Naphthenic acids, lithium salts

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as an unpublished report.

Justification for type of information:

See IUCLID section 13 for read across justification

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: OECD 301B, CO2 evolution test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Not applicable

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

Pre-solubility/dispersion work was conducted in order to determine the most suitable method of preparation. For the purpose of the test, the test item was dispersed directly into the test media with the aid of high shear mixing (approximately 7500 rpm, 15 minutes) prior to dispersal in the inoculated mineral medium.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source of inoculum: Aeration stage of sewage treatment plant which treats predominantly domestic sewage.
- Storage conditions: Continuously aerated at 21°C
- Storage length: Used on day of collection
- Preparation of inoculum for exposure: Washed twice by settlement and resuspension. Test vessels containing 2400 mL mineral medium and 24.3 mL inoculum were aerated with CO2 free air for approximately 24 hours prior to the addition of the test and reference items.
- Concentration of sludge: 30 mg suspended solids/L

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

28 d

Post exposure observation period:

No data reported

Hardness:

No data reported

Test temperature:

- Temperature: 21°C

pH:

- pH adjustment: If necessary, the pH was adjusted to 7.4 ± 0.2 using dilute hydrochloric acid or sodium hydroxide solution prior to aeration of the mineral medium.

Dissolved oxygen:

No data reported.

Salinity:

Not applicable

Nominal and measured concentrations:

- Nominal concentration: 10 mg Carbon/L, equivalent to 13 mg/L lithium behenate

Details on test conditions:

- Composition of medium: OECD guideline mineral medium
- Vessels: 5 L test culture vessels each containing 3 L of solution
- Lighting: Continuous darkness
- Aeration: Test vessels were sealed and CO2-free air was bubbled through the solutions at rates of 30 to 100 mL/minute/vessel. CO2-free air was produced by passing compressed air through a glass column containing self-indicating lime soda.
- Agitation: Continuous stirring by magnetic stirrer
- CO2 trap: Two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH solutions with purified degassed water.
- Toxicity control: Sodium benzoate stock solution and inoculated mineral medium to a final concentration of 10 mg carbon/L
- Replicates: Control 2, reference susbtance 2, test substance 2, toxicity control 1.
- Sampling: 2 mL samples were taken from the first CO2 absorber vessels on days 0, 2, 6, 8, 10, 14, 21, 28 and 29 and analysed for CO2 immediately using TOC analysers. Second absorber vessels were sampled on days 0 and 29.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Results: The toxicity to micro-organisms result was taken from the inhibition control of a ready biodegradability test. At the only concentration of lithium behenate tested (13 mg/L), the toxicity control flask showed no inhibition of the micro-organisms.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

No data reported

Validity criteria fulfilled:

not applicable

Conclusions:

The No Observed Effect Concentration (NOEC) of lithium behenate to aquatic micro-organisms is 13 mg/L.

Executive summary:

The No Observed Effect Concentration (NOEC) of lithium behenate to aquatic micro-organisms is 13 mg/L. The toxicity of lithium behenate to activated sewage sludge was taken from the inhibition control of a GLP-compliant ready biodegradability test following OECD guideline 301B (Harlan 2013).

Reference 2

Endpoint:

toxicity to microorganisms

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The report was a well-documented study that meets basic scientific principles.

Justification for type of information:

See IUCLID section 13 for read across justification

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Microtox analysis was performed on eight carboxylic acids used as surrogates for individual Naphthenic Acids (NAs). Four (hexanoic acid, cyclohexanecarboxylic acid, decanoic acid, and cyclohexanepentanoic acid) contained one carboxyl functional group and had the common naphthenic acid general formula of CnH2n+zO2. The other four surrogates (succinic acid, adipic acid, cyclohexanedicarboxylic acid, and cyclohexylsuccinic acid) differed only by the presence of an additional carboxylic group. The toxicity of the NA-like surrogates was assessed by measuring bioluminescence of V. fischeri following 15 min exposure using three replicates.

