Alkanes, C14-17, chloro

Endpoint summary

Description of key information

[Note: See CSR for complete text, several figures could not be uploaded into IUCLID]

The biodegradation of MCCP has been evaluated by numerous studies on both commercial products and specifically identified components of interest, in particular different chlorinated version of C₁₄(tetradecane) and C₁₅(pentadecane). The ability to test the biodegradation of MCCP is high dependent upon the testing system set up given the very low water solubility of MCCP and its high potential to sorb onto organic matter and vessel walls in the test system. Studies that have taken this into account and made MCCP more bioavailable have demonstrated that MCCP up to 50% chlorination by weight is readily biodegradable and that MCCP in the 50-60% chlorination (by wt.) is inherently biodegradable.

 

A series of GLP Closed Bottle Test (CBT), OECD 301D guideline, studies were conducted in 2009 and 2010 to evaluate the ready biodegradation of MCCP and its constituents (van Ginkel 2010 a,b,c,d,e). In these CBT studies 2 mg/L of the test material was incubated in the dark with either river water or secondary activated sludge derived from a plant treating predominantly domestic wastewater and oxygen consumption was measured by analyzing for dissolved oxygen content. These studies were conducted using procedures to improve the bioavailability of the test material to the inoculum, in accordance with the guideline. In the first study, a chlorinated tetradecane (C14) at 45% Cl (wt.) showed biodegradation on days 0, 7, 14, 21, 28 and 42 of 0, 3, 14, 32, 64, and 67%, respectively. Therefore, this test material is considered “readily biodegradable” (van Ginkel, 2010a). Subsequently, three MCCP commercial products (46.5, 51.7% and 63.2% Cl wt.) were assessed in the CBT (van Ginkel, 2010b,c,d). Lastly, a comparative study of the biodegradation rates of chlorinated paraffins by chlorination levels was conducted using tetradecane chlorinated across the range from approximately 40% to 60% Cl (wt.) (van Ginkel 2010e). Table 1 below summarizes the results of these studies.

 

Table 1: Percent Degradation by Test Material from van Ginkel 2010 CBT Studies

Test Material	C14, 45% Cl^a	MCCP, 45.6% Cl^b	MCCP, 51.7% Cl^d	MCCP, 63.2% Cl^c	C14, 41.4% Cl^e	C14, 45.4% Cl^e	C14, 50.0% Cl^e	C14, 55.0% Cl^e	C14, 60.2% Cl^e
Sampling Day
7	3	0	7	0	3	5	1	0	4
14	14	9	10	5	34	28	13	4	11
21	32	31	17	14	56	64	46	18	22
28	64	51	27	5	62	73	54	30	28
42	67	63	47	5	74	75	71	50	39
56					83	73	78	57	49
60			57	10

a: van Ginkel 2010a

b: van Ginkel 2010b

c: van Ginkel 2010c

d: van Ginkel 2010d

e: van Ginkel 2010d

 

Additional OECD guideline 301D CBT studies (van Ginkel 2014a,b and van Ginkel 2018a,b,c, d) were conducted on polychlorinated tetradecane (C14, 50, 55, 60% Cl by wt.) and pentadecane (C15, 51% Cl by wt.) as these test substances were identified by ECHA and the U.K. Environment Agency for additional evaluation as a part of an overall substance evaluation of MCCP. The 2014 studies on C15 (51% Cl wt.) found that the test material degraded up to 63% on day 60, indicating that the test material is inherently biodegradable and not persistence. The 2018 CBT studies included additional chemical analysis of the test materials beyond the standard biodegradation parameter, oxygen consumption in the case of the OECD 301D, and were conducted over an extended period of 120 days. The chemical analysis portion of this testing, conducted by Vrije Universitat (VU) Amsterdam, compared two different high-resolution methods of chemical analysis – GCxGC-ECD and APCI-TOFMS - to determine their abilities to measure and distinguish chloroalkane congener groups (e.g. C₁₄H₂₆Cl₄) within the test materials. Both methods performed well and were able to quantify C₁₄congener groups from Cl₄to Cl₁₁, which represent a range of approximately 35-68% chlorine by weight. This is the first time that such a congener group analysis has been applied to the biodegradation testing of MCCP. It should be noted that whilst the test materials are chlorinated to specific levels of chlorine by weight, they all contain generally the same congener groups just in different proportions. The removal of the parent compounds after 120 days by congener group for the chlorinated tetradecane studies (van Ginkel 2018a,b,c) are provided in the table below, including a comparison of the two analytical methods used, except in the case of the C14, 60% Cl test material where the results of a second experiment were included given the significant removal results from the first experiment.

