Fatty acids, C12-14 (even numbered), methyl ester

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

28-days uptake/4-days depuration study with Lepomis macrochirus under flow-through conditions.

GLP compliance:

no

Radiolabelling:

yes

Details on sampling:

- Sampling intervals/frequency for test organisms: Sampling of fish was performed at 0.5, 1, 2, 4, 8, 14, 21 and 28 days during exposure phase. During clearance phase, fish were sampled at 1, 2 and 4 days.
- Sampling intervals/frequency for test medium samples: Determination of total radioactivity in water samples was made daily. For analysis of parent compound and metabolites water samples from exposure aquarium were analysed at least once a week.
- Sample storage conditions before analysis:
- Details on sampling and analysis of test organisms and test media samples: Water samples were analysed using Liquid Scintillation (LS) counting method. Fish tissue samples were analyzed for radioactivity by combusting.

Vehicle:

yes

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: bluegill
- Length at study initiation (lenght definition, mean, range and SD): 3 - 4.5 cm
- Weight at study initiation (mean and range, SD): 0.5 - 0.7 g
- Feeding during test: yes
- Food type: synthetic diet


ACCLIMATION
- Acclimation period: 21 days
- Acclimation conditions (same as test or not): same

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

4 d

Hardness:

73 - 76 mg/L

Test temperature:

16.3 - 17.9 °C

pH:

7.7 - 8.1

Dissolved oxygen:

8.1 - 9.6 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, fill volume: glass aquaria, fill volume 4 Litre
- Aeration: no, flow through test
- Renewal rate of test solution: 6 volume changes every 24 h
- No. of vessels per concentration: 1
- No. of vessels per control: 1

OTHER TEST CONDITIONS
- Adjustment of pH: with CO2 to approx. pH 8

Nominal and measured concentrations:

Nominal: 0 (vehicle control), 0.33 µg/L
Measured: < LOD, 0.29 µg/L (average)

Reference substance (positive control):

no

Details on estimation of bioconcentration:

BASIS FOR CALCULATION OF BCF
- Estimation: Tow compartment model is used to describe the uptake and elimination of xenobiotcs by fish.

Key result

Type:

BCF

Value:

< 17 dimensionless

Basis:

not specified

Time of plateau:

28 d

Calculation basis:

kinetic

Remarks on result:

other: Conc.in environment / dose:0.29 µg/L

Metabolites:

Haloxyfop, polar metabolites 1 and 2.

Bluegill exposed to 14C haloxyfop-methyl for 28 days were found to rapidly absorb the ester from water which was then biotransformed at an extremely fast rate within the fish such that essentially no haloxyfop-methyl was detected in the fish. The estimated bioconcentration factor for the haloxyfop-methyl in whole fish was < 17, based upon the detection limit for ester in fish and the average concentration of haloxyfop-methyl in exposure water. The total 14C residue level within whole fish averaged about 0.27 µg/g equivalents over the course of the uptake phase. The principal component of the 14C residue was haloxyfop, which accounted for an average of about 60% of the radioactivity. Two other polar metabolites were detected in the fish which accounted for an average of about 14% of the radioactivity and an average of about 25% of the radioactivity. Once the fish were transferred to clean water, all metabolites cleared quickly with similar clearance rates. A simulation model estimated the uptake rate constant of haloxyfop-methyl from water to be about 720 mL/g/day. The rate constants for biotransformation of haloxyfop-methyl and the clearance of metabolites formed were estimated to be 200 days-1 (t1/2 = 5 min) and 0.82 day-1 (t1/2 = 0.8 days), respectively. The high rate of biotransformation of the parent compound within the fish demonstrates the importance of basing the bioconcentration factor upon the actual concentration of parent material within the organisms rather than the total radioactive residue levels for radiolabeled bioconcentration studies.

Reference 2

Endpoint:

bioaccumulation in aquatic species, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The concept “Environmental Hazard Profile” investigated concentration factors in activated sludge, in algae, and fish. The microbial degradation of chemicals to CO2 in activated sludge and the decomposition to CO2 under artificial light were determined.

GLP compliance:

not specified

Radiolabelling:

yes

Test organisms (species):

other: Leuciscus idus melanotus, Chlorella fusca var.vacuolata and activated sludge

Route of exposure:

aqueous

Test type:

static

Water / sediment media type:

natural water: freshwater

Nominal and measured concentrations:

Nominal concentration: 0.05 mg/L

Details on estimation of bioconcentration:

- Fish: bioconcentration was measured as the concentration of chemical in the fish/average concentration of chemical in water (µg/g)
- Algae and activated sludge: bioconcentration was measured as the concentration of chemical in algae or sludge/final concentration of chemical in water (µg/g)

Key result

Type:

BCF

Value:

< 10

Basis:

whole body w.w.