GLP compliance:

not specified

Remarks:

Published literature - GLP status of laboratory not specified

Specific details on test material used for the study:

- Collection, Extraction, Purification, and Fractionation of a Naphthenic Acid (NA) Extract: Approximately 3000 L of oil sands pond water (OSPW) was collected from the West In-pit settling basin of Syncrude Canada Ltd. in Fort McMurray, Alberta, Canada, in June 2005. NAs were extracted from the OSPW and partially purified. The water was acidified to pH 2, the aqueous layer was decanted, and the acid precipitate was centrifuged to remove water. The organic acid pellet was then dissolved in 0.1 M NaOH and centrifuged to remove sediment that had cosettled with the acid precipitate. Next, the organic acid solution was filtered through diethylaminoethyl (DEAE) cellulose to remove humic-like substances, resulting in an organic acid filtrate composed primarily of NAs and NA-like compounds. The filtered NA extract was then washed with dichloromethane (DCM) to remove neutral organics such as polycyclic aromatic hydrocarbons (PAHs). The NA extract was again acidified and subsequently filtered through a 0.2 μmTeflon filter to remove any trace DCM. The NA precipitate was then redissolved in 0.05 M NaOH, and this final NA extract was dispensed into 1-L amber glass bottles (Fisher Scientific) and stored at 4°C. The NA mixture was methylated using diazomethane, and Kugelrohr distillation fractionated the methylated NAs into five fractions of increasing molecular weight.
- Naphthenic acids (NA)-Like Surrogates: Eight carboxylic acids were purchased (Sigma-Aldrich, Oakville, ON) to be used as surrogates for individual NAs. Four (hexanoic acid, cyclohexanecarboxylic acid, decanoic acid, and cyclohexanepentanoic acid) contained one carboxyl functional group and had the common naphthenic acid general formula of CnH2n+zO2. The other four surrogates (succinic acid, adipic acid, cyclohexanedicarboxylic acid, and cyclohexylsuccinic acid) differed only by the presence of an additional carboxylic group. The addition of an extra carboxylic group altered the naphthenic acid formula to CnH2n+z-fO2+f, where f is related to the number of additional carboxylic groups present within the structure (f=2, one additional COOH; f=4, two additional COOH; etc.).

Analytical monitoring:

yes

Details on sampling:

- Preparation: Following distillation, the fractions, dissolved in dichloromethane (DCM, Caledon, Georgetown, ON), were blown down to constant weight under N2 gas to remove the solvent. A small aliquot was collected from each blown down, methylated NA fraction and dissolved in deuterated DCM (CD2Cl2).

Vehicle:

not specified

Details on test solutions:

- Preparation: The four monocarboxyl naphthenic acid (NA)-like surrogates were dissolved in 0.01 N NaOH, and the four dicarboxyl NA-like surrogates were dissolved in 0.1 N NaOH to produce sodium salts that were consistent with NAs in alkaline OSPW.

Test organisms (species):

Vibrio fisheri

Details on inoculum:

- Equipment: Microtox analysis was performed on a Microtox Model 500 Analyzer using the Basic Test provided by Azur Environmental.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

15 min

Post exposure observation period:

No details reported

Hardness:

No details reported

Test temperature:

No details reported

pH:

The pH of the surrogates was adjusted to 7.5 (0.1 using HCl and NaOH).

Dissolved oxygen:

No details reported

Salinity:

No details reported

Conductivity:

No details reported

Nominal and measured concentrations:

Nominal concentrations (Table S1, Supporting Information) were serially diluted to 100%, 50%, 25%, and 12.5% of the initial test concentration for each of the NA-like surrogates.

Details on test conditions:

- Method: Following the pH titration and the 10% addition of osmotic adjusting solution, the toxicity of the NA-like surrogates was assessed by measuring bioluminescence of V. fischeri following 15 min exposure using three replicates.