 

Table 2: Removal of Chlorinated Congener Group in CBT Studies (van Ginkel 2018a-d) After 120 Days

Test Material:	C14 50%Cl		C14 55%Cl		C14 60%Cl		C15 51%Cl
Congener Group	GCxGC-ECD	APCI-TOFMS	GCxGC-ECD	APCI-TOFMS	APCI-TOFMS 1^stExperiment	APCI-TOFMS -2nd Experiment*	GCxGC-ECD	APCI-TOFMS
Cl4	92%	99%	74%	76%	92%	86%	93%	-**
Cl5	92%	97%	74%	71%	92%	82%	89%	96%
Cl6	90%	93%	75%	68%	92%	81%	89%	95%
Cl7	83%	87%	61%	62%	91%	80%	84%	88%
Cl8	83%	75%	64%	48%	91%	78%	78%	76%
Cl9	83%	60%	57%	19%	89%	76%	76%	66%
Cl10	73%	57%	46%	-	86%	70%	72%	61%
Cl11	79%	71%	49%	-	80%	60%	65%	56%
Cl12	76%	82%	27%	-	75%	50%	41%	52%
Total reduction	86%	85%	65%	50%	89%	77%	83%	86%

* A second experiment was conducted and analyzed given the greater than expected removals in the first experiment.

** Reliable results were not determined for this congener group using the APCI-TOFMS method.

 

For the C14, 50% and 55% Cl (by wt.) test materials the Day 60 results are very similar to the Day 120 results (see Figure 1 below). For the C14, 60% Cl (by wt.) test material there continued to be significant biodegradation from Day 60 to Day 120. These results may be indicative of the slower biodegradation rates of more highly chlorinated congener groups, which will have more highly chlorinated metabolites. The more extensive biodegradation of the C14, 60% Cl (by wt.) test material compared to the C14, 55% Cl (by wt.) was an unexpected result, though a second experiment confirmed the extensive biodegradation of this test material.

 

Figure 1: Test Material Removal Rates for C14 Test Materials in Closed Bottle Tests (van Ginkel 2018a-c)

 

 

An attempt was made to analyze for metabolites in these CBT studies. Whilst metabolites were detected, their specific identification and quantification is difficult without having established standards. The APCI-TOFMS method was felt to be superior for metabolite detection since the metabolite and CP could still be clustered into congener groups. Figure 2 shows the Day 0 and Day 120 mass spectrometry analysis of the C14, 50% Cl test material. These results suggest that essentially all of the Cl₄, Cl₅and most of the Cl₆test material and metabolites are completely mineralized but that there will be metabolites and CP test material remaining from Cl₇and up. The fact that the Cl₇peaks are about the same and Cl₈and Cl₉peaks are higher is being interpreted as a sign of the formation of metabolites in these congener groups from higher (i.e. C₁₀₊) congener groups in the test material.

 

Figure 2:APCI-TOFMS: CP and Metabolite Detection Using C14, 50% Cl Test Material

 

 

In earlier studies, the degradation of four C14-17 chlorinated paraffins (with extent of chlorination ranging from 40 to 58%) has been determined by measuring oxygen consumption during incubation with non-acclimated microorganisms for up to 25 days at concentrations of 2, 10 and 20 mg/L. The extent of biodegradation varied from 15% (for the 40% chlorinated paraffin) to 0% (for the 58% chlorinated paraffin) at day 25. The 5-day biochemical oxygen demand (BOD5) values ranged from 0.02 g O2/g of a 40% chlorinated C14-17 paraffin to “no significant oxygen uptake” for a 58% chlorinated C14-17 paraffin (Madeley and Birtley, 1980).