Remarks on result:

other: Fish (3 days)

Remarks:

Conc.in environment / dose:0.05 mg/L

BCF values for algae and activated sludge were 28600 and 470 respectively. The differences observed between fish and algae/activated sludge values respectively suggests a detoxification process in higher developed organisms as fish (metabolism).

Reference 3

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study reports in vitro metabolic transformation tests using fish S9 fractions to refine bioconcentraion factor (BCF) computer model estimates.

GLP compliance:

no

Test organisms (species):

other: Oncorhynchus mykiss

Route of exposure:

other: in vitro liver S9 fraction

Test type:

other: in vitro

Water / sediment media type:

natural water: freshwater

Type:

BCF

Value:

63 dimensionless

Calculation basis:

other: Predicted BCF incorporating kMET based on total hepatic blood flow

Remarks on result:

other: Haloxyfop ME

Type:

BCF

Value:

56 dimensionless

Calculation basis:

other: Predicted BCF incorporating kMET based on total hepatic blood flow

Remarks on result:

other: Fluroxypyr ME

Table: Comparison of predicted BCF and measured BCF using kMET (whole body metabolic transformation rate constant) estimated from trout S9 in vitro los data.

Chemical (log Kow)	Measured in vitro loss rate (µmol of parent lost g active protein h)	Modeled kMET (d-1) assuming only arterial hepatic blood flow	Modeled kMET (d-1) assuming total arterial hepatic blood flow	Predicted BCF Using log Kow only	Predicted BCF incorporating kMET based on arterial hepatic blood flow	Predicted BCF incorporating kMET based on total hepatic blood flow	Measured BCF (OECD 305)
Haloxyfop – ME (3.5)	12.6	0.212	1.22	313	186	63	13
Fluroxypyr - MHE (4.7)	444	0.257	1.76	4288	355	56	6

The results demonstrate that refined BCF estimates when metabolism rates are included were more similar the measured BCF values for the chemicals tested than the modeled BCF values based in Kow alone. Furthermore, when arterial blood flow was used to represent hepatic blood flow, the refined BCF estimates were very close to the measured BCFs for the chemicals that are primarily metabolized by hepatic Phase I enzymes. And let to overestimates of BCF for chemicals that can be extensively metabolized in the gut and/or by more ubiquitous enzymes such as esterases, and ehterases. This implies that use of arterial blood flow to the liver in the scale-up equations will produce a conservative estimate of the BCF, which depending the loss rate of the chemical in the in vitro test, could be sufficient to indicate that the chemical does not have bioaccumulation potential above any criteria for concern.

Reference 4

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Category justification document is attached in chapter 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Conc. / dose:

0.29 µg/L

Type:

BCF

Value:

< 17

Basis:

whole body w.w.

Time of plateau:

28 d

Calculation basis:

kinetic

Remarks on result:

other: CAS 69806-40-2

Key result

Conc. / dose:

0.05 mg/L

Type:

BCF

Value:

< 10

Basis:

whole body w.w.

Time of plateau:

3 d

Calculation basis:

steady state

Remarks on result:

other: CAS 67-56-1

Reference 5

Endpoint:

bioaccumulation in aquatic species, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

QMRF and QPRF are attached.

Principles of method if other than guideline:

Calculation based on BCFBAF v3.01, Estimation Programs Interface Suite™ for Microsoft® Windows v 4.10. US EPA, United States Environmental Protection Agency, Washington, DC, USA.

GLP compliance:

no

Test organisms (species):

other: Fish

Route of exposure:

aqueous

Test type:

other: calculation

Water / sediment media type:

natural water: freshwater

Details on estimation of bioconcentration:

BASIS FOR CALCULATION OF BCF
- Estimation software: BCFBAF v3.01
- Result based on calculated log Kow of: 6.41 (KROP, HB ET AL. 1997)

Key result

Type:

BCF

Value:

201 L/kg

Basis:

whole body w.w.

Remarks on result:

other: Arnot-Gobas (including biotransformation rate estimates, upper trophic)

Type:

BAF

Value:

215.1 L/kg

Basis:

whole body w.w.

Remarks on result:

other: Arnot-Gobas (including biotransformation rate estimates, upper trophic)

Details on results:

For detailed description on the model and its applicability, see "Any other information on materials and methods incl. tables".