Reference substance (positive control):

not specified

Duration:

15 min

Dose descriptor:

EC50

Effect conc.:

19.12 other: mM

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

not specified

Remarks on result:

other: Hexanoic acid

Duration:

15 min

Dose descriptor:

EC50

Effect conc.:

9.62 other: mM

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

not specified

Remarks on result:

other: Cyclohexane carboxylic acid

Duration:

15 min

Dose descriptor:

EC50

Effect conc.:

0.33 other: mM

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

not specified

Remarks on result:

other: Decanoic acid

Duration:

15 min

Dose descriptor:

EC50

Effect conc.:

0.07 other: mM

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

not specified

Remarks on result:

other: Cyclohexane pentanoic acid

Duration:

15 min

Dose descriptor:

EC50

Effect conc.:

627.31 other: mM

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

not specified

Remarks on result:

other: Succinic acid

Duration:

15 min

Dose descriptor:

EC50

Effect conc.:

468.06 other: mM

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

not specified

Remarks on result:

other: Adipic acid

Duration:

15 min

Dose descriptor:

EC50

Effect conc.:

465.71 other: mM

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

not specified

Remarks on result:

other: 1,4 -Cyclohexanedicarboxylic acid

Duration:

15 min

Dose descriptor:

EC50

Effect conc.:

26.09 other: mM

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

not specified

Remarks on result:

other: Cyclohexylsuccinic acid

Key result

Duration:

15 min

Dose descriptor:

EC50

Effect conc.:

13 mg/L

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

not specified

Remarks on result:

other: cyclohexane carboxylic acid

Details on results:

The 15 minutes EC50 for V fisheri for NA-like surrogates ranged from 0.07 to 627.31 mM.

Reported statistics and error estimates:

The EC50 values were calculated as the concentration of NA required to reduce bioluminescence by 50% in relation to a 0% NA blank solution. A fourparameter logistic model was used to fit regression lines to the data.

Because of the limited availability of larger compounds, the NA-like surrogates investigated in this study had relatively low MWs (116.16-200.23 Da) in comparison to the NAs observed in OSPW (142-500 Da 13, 14). Also, the nonbranched acyclic surrogate structures were likely simpler than OSPW NAs, which are highly branched. However, the goal of this study was to determine the effect that size, structure, and carboxylic acid content had on NA toxicity; therefore, the relationships observed from the studied surrogates in this study could exist for larger compounds more commonly found in OSPW NA mixtures.

Results

NA-like surrogate	Observed EC50 for 15 -min V. fisheri assay(mM)	Observed LC50 for 48hr D. Magna acute lethality assay (mM)
Hexanoic acid (HA)	19.12 +/-2.6	10.04 +/- 1.3
Cyclohexane carboxylic acid (CHCA)	9.62 +/- 1.9	6.67 +/- 0.8
Decanoic acid (DA)	0.33 +/- 0.04	1.27 +/- 0.4
Cyclohexane pentanoic acid (CHPA)	0.07 +/- 0.01	0.59 +/- 0.2
Succinic acid (SA)	627.31 +/- 29	27.3 +/-2.2
Adipic acid (AP)	468.06 +/- 214	20.51 +/- 1.1
1,4 -Cyclohexanedicarboxylic acid (CHDCA)	465.71 +/-21	15.28 +/- 0.7
Cyclohexylsuccinic acid (CHSA)	26.09 +/- 1.9	6.71 +/- 1.2

Conclusions:

Based on the microtox assay with NA-like surrogates, the lowest EC50 for Vibrio fisheri was 0.07 mM for cyclohexane carboxylic acid, which coresponds to 13.0 ± 1.6 mg/L.

