Naphthenic acids

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

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:

equivalent or similar to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Deviations:

yes

Remarks:

no information regarding a reference substance and enrichment culture based on process affected waters

Principles of method if other than guideline:

The method followed is close to the OECD method 310.
Cul

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

other: process affected water

Details on inoculum:

Enrichment cultures of NAs-degraders were established with each of the commercial NAs preparations. The source of microorganisms was process-affected water from the Mildred Lake Settling Basin, a containment pond for oil sand extraction tailings at the Mildred Lake site of Syncrude Canada Ltd. The enrichment cultures were established in 500-mL Erlenmeyer flasks that contained 200 mL of sterile modified Bushnell-Haas mineral salts medium (pH approximately 7), inoculated with 20 mL of the tailings pond water. The NAs preparations (about 100 mg/L) served as the sole carbon source. The enrichment cultures were incubated under aerobic conditions in the dark on a shaker at room temperature (about 21 °C). At monthly intervals, over several months, portions of the cultures were transferred (1% v/v) to fresh medium and the corresponding NAs preparation.

Duration of test (contact time):

45 d

Initial conc.:

98 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

inorg. C analysis

Parameter followed for biodegradation estimation:

CO2 evolution

Parameter:

% degradation (inorg. C analysis)

Remarks:

and CO2 in the headspace have been analysed

Value:

ca. 60

Sampling time:

22 d

Remarks on result:

other: According to a graph shown in the article the CO2 emitted in viable cultures reaches 60% after about 22 days in the case of the Kodak naphthenic acid sodium salt

Parameter:

% degradation (inorg. C analysis)

Remarks:

and CO2 in the headspace have been analysed

Value:

ca. 60

Sampling time:

17 d

Remarks on result:

other: In the case of the Merichem 60% CO2 is reached after 17 days.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

This work showed that two commercial NAs preparation can be biodegraded extensively under laboratory conditions, using aerated cultures with an abundant supply of inorganic nutrients including phosphorus and fixed nitrogen.
Upon degradation the GC-MS fingerprints are changed dramatically. The napththenic acids concentration was decreased to level approaching zero by day 14.
Decomposition products of the nathphthenic acid include palmitic acid and stearic acid.
Further the decrease in toxicity upon aerobic degradation has also been shown. Merichem acids were detoxified by day 17. The extent of detoxification of the Kodak salts was not observed until day 43.

Executive summary:

This work showed that two commercial NAs preparation can be biodegraded extensively under laboratory conditions, using aerated cultures with an abundant supply of inorganic nutrients including phosphorus and fixed nitrogen. Upon degradation the GC-MS fingerprints are changed dramatically. The napththenic acids concentration was decreased to level approaching zero by day 14. Decomposition products of the nathphthenic acid include palmitic acid and stearic acid. Further the decrease in toxicity upon aerobic degradation has also been shown. Merichem acids were detoxified by day 17. The extent of detoxification of the Kodak salts was not observed until day 43.

Reference 2

Endpoint:

biodegradation in water: screening tests

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:

Four experiments were run:
1) Evaluation of mineralization of naphthenic acids sodium salts (NAS) and oil sands tailings extracts of naphthenic acids (TEX),
2) Evaluation of mineralization of four model naphthenic acid compounds, cyclohexane carboxylic acid (CCA), cyclohexane pentanoic acid (CPA) 2-methyl-1-cyclohexane carboxylic acid (2MCCA), and trans-4-pentylcyclohexane carboxylic acid (4PCCA),
3) Gas chromatographic analysis of NAS and TEX biodegradation, and
4) Respirometry measurements of cyclohexane pentanoic acid, NAS, and TEX in tailings microcosms.

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

other: enrichement cultures derived from oil sands tailings waters

Details on inoculum:

Inoculum: Inoculum used in the biodegradation experiments was NAS- and TEX- degrading enrichment cultures derived from oil sands tailings water. These cultures were created by diluting a 10-ml sample of oil sands tailing into 90 ml of mineral salts medium that contained either NAS (100 mg/l) or TEX (1:50 dilution). The mineral salts medium was modified Bushnell-Haas medium. Successive transfers 1% v/v) of the enrichment culture into fresh NAS- to TEX-containing medium were on monthly basis and incubated at room temperature on a gyratory shaker (100 rpm). The viable cell number within each enrichment culture was estimated using the plate count technique.

TEX=Tailings extracts of naphthenic acids

CCA= Cyclohexane carboxylic acid

CPA=cyclohexane pentanoic acid

2MCCA=2 -methyl-1-cyclohexane carboxylic acid

4PCCA=trans-4 -pentylcyclohexane carboxylic acid

Results of Experiment No. 1. The mineralization studies showed that the NAS- and TEX-degrading enrichment culture was capable of mineralizing components within both the NAS and TEX mixtures. The percentage of organic carbon converted to CO2 by the NAS-degrading culture was 48% (day 24) in the NAS bottles and 20% (day 20) in the TEX bottles. The percentage of organic carbon converted to CO2 by the TEX-degrading culture was 34% (day 30) for the TEX bottles and 20% (day 25) for the NAS bottles.