Estimated Log BCF (mid trophic)  = 2.441 (BCF = 276 L/kg wet-wt)

Estimated Log BAF (mid trophic)  = 2.781 (BAF = 603.9 L/kg wet-wt)

Estimated Log BCF (lower trophic) = 2.483 (BCF = 304.2 L/kg wet-wt)

Estimated Log BAF (lower trophic) = 3.328 (BAF = 2128 L/kg wet-wt)

 

Arnot-Gobas BCF & BAF Methods (assuming a biotransformation rate of zero):

Estimated Log BCF (upper trophic) = 4.292 (BCF = 1.957e+004 L/kg wet-wt)

Estimated Log BAF (upper trophic) = 6.780 (BAF = 6.027e+006 L/kg wet-wt)

Biotransformation Rate Constant:

kM (Rate Constant): 0.9449 /day (10 gram fish)

kM (Rate Constant): 0.5314 /day (100 gram fish)

kM (Rate Constant): 0.2988 /day (1 kg fish)

kM (Rate Constant): 0.168 /day (10 kg fish)

Biotransformation Half-Life (days) : 0.734 (normalized to 10 g fish)

 

Validity criteria fulfilled:

yes

Reference 6

Endpoint:

bioaccumulation in aquatic species, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

QMRF and QPRF are attached.

Principles of method if other than guideline:

Calculation based on BCFBAF v3.01, Estimation Programs Interface Suite™ for Microsoft® Windows v 4.10. US EPA, United States Environmental Protection Agency, Washington, DC, USA.

GLP compliance:

no

Test organisms (species):

other: Fish

Route of exposure:

aqueous

Test type:

other: calculation

Water / sediment media type:

natural water: freshwater

Details on estimation of bioconcentration:

BASIS FOR CALCULATION OF BCF
- Estimation software: BCFBAF v3.01
- Result based on calculated log Kow of: 5.41 (KROP, HB ET AL. 1997)

Key result

Type:

BAF

Value:

154.3 L/kg

Basis:

whole body w.w.

Remarks on result:

other: Arnot-Gobas (including biotransformation rate estimates, upper trophic)

Key result

Type:

BCF

Value:

154 L/kg

Basis:

whole body w.w.

Remarks on result:

other: Arnot-Gobas (including biotransformation rate estimates, upper trophic)

Details on results:

For detailed description on the model and its applicability, see "Any other information on materials and methods incl. tables".

Arnot-Gobas BCF & BAF Methods (including biotransformation rate estimates):

Estimated Log BCF (mid trophic)  = 2.322 (BCF = 210 L/kg wet-wt)

Estimated Log BAF (mid trophic)  = 2.350 (BAF = 223.8 L/kg wet-wt)

Estimated Log BCF (lower trophic) = 2.363 (BCF = 230.8 L/kg wet-wt)

Estimated Log BAF (lower trophic) = 2.527 (BAF = 336.8 L/kg wet-wt)

Arnot-Gobas BCF & BAF Methods (assuming a biotransformation rate of zero):

Estimated Log BCF (upper trophic) = 4.151 (BCF = 1.414e+004 L/kg wet-wt)

Estimated Log BAF (upper trophic) = 5.675 (BAF = 4.733e+005 L/kg wet-wt)

 

Biotransformation Rate Constant:

kM (Rate Constant): 1.816 /day (10 gram fish)

kM (Rate Constant): 1.021 /day (100 gram fish)

kM (Rate Constant): 0.5743 /day (1 kg fish)

kM (Rate Constant): 0.323 /day (10 kg fish)

Biotransformation Half-Life (days) : 0.382 (normalized to 10 g fish)

Validity criteria fulfilled:

yes

Description of key information

Fatty acids, C12-C14 (even numbered), methyl esters is expected to show low bioaccumulation potential

Additional information

5.3.1 Aquatic bioaccumulation- SCAE Me category

 No experimental data evaluating the bioaccumulation potential of the SCAE Me category members are available. All substances within the SCAE Me category have log Kow values above 3, suggesting potential to bioaccumulate in biota, with the exception of methyl hexanoate (CAS No. 106-70-7). The log Kow value estimated for this substance is 2.3 and therefore, according to Regulation (EC) No. 1907/2006, Annex IX, Column 2, 9.3.2, this substance can be considered to have low bioaccumulation potential (log Kow ≤ 3). Regarding the rest of the SCAE Me category members, the information gathered on environmental behaviour and metabolism in combination with the QSAR-estimated BCF values provide enough evidence (in accordance to the REACh Regulation (EC) No 1907/2006, Annex XI General rules for adaptation of the standard testing regime set out in Annexes VII to X, 1.2, to cover the data requirements of Regulation (EC) No. 1907/2006, Annex IX) to state that these substances are likely to show low bioaccumulation potential as well.