Executive summary:

The acute toxicity of naphthenic acids and naphthenic acid like surrogates to microorganisms was taken from peer-reviewed published literature (Frank 2009). The report was a well-documented study that meets basic scientific principles and has been assigned a Klimisch score of 2, reliable with restriction. Microtox analysis was performed on eight carboxylic acids used as surrogates for individual Naphthenic Acids (NAs). Four (hexanoic acid, cyclohexanecarboxylic acid, decanoic acid, and cyclohexanepentanoic acid) contained one carboxyl functional group and had the common naphthenic acid general formula of CnH2n+zO2. The other four surrogates (succinic acid, adipic acid, cyclohexanedicarboxylic acid, and cyclohexylsuccinic acid) differed only by the presence of an additional carboxylic group. The toxicity of the NA-like surrogates was assessed by measuring bioluminescence of V. fischeri following 15 min exposure using three replicates. The bioassays with the NA-like surrogates indicate that toxicity is likely a function of hydrophobicity. As the MW of the surrogates increased, so too did the acute toxicity, while if an additional carboxylic group was present in the compound, the toxicity was significantly decreased. Based on the microtox assay, the lowest EC50 found for Vibrio fisheri for the NA surrogates was 0.07 mM for cyclohexane carboxylic acid, which corresponds to 13.0 ± 1.6 mg/L. The 15 minutes EC50 for V fisheri for NA-like surrogates ranged from 0.07 to 627.31 mM.

Description of key information

The toxicity to microorganisms endpoint has been waived on the basis that lithium naphthenate is not soluble in water. However, lithium naphthenate consists of lithium salts of naphthenic acids and, therefore, data have been presented for both the organic anion and the lithium cation. Studies are included for naphthenic acids as well as lithium salts of docosanoic acid (C22). Frank (2009) showed that, based on the Microtox assay, the lowest 15 minute EC50 for Vibrio fisheri for the NA surrogates was for cyclohexane carboxylic acid, with 13 mg/L. Harlan (2013) gave a 28 day NOEC of lithium docosanoate to aquatic micro-organisms of 13 mg/L. Supporting data for naphthenic acids were also available. Herman (1994) showed the initial Microtox EC50 was 30 mg/L for NAS and 0.86 - 1.25 mg/L for TEX (43% of 1:50 dilution and 25% of 1:20 dilution) but the toxicity decreased after degradation so that the degraded Microtox EC50 was 100 mg/L for NAS and 1.64 – 2.5 mg/L for TEX (82% of 1:50 dilution and 50% of 1:20 dilution). Herman (1994) concluded the 20 day NOEC for the toxicity of naphthenic acids to the identified microorganism species was 100 mg/L. Del Rio (2004) concluded that the identified microorganism species, Pseudomonas putida and fluorescens, seemed not to be affected by the presence of naphthenic acid and the NOEC was therefore concluded to be 0.04% naphthenic acids.

Taking a worst-case approach for the results of the key studies, the toxicity of lithium naphthenate to microorganisms is determined to give a 15 minute EC50 of 13 mg/L and a 28 day NOEC of 13 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

13 mg/L

EC10 or NOEC for microorganisms:

13 mg/L

Additional information

Adaptations

The toxicity to microorganisms study does not need to be conducted because the substance is highly insoluble in water, hence indicating that aquatic toxicity is unlikely to occur. No determination of water solubility was feasible for the test item. This was due to the test item, representative of the chemical class of anionic surfactants, showing significant surface-active properties in an aqueous environment. When attempting to generate saturated solutions, a colloidal suspension (i.e. containing micelles < 0.2 µm) was formed. As this dispersed, excess, undissolved material could not be satisfactorily removed by either centrifugation of filtration techniques, it was not possible to isolate a genuine saturated aqueous solution of test item suitable for analysis and quantification.

In most cases the reactions to form the grease thickener occur in situ during the grease manufacturing process and consequently these grease thickeners normally only exist in the base oil matrix. In realistic use scenarios, the thickeners will be contained in base oil, with the formulated greases specifically designed to minimise the leaching of the thickener. The thickeners would not be bioavailable and therefore, toxicity to aquatic micro-organisms is not considered to be relevant. Additionally, the disposal of greases via sewage treatment works is not applicable as the majority of greases are used within sealed parts or as total loss lubricants in applications and therefore the amount of grease which would enter sewage treatment plants is expected to be limited.

