary bioaccumulation 

for higher chlorinated, higher KOW MCCP constituents is also consistent and explained by the measured 

reduced dietary uptake efficiency for very hydrophobic organics (see, e.g., [6, 7, 21, 29, 30].
22

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