Naphthenic acids, zinc salts

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge nitrification inhibition testing

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Justification for type of information:

Please see section 13 for the read across justification

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

ISO 9509 (Toxicity test for assessing the inhibition of nitrification of activated sludge microorganisms)

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge from a municipal wastewater treatment plant

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

4 h

Test temperature:

20°C

pH:

6.5-7.5

Dissolved oxygen:

5-6 mg/L

Nominal and measured concentrations:

between 0-1.2 mg Zn/L.

Reference substance (positive control):

no

Duration:

4 h

Dose descriptor:

other: IC20

Effect conc.:

0.16 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

inhibition of nitrification rate

Duration:

4 h

Dose descriptor:

IC50

Effect conc.:

0.35 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

inhibition of nitrification rate

Duration:

4 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

inhibition of nitrification rate

Key result

Duration:

4 h

Dose descriptor:

NOEC

Effect conc.:

0.7 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Recalculated for zinc naphthenate

Basis for effect:

inhibition of nitrification rate

Details on results:

A study conducted by Juliastuti et al. (2003) reported a NOEC of 0.1 mg Zn/L in an ISO 9509 nitrification inhibition test. Applying the rules for PNEC setting (ECHA R.10, table R.10.6) this result yields a PNEC STP of 100 µg Zn/L (AF of 1). These results have been recalculated so the STP PNEC of 100 µg Zn/L is equivalent to 689.7 µg/L (or 0.7 mg/L) for zinc naphthenate.

Validity criteria fulfilled:

yes

Conclusions:

A study conducted by Juliastuti et al. (2003) reported a NOEC of 0.1 mg Zn/L in an ISO 9509 nitrification inhibition test. Applying the rules for PNEC setting (ECHA R.10, table R.10.6) this result yields a PNEC STP of 100 µg Zn/L (AF of 1). These results have been recalculated so the STP PNEC of 100 µg Zn/L is equivalent to 689.7 µg/L (or 0.7 mg/L) for zinc naphthenate.

Executive summary:

Test done according to standard protocol and considered useful for PNEC derivation for STP.

Description of key information

Zinc naphthenate consists of zinc salts of naphthenic acids and, therefore, data have been presented for both the organic anions and the zinc cations. Studies are included for naphthenic acids as well as zinc substances.

A nitrification inhibition test with Zn sulfate on activated sludge originating from a municipal wastewater treatment plant was performed leading to a NOEC of 100 µg Zn/L. These results have been recalculated so the STP PNEC of 100 µg Zn/L is equivalent to 689.7 µg/L (or 0.7 mg/L) for zinc naphthenate.

Frank (2009) showed that, based on the Microtox assay, the lowest 15 minute EC50 for Vibrio fisheri for the naphthenic acid surrogates was for cyclohexane carboxylic acid, with 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 zinc naphthenate to microorganisms has been recalculated from the data for the zinc cation, so the STP PNEC would be 0.7 mg/L for zinc naphthenate.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

0.7 mg/L

Additional information

In most cases the reactions to form the grease thickener occurin situduring 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.

Zinc naphthenate consists of zinc salts of naphthenic acids. Therefore, data have been presented for both the organic anion and the zinc cation. Studies are included for naphthenic acids as well as zinc substances.

Zinc

Several data were available for this endpoint. Formerly (ECB 2008), and in the REACH-registrations of a number of zinc substances of November 2010, a PNEC of 52 µg/L for STP was derived, based on the lowest EC50 of 5.2 mg Zn/L observed in a sludge respiration inhibition test (Dutka et al. 1983). According to the guidance (ECHA 7.8.17.1) preference should be given to nitrification inhibition because respiration inhibition is a less sensitive endpoint. A study conducted by Juliastuti et al. (2003) reported a NOEC of 0.1 mg Zn/L in an ISO 9509 nitrification inhibition test. Applying the rules for PNEC setting (ECHA R.10, table R.10.6) this result yields a PNEC STP of 100 µg Zn/L (AF of 1).

These results have been recalculated so the STP PNEC of 100 µg Zn/L is equivalent to 689.7 µg/L (or 0.7 mg/L) for zinc naphthenate.

Naphthenic acids

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 forV fisherifor NA-like surrogates ranged from 0.07 to 627.31 mM.

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

Zinc naphthenate consists of zinc salts of naphthenic acids. Therefore, data have been presented for both the organic anion and the zinc cation. Studies are included for naphthenic acids as well as zinc substances.

A nitrification inhibition test with Zn sulfate on activated sludge originating from a municipal wastewater treatment plant was performed leading to a NOEC of 100 µg Zn/L. These results have been recalculated so the STP PNEC of 100 µg Zn/L is equivalent to 689.7 µg/L (or 0.7 mg/L) for zinc naphthenate.

Frank (2009) showed that, based on the Microtox assay, the lowest 15 minute EC50 forVibrio fisherifor the naphthenic acid surrogates was for cyclohexane carboxylic acid, with 13 mg/L. Supporting data for naphthenic acids were also available.

Taking a worst-case approach for the results of the key studies, the toxicity of zinc naphthenate to microorganisms has been recalculated from the data for the zinc cation, so the STP PNEC would be 0.7 mg/L for zinc naphthenate.