Experimental and read across data

Lithium naphthenate consists of lithium salts of naphthenic acids. Therefore, data have been presented for both the organic anion and the lithium cation. Studies are included for naphthenic acids as well as lithium salts of docosanoic acid (C22).

Lithium naphthenate is produced through the reaction of naphthenic acids with lithium hydroxide. Naphthenic acids consist of a main acidic (naphthenic) fraction (70-95%) and a smaller non-acidic (petroleum) fraction (5-30%). The acidic fraction contains typically C8-C20 with 0-3 rings and the non-acidic fraction has a carbon range mainly of C12-C22 and no single constituent >10%. The lithium hydroxide added to the naphthenic acids will neutralise the components within the acid fraction to form lithium salts while any components present in the non-acidic fraction of naphthenic acids (the unsaponifiable matter) will be unchanged by the manufacturing process. Therefore, lithium naphthenate is expected to consist of 70 - 95% lithium salts of naphthenic acids (C8-C20, 0-3 rings), with 5 - 30% unsaponifiable matter unchanged from the naphthenic acids starting material.

Lithium docosanoate (C22) has a longer chain length as the longest chain length acids present in the acidic fraction of lithium naphthenate (C20). Therefore, lithium docosanoate, as a lithium salt of a carboxylic acid two longer than those present in lithium naphthenate, brackets the other end of the range of potential properties of constituents present in the UVCB.

Frank (2009)

The acute toxicity of naphthenic acids and naphthenic acid like surrogates to microorganisms was taken from peer-reviewed published literature (Frank 2009). Microtox analysis was performed on eight carboxylic acids used as surrogates for individual Naphthenic Acids (NAs). Four (hexanoic acid, cyclohexanecarboxylic acid, decanoic acid, and cyclohexanepentanoic acid) contained one carboxyl functional group and the other four surrogates (succinic acid, adipic acid, cyclohexanedicarboxylic acid, and cyclohexylsuccinic acid) had an additional carboxylic group. The toxicity of the NA-like surrogates was assessed by measuring bioluminescence of V. fischeri following 15 min exposure using three replicates. The bioassays with the NA-like surrogates indicate that toxicity is likely a function of hydrophobicity. As the MW of the surrogates increased, so too did the acute toxicity, while if an additional carboxylic group was present in the compound, the toxicity was significantly decreased. Based on the microtox assay, the lowest EC50 found for Vibrio fisheri for the NA surrogates was 0.07 mM for cyclohexane carboxylic acid, which corresponds to 13.0 ± 1.6 mg/L. The 15 minutes EC50 for V fisheri for NA-like surrogates ranged from 0.07 to 627.31 mM.

Harlan (2013)

The No Observed Effect Concentration (NOEC) of lithium docosanoate to aquatic micro-organisms is 13 mg/L. The toxicity of lithium docosanoate to activated sewage sludge was taken from the inhibition control of a GLP-compliant ready biodegradability test following OECD guideline 301B (Harlan 2013).

Herman (1994)

Four experiments were run using the following materials: 1) Tailings water extract (TEX), 2) commercial sodium naphthenate mixture (NAS), and 3) pure compound naphthenic acids, cyclohexane carboxylic acid (CCA), cyclohexane pentanoic acid (CPA), 2-methyl-1-cyclohexane carboxylic acid (2MCCA), and trans-4-pentylcyclohexane carboxylic acid (4PCCA). The four experiments were:

1) Evaluation of mineralizaton 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.

Biodegradation tests have shown that enrichment cultures mineralize components within commercial and oil sands tailings naphthenic acids. The enrichment cultures contained initially between 21 to 60 mg/L organic carbon (60 mg/L being equivalent to a concentration of 100 mg/L naphthenic acid in the case of commercial naphthenic acid). One enrichment culture contained a colony of Pseudomonas stutzeri and Alcaligens denitrificans. The other one contained Acinetobacter calcoaceticus and a member of the Pseudomonas fluorescens group that thus seem to be able to stand such concentrations of naphthenic acid. The NOEC for the toxicity of naphthenic acids to the identified microorganism species was concluded to be 100 mg/L.