Results of Experiment No. 2. The following results were obtained:. Mineralization by day 24, % organic C converted to CO2:

Substrate	NAS-degraders	TEX-degraders	TPW
CCA	41	56	57
CPA	45	57	58
2MCCA	47	7	67
4PCCA	6	24	24

Results of Experiment No. 3. Chromatographic analysis of solution from the control flasks revealed an unresolved series of many overlapping peaks that created a hump in the GC profile. When the mixture that was inoculated with NAS-enrichment culture, a reduction in the size of the hump was evident within 4 days, indicating that components within the naphthenic acid mixture were being degraded. Chromatographic analysis of the TEX samples revealed a similar hump of many overlapping peaks that appeared in the NAS GC profile. Biodegradation of TEX by the NAS-degrading culture did not result in a noticeable reduction in the size of the hump associated with TEX, despite evidence of mineralization of components within the mixture.

Results of Experiment No. 4. The addition of CPA to TPW resulted in increased microbial activity, as indicated by greater levels of CO2 production and O2 utilization when compared with TPW alone. Sterilized TPW demonstrated no CO2 production or O2 utilization. Even greater levels of microbial activity were evident when N and P were added in addition to CPA, indicating that mineralization could be enhanced by the addition of mineral nutrients. GC analysis of CPA in TPW microcosms after 35 d of incubation revealed that the concentration of CPA was below the level of detection in 2/3 microcosms and reduced 10-fold in the third microcosm. There was no detectable CPA in the three N and P-amended microcosms. Similarly, NAS and TEX additions to microcosms increased microbial activity in TPW, although microbial activity was enhanced by the addition of N and P. Increases in both CO2 evolution and O2 utilization were seen.       

Interpretation of results:

inherently biodegradable

Conclusions:

This investigation showed that naphthenic acids, either as a commercial preparation of sodium salt (NAS) or natural extracts from oil sands tailing water (TEX) are capable of being utilized by natural assemblages of microorganisms. Addition of nitrogen and phosphorus enhances the utilization of these substrates by the microbes.

Executive summary:

This investigation showed that naphthenic acids, either as a commercial preparation of sodium salt (NAS) or natural extracts from oil sands tailing water (TEX) are capable of being utilized by natural assemblages of microorganisms. Addition of nitrogen and phosphorus enhances the utilization of these substrates by the microbes.

The microcosm studies with NAS-amended TPW indicated that microbial activity resulted in a reduction in acute toxicity to the level of TPW alone (see attached figure). In contrast, and despite the evidence of microbial activity, there was no evidence of a reduction in the acute toxicity within the TEX-amended TPW.

Reference 3

Endpoint:

biodegradation in water: screening tests

Type of information:

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

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:

Herman et al. (1993) conducted four experiments on the biodegradation of specific cycloalkane carboxylic acids:

GLP compliance:

not specified

Specific details on test material used for the study:

Naphthenic acids being a complex mixture of carboxylic acids model compounds have bene chosen to model the biodegradability

Oxygen conditions:

aerobic

Inoculum or test system:

other: enrichment culture from tailings ponds water

Details on inoculum:

Tailings pond bacteria were isolated on agar-hardened half-strength brain heart infusion (BHI) plates, and representative colony types were examined for their ability to utilize carboxylated cycloalkanes as their sole carbon source. Individual colonies were inoculated into a solution of carboxylated cycloalkanes (1000mg/L) in modified Bushnell and Haas (MBH) minimal salts medium. The ability of the isolate to utilize the carbon source was monitored by GC analysis. As well, a carboxylated cycloalkane-degrading mixed bacterial culture was enriched from the tailings pond sample, using standard procedures. The mixed bacterial culture was maintained on a mixture of CCP, 1MCCH, and MCCH (500mg/L each) in MBH with yeast extract (1000mg/L) added as a supplemental carbo source.

Duration of test (contact time):

40 d

Initial conc.:

ca. 1 000 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Results of Experiment 1. The bacterial populations of oil sands tailings was shown to have the metabolic capability of degrading carboxylated cycloalkanes as shown in the following table of results.

Table: Percent Remaining

	CCP	CCP	CCH	CCH	1MCCH	1MCCH	2MCCH	2MCCH
Day	NP-	NP+	NP-	NP+	NP-	NP+	NP-	NP+
0	100	42	100	68	100	100	100	100
6	100	5	100	12	100	100	100	100
10	100	0	100	1	100	100	100	100
16	100	0	100	0	100	100	100	100
26	100	0	100	0	100	100	100	49
40	100	0	100	0	100	100	100	0

CCP= cyclopentane carboxylic acid, CCH= cyclohexane carboxylic acid, 1MCCH=1-methyl-1-cyclohexane carboxylic acid, 2MCCH=2-methyl-1-cyclohexane carboxylic acid

Using tailings pond water as a growth medium, degradation of CCP, CCH, and 2MCCH was achieved only if nutrients were added to the medium. CCP and CCH were degraded rapidly, within one week, while methylated carboxylic acids were more resistant to biodegradation. 2MCCH was degraded within 40 days, but no degradation was observed for 1MCCH.