 

The degradation of two MCCPs has been studied by Omori et al. (1987) using a variety of bacterial cultures. The MCCPs studied had averages of C14.5 (43.5% chlorinated) and C15.4 (50% chlorinated) and degradation was studied by monitoring the release of chloride ion. Firstly, degradation of the 50% chlorinated paraffin was studied using resting cell cultures of Pseudomonas aeroginosa, Achromobacter delmarvae, A. cycloclastes, Micrococcus sp. and Corynebacterium hydrocarboclastus grown on glycerol. Little or no degradation (chloride release) was observed when the C15 chlorinated paraffin was incubated with the bacteria for 24 hours at 30oC. In addition, cometabolic biodegradation experiments were carried out with the C14.5 and the C15.4 chlorinated paraffins (43.5% and 50% chlorinated) at a concentration equivalent to 180 mg Cl/1.2 L solution, using a mixed bacterial inoculum (containing strains HK-3, HK-6, HK-8 and HK-10) incubated at 30oC for 48 hours. Bacterial strains were isolated from soil (no further details on soil characteristics given in paper) using an enrichment culture containing n-hexadecane as the sole carbon source. Both paraffins showed significant degradation, with 77 mg/L and 85 mg/L of chloride being released from the 50% and 43.5% chlorinated paraffins, respectively, after 36 hours. As the starting MCCP concentration was 180 mg Cl/1.2 L solution, about 51 and 57% of the chlorine present was released during the degradation, respectively. These findings suggest the potential for biodegradation appears to increase with decreasing chlorine content (Omori et al. 1987). The authors concluded that the degradation seen was consistent with that previously observed for other chlorinated alkanes in that a variety of enzymes are required to degrade chlorinated paraffins and that the most likely mode of degradation involves firstly dechlorination of the terminal methyl groups, with subsequent oxidation to form chlorinated fatty acids, which are then broken down to 2- or 3-chlorinated fatty acids via β-oxidation (EU, 2005).

 

A recent analysis of the likely biochemical biodegradation pathways was conducted on a series of theoretical constituents in MCCP (Federle 2017). This assessment identified multiple biodegradation pathway options that may exist for a chlorinated paraffin (CP) molecule and that all of these pathway options involve conversion of the CP to a chlorinated fatty acid, shortening of the carbon chain and dechlorination. Each CP isomer will be degraded by a somewhat different pathway but overall these pathways lead to increasingly polar and less toxic metabolites. Pathway options decline as chlorination levels and occurrence of vicinal chlorine substitutions increase (Federle 2017 – attached in Section 13.2 of the dossier). One important conclusion from this work is that MCCP constituents cannot degrade into SCCP constituents as the chain-shortening reaction will involve oxidation of the molecule to a fatty acid.

 

The half-lives of C16 chlorinated paraffins were estimated at about 12 days (35% chlorinated) and 58 days (69% chlorinated), respectively, in an aerobic sediment system (from a freshwater lake) containing oligochaete worms (Lumbriculus variegatus). The extent of degradation was determined at day 0 and day 14 of the experiments based on the difference between toluene-extractable 14C measurements (taken to represent unchanged chlorinated paraffin) and total 14C measurements (Fisk et al. 1998). These substances represent the extremes of the typical chlorine contents of MCCPs in commercial production. The RAR (EU, 2005) notes that the “results of this test should be treated with caution as the identity of the 14C present in the samples was not determined, and it was only assumed that the non-extractable 14C represented metabolites”. A new OECD 308 sediment simulation study was conducted at EAG Laboratories with analysis by Vrije Universitat (VU) Amsterdam using the same analytical methods used in the CBT studies (van Ginkel 2018a-d). The test was conducted using two sediments and their associated waters. Test systems were dosed with C₁₄, 50% Cl (by wt.) test material at a nominal concentration of 5 µg/g dry weight of sediment. Test systems were housed on a shaker table in an incubator set to maintain a temperature of 12 ºC for up to 120 days. The initial results from this study are inconclusive as the recoveries of the test material from the sediment have varied greatly, likely due to the interference of the organic matter in the sediment. Additional analysis is ongoing, but there are currently no conclusions from this study.

 

Adsorption onto sludge is likely to be the major removal mechanism for MCCPs during waste water treatment processes. A removal rate of 97.1% by adsorption onto sewage sludge during waste water treatment was obtained using the SIMPLE TREAT 4.0 model on the influent values reported by Coelhan 2010 (see analysis in CSR).

 

There is clear evidence that microorganisms are capable of degrading MCCPs in the environment based on the extensive biodegradation observed in the CBTs and that the rate of biodegradation decreases with increasing chlorine content. However, given the range of chlorination levels in this assessment a worst-case approach of no biodegradation was assumed in the PEC calculations. 

 

Hydrolysis is not expected to be a significant degradation process for MCCPs in the environment. An atmospheric half-life of 1-2 days is estimated for MCCPs for reaction with hydroxyl radicals. A value for the rate constant for the reaction (kOH) of 8.10-12 cm3 molecule-1 s-1 will be used for the environmental modelling in the risk assessment (EU, 2005).