In mineral salts medium, NAS (100 mg/L) had an initial Microtox EC50 of 30% (v/v). When inoculated with the NAS-degrading enrichment culture, the Microtox EC50 value of NAS rapidly increased to a point where 100% (v/v) of the biodegraded NAS solution had no effect on bioluminescence, indicating a loss of acute toxicity. The TEXa dilutions had an initial Microtox EC50 of 43% (v/v) for the 1:50 dilution and 25% (v/v) for the 1:20 dilution. Microtox analysis of biodegraded TEXa revealed that microbial activity had reduced the acute toxicity, as indicated by an increase in Microtox EC50 values to 82% (v/v) for the 1:50 dilution and 50% (v/v) for the 1:20 dilution, when averaged over the last three sampling times when mineralization had reached a plateau. Microbial activity reduced the toxicity of a by approximately one half but did not completely remove the acute toxicity as was evident with NAS. There was little change in the toxicity of the control bottles compared with the initial estimate.

Del Rio (2004)

Del Rio (2004) studied eleven wetlands, both natural and process-affected, and one tailings settling pond in Northern Alberta. Enrichment cultures were obtained from an active tailings settling pond, using commercially available NAs as the sole carbon source. The extent of NA degradation by cultures incubated with 0.04% (w/v) of Kodak's NA for 4 weeks was determined by GC-MS of culture supernatants. The acute toxicity of the free water was determined using the bacterial Microtox bioassay. In addition, phenotypic identification was performed using API 20 NE identification strips and telephone database. Enrichment cultures resulted in the isolation of a co-culture containing Pseudomonas putida and Pseudomonas fluorescens. Quantitative GC–MS analysis showed that the co-culture removed >95% of the commercial NAs, and partially degraded the process NAs from OSPW with a resulting NA profile similar to that from ‘aged wetlands’. Pseudomonas putida and fluorescens seem thus not to be affected by the presence of naphthenic acid and the NOEC was therefore concluded to be 0.04% naphthenic acids.

Conclusion

The toxicity to microorganisms endpoint has been waived on the basis that lithium naphthenate is not soluble in water.

Lithium naphthenate consists of lithium salts of naphthenic acids. Therefore, data have been presented for both the organic anion and the lithium cation. Studies are included for naphthenic acids as well as lithium salts of docosanoic acid (C22).

Frank (2009) showed that, based on the Microtox assay, the lowest 15 minute EC50 for Vibrio fisheri for the NA surrogates was for cyclohexane carboxylic acid, with 13 mg/L. Harlan (2013) gave a 28 day NOEC of lithium docosanoate to aquatic micro-organisms of 13 mg/L.

Supporting data for naphthenic acids were also available. Herman (1994) showed the initial Microtox EC50 was 30 mg/L for NAS and 0.86 - 1.25 mg/L for TEX (43% of 1:50 dilution and 25% of 1:20 dilution) but the toxicity decreased after degradation so that the degraded Microtox EC50 was 100 mg/L for NAS and 1.64 – 2.5 mg/L for TEX (82% of 1:50 dilution and 50% of 1:20 dilution). Herman (1994) concluded the 20 day NOEC for the toxicity of naphthenic acids to the identified microorganism species was 100 mg/L. Del Rio (2004) concluded that the identified microorganism species, Pseudomonas putida and fluorescens, seemed not to be affected by the presence of naphthenic acid and the NOEC was therefore concluded to be 0.04% NA.

Taking a worst-case approach for the results of the key studies, the toxicity of lithium naphthenate to microorganisms is determined to give a 15 minute EC50 of 13 mg/L (Frank 2009) and a 28 day NOEC of 13 mg/L (Harlan 2013).