Results of Experiment No. 2. Biodegradation of CCP was complete within the first week. No biodegradation of 1MCCH was evident after six weeks. At the six-week period, nitrogen and phosphorus was added whereby complete biodegradation of 1MCCH was noted following between the 6 and 9-week sampling. No 1MCCH was measured at 9 weeks. Neither CCP nor 1MCCH was degraded in the control microcosms.

Results of Experiment No. 3. Of 10 separate colony types isolated from oil sands tailings, one colony type was found to utilize CCP and CCH as its sole carbon source. The isolate was a Gram negative, non-motile, catalase positive, oxidase negative, non-fermenting, aerobic rod, and was identified as an Acinetobacter sp. The isolate rapidly degraded CCP and CCH, with complete loss of substrate from the medium within 2 weeks of incubation. However, this isolate was unable to degrade methyl-substituted cyclohexane carboxylic acids. The mixed bacterial culture enriched from the tailings pond sample on a mixture of carboxylated cycloalkanes was found to degrade 1MCCH and 2MCCH, but only when the medium was supplemented with yeast extract. After a 2-week incubation period, the mixed culture had degraded 100% of the 1MCCH and 67% of the 2MCCH.

Results of Experiment No. 4. The results of hexadecane mineralization within oil sands tailings showed that the biodegradation of an n-alkane was nutrient limited. Percent biodegradation reached 50% by day 16 and maintained a plateau through day 40.

Interpretation of results:

inherently biodegradable

Conclusions:

This study showed the potential for biodegradation of naphthenic acids by investigating the biodegradation of both carboxylated cycloalkanes and hexadecane. Although natural naphthenic acids present in oil sands tailings have greater structural complexity than the compounds examined in this study, the results show the potential for both for biodegradation of the alkyl side chain and the carboxylated cycloalkane ring components of naphthenic acids. Biodegradation potential was reduced by methyl substitution on the cycloalkane ring, although these compounds could be degraded with the addition of mineral nutrients

Executive summary:

Herman et al. (1993) conducted four experiments on the biodegradation of specific cycloalkane carboxylic acids:

Experiment No. 1. Biodegradation of four naphthenic acid compounds (cyclopentane carboxylic acid, CCP; cyclohexane carboxylic acid, CCH; 1-methyl-1-cyclohexane carboxylic acid, 1MCCH; and 2-methyl-1-cyclohexane carboxylic acid, 2MCCH) was measured in pore water from Athabasca oil sands tailings ponds.

Experiment No. 2. Triplicate tailings pond microcosms were created using 200 ml of the tailings sample (as inoculum and medium).

Experiment No. 3: Tailings pond bacteria were isolated on agar plates and colony types were examined for their ability to utilize carboxylated cycloalkanes as their sole carbon source.

Experiment No. 4. Radiolabeled hexadecane was spiked into the maltene fraction of pure bitumen. Hexadecane mineralization experiments were performed using 5 ml of oil sands tailings in 60-ml serum vials and inoculated with 10 ul of spiked maltene.

Although natural naphthenic acids present in oil sands tailings have greater structural complexity than the compounds examined in this study, the results show the potential for both for biodegradation of the alkyl side chain and the carboxylated cycloalkane ring components of naphthenic acids. Biodegradation potential was reduced by methyl substitution on the cycloalkane ring, although these compounds could be degraded with the addition of mineral nutrients.

Description of key information

A study of Clemente et al (2004) indicated that naphtenic acids are readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

One of the main goals of testing ready biodegradability is to estimate the risk of long term adverse effects on biota. Although no standardized ready or inherent biodegradation studies were available for naphthenic acids, research has shown these materials to be amenable to microbial utilization similar to other hydrocarbon compounds. Studies have demonstrated that microorganisms indigenous to oil sands tailings were capable of degrading complex mixtures of commercial sodium salts of naphthenic acids as well as mixtures of organic acids extracted from oil sands tailings (Herman et al., 1993, 1994). Although rates of biodegradation may be affected by steric factors related to the numbers of cycloalkane rings or the alkyl constituents on the ring structure, microbial populations respond to naphthenic acid substrates through increased CO2 production, O2 consumption, and enhancement of metabolism with the addition of nutrients. Another study (Clemente et al., 2004) has demonstrated the biodegradability of commercial mixture of naphthenic acid sodium salt and naphthenic acids of about 60% in 10 days. In the same study the toxicity has been monitored. A decrease in toxicity with time upon biodegradation with solutions of commercial naphthenic acid or naphthenic acid sodium salt being completely detoxified in 17 (Merichem acid) or 43 (Kodak naphthenatic acid sodium salt) days respectively.

The studies have shown that naphthenic acid are biodegradable and that a decrease in toxicity linked to biodegradation occurs over time.

Value to be used for CSA: depending on the composition naphthenic acids mixtures are at least inherently biodegradable, with some naphthenic acids mixtures being readily biodegradable.