 

Intrinsic properties and fate

 All substances included in the SCAE Me category are readily biodegradable. According to the Guidance on information requirements and chemical safety assessment, Chapter R.7b, readily biodegradable substances can be expected to undergo rapid and ultimate degradation in most environments, including biological Sewage Treatment Plants (STPs)(ECHA, 2008). Therefore, after passing through conventional STPs, only low concentrations of these substances are likely to be (if at all) released into the environment.

 

Once available in the water phase, the estimated log Koc values (< 3, KOCWIN v2.00) of methyl hexanoate (C6 FA, CAS No. 106-70-7) and methyl octanoate (C8 FA, CAS No. 111-11-5) indicate low adsorption potential of these substances to sediment and organic particles, and therefore, they will be available for uptake by aquatic organisms such as fish mainly via water. Nevertheless, since the reported log Kow values for these two substances are 2.3 and 3.3 (CAS No. 106-70-7 and CAS No. 111-11-5), a low degree of actual bioaccumulation in aquatic organisms can be expected. According to the Guidance on information requirements and chemical safety assessment, Chapter R.11, organic substances with a log Kow value below 4.5 are assumed to not exceed the B criterion (due to an insufficient affinity for lipids)(ECHA, 2008).

 

On the other hand, the remaining substances in the SCAE Me category have log Kow values > 4 and log Koc values > 3. The Guidance on information requirements and chemical safety assessment, Chapter R7.B (ECHA, 2008) states that once insoluble chemicals enter a standard STP, they will be extensively removed in the primary settling tank and fat trap and thus, only limited amounts will get in contact with activated sludge organisms. Nevertheless, once this contact takes place, these substances are expected to be removed from the water column to a significant degree by adsorption to sewage sludge (Guidance on information requirements and chemical safety assessment, Chapter R.7a, (ECHA, 2008)) and the rest will be extensively biodegraded (due to ready biodegradability). Thus, discharged concentrations of these substances into the aqueous compartment are likely to be very low. Should the substances be released into the water phase, due to their hydrophobicity and expected high adsorption potential, they will tend to bind to sediment and other particulate organic matter, and therefore, the actual dissolved fraction available to fish via water will be reduced (Mackay and Fraser, 2000). Thus, the main route of exposure for aquatic organisms such as fish will be via food ingestion or contact with suspended solids.

 

QSAR data

Additional information on the bioaccumulation of SCAE Me in fish species is available. Estimated bioconcentration (BCF) and bioaccumulation (BAF) values were calculated for all substances using the BCFBAF v3.01 program (Estimation Programs Interface Suite™ for Microsoft® Windows v 4.10., US EPA), including biotransformation rates (Arnot-Gobas method). In the case of the UVCB substances, the calculations were performed on the main fatty acid components, as representative structures for each UVCB. All SCAE Me substances (or at least their main FA components) are within the applicability domain of the QSAR model (log Kow 0.31-8.70) and therefore, provide valid supporting information to be considered in the overall bioaccumulation assessment of these substances.

 

Within the category, BCF values tend to increase with increasing fatty acid C-chain lengths from C6 (10 L/kg) up to C14 (201 L/kg), to decrease again at longer C-chain lengths (C16, 95.6 L/kg and C18, BCF 23.3-29 L/kg), with the exception of the C18 unsaturated fatty acid component, with a BCF of 117.8 L/kg. On the other hand, BAF values increase as the fatty acid C-chain length increases, ranging from 10 L/kg (C6) up to 500 L/kg (C18 unsaturated).

The difference in the pattern for BCF and BAF values across the category can be explained by the exposure route(s) considered for the estimation: BCF calculations reflect the bioaccumulation potential after uptake via water, whereas the BAF gives an indication of the bioaccumulation when all exposure routes (water, food, etc.) are taken into account.

The obtained results indicate that the members of the SCAE Me category are likely to show low (shorter C-chain lengths) to moderate (longer C-chain lengths) bioaccumulation potential. According to Regulation (EC) No. 1907/2006, Annex XIII, 1.1.2, a substance only fulfils the bioaccumulation criterion (B) when BCF values are > 2000. Even though this condition is preferred to be confirmed with experimental data, in this case the estimated QSAR-based BCFs provide sufficient reliable evidence which suggests that the SCAE Me category members will not be bioaccumulative.

 

Biotransformation and metabolism

 

After lipid content, the degree of biotransformation seems to be the most relevant factor regarding the bioaccumulation of organic chemicals in aquatic organisms (Katagi, 2010). Biotransformation consists in the conversion of a specific substance into another/others (metabolites) by means of enzyme-catalyzed processes (ed. van Leeuwen and Hermens, 1995).

 

Carboxylesterases are a group of ubiquitous and low substrate specific enzymes, involved in the metabolism of ester compounds in both vertebrate and invertebrate species, including fish (Leinweber, 1987; Barron et al., 1999). Fatty acid methyl esters are hydrolysed to the corresponding alcohol (methanol) and fatty acid by esterases (Fukami and Yokoi, 2012). Particularly in fish rapid metabolism rates of two methyl esters (haloxyfop methyl ester and fluroxypyr methylheptyl ester) have been observed in vitro (fish liver homogenates) with half-lives of 5 and 1 minute respectively (Murphy and Lutenske, 1990; Cowan-Elsberry et al., 2008). Furthermore, in vivo studies conducted with esters, including a methyl ester (according to OECD 305) resulted in experimental fish BCFs ranging from 1 to 70, even when the log Kow values of these substances are above 3, indicating once again rapid metabolism (Rodger and Stalling, 1972; Barron et al., 1989; Barron et al., 1990).

 

According to the Guidance on information requirements and chemical safety assessment, Chapter R.7c (ECHA, 2008), even though ready biodegradability does not per se preclude bioaccumulation potential, generally (depending on exposure and uptake rates) ready biodegradable substances are likely to be rapidly metabolised, and therefore, concentrations stored in aquatic organisms will tend to be low.

 

Regarding the biotransformation products of SCAE Me(s), methanol will partially tend to evaporate from water surfaces (Henry's Law Constant of 4.55x10-6 atm m3/mole (Gaffney et al., 1987)) or stay in the water phase (low adsorption potential to sediment and organic particles according to a Koc of 2.75 (Schuurmann et al, 2006)), in which rapid biodegradation is expected to occur (92% biodegradation in 14 days; NITE, Japan, 2012). Therefore, its bioavailability to aquatic organisms will be generally low.

Methanol is a naturally occurring compound in living organisms. It is known to be metabolised and further excreted in the form of CO2 and H2O in several species such as mammals. The log Kow value of this substance (-0.77, Hansch et al., 1995) indicates that bioaccumulation in biota is not expected. In fish (Leuciscus idus), this was confirmed by a test in which a measured BCF < 10 was obtained for methanol (Freitag et al., 1985).

 

On the other hand, fatty acids are naturally occurring components in living organisms (mammals, aquatic organisms, earthworms, plants, etc.), which are known to be metabolised quickly and participate in ubiquitous standard physiological processes (e. g. citric acid cycle, sugar synthesis and lipid synthesis)(Hochachka et al., 1977; Jump, 2002). In fish species, fatty acids are the most important energy source resulting in the release of acetyl CoA and NADH (through β-oxidation) and eventually, via the tricarboxylic cycle, the production of metabolic energy in the form of ATP. This fatty acid-catabolism pathway is the predominant source of energy related to growth, reproduction and development from egg to adult fish (Tocher, 2003). A similar metabolic pathway is observed in mammals (see section 7.1.1 Basic toxicokinetics).

 

Conclusion

The substances included in the SCAE Me category are not expected to be bioaccumulative. Due to their readily biodegradable nature, extensive degradation of these substances in conventional STPs will take place and only low concentrations are expected to be released (if at all) into the environment. Once present in the aquatic compartment, further biodegradation will occur and, depending on their log Kow, water solubility and adsorption potential, the SCAE Me(s) will be bioavailable to aquatic organisms such as fish mainly via water or on the other hand via feed and contact with suspended organic particles. After uptake by fish species, extensive and fast biotransformation of the SCAE Me(s) by carboxylesterases into fatty acids and methanol is expected. Fatty acids will be further used by these organisms as their main source of energy throughout all the different life stages (early development, growth, reproduction,etc.). Rapid metabolism of analogue ester compounds (involving hydrolysis into fatty acids and methanol) in fish has been observed in vitro, with half-lives in fish liver homogenates below 6 minutes. In vivo fish tests reported BCF values ranging from 1 to 70 for similar ester substances, supporting the argument that rapid metabolism takes place even when log Kow values are above the trigger value of 3. The supporting BCF/BAF values estimated with the BCFBAF v3.01 program also indicate that these substances will not be bioaccumulative (all well below 2000).

 

The information above provides strong evidence supporting the statement that rapid metabolism and low bioaccumulation potential can be expected for the members of the SCAE Me category (detailed information on the results of a more extensive literature search can be found as an attachment).

 

A detailed reference list is provided in the technical dossier (see IUCLID, section 13) and within CSR.