Toxicity of Individual Naphthenic Acids to Vibrio fischeri

ARTICLE pubs.acs.org/est

Toxicity of Individual Naphthenic Acids to Vibrio fischeri David Jones, Alan G. Scarlett, Charles E. West, and Steven J. Rowland* Petroleum and Environmental Geochemistry Group, Biogeochemistry Research Centre, University of Plymouth, Drake Circus, Plymouth, PL4 8AA, United Kingdom

bS Supporting Information ABSTRACT: Numerous studies have suggested that the toxicity of organic compounds containing at least one carboxylic acid group and broadly classified as “naphthenic acids”, is of environmental concern. For example, the acute toxicity of the more than 1 billion m3 of oil sands process-affected water and the hormonal activity of some offshore produced waters has been attributed to the acids. However, experimental evidence for the toxicity of the individual acids causing these effects has not been very forthcoming. Instead, most data have been gathered from assays of incompletely characterized extracts of the water, which may contain other toxic constituents. An alternative approach is to assay the individual identified toxicants. Since numerous petroleum-derived naphthenic acids and some in oil sands process water, have recently been identified, we were able to measure the toxicity of some individual acids to the bioluminescent bacterium, Vibrio fischeri. Thirty-five pure individual acids were either synthesized or purchased for this purpose. We also used the US EPA ECOSAR computer model to predict the toxicity of each acid to the water flea, Daphnia magna. Both are wellaccepted toxicological screening end points. The results show how toxic some of the naphthenic acids really are (e.g., V. fischeri Effective Concentrations for 50% response (EC50) 0.004 to 0.7 mM) and reveal the influence of hydrophobicity and aqueous solubility on the toxicities. Comparison with measured toxicities of other known, but more minor, constituents of oil sands process water, such as polycyclic aromatic hydrocarbons and alkylphenols, helps place these toxicities into a wider context. Given the reported toxicological effects of naphthenic acids to other organisms (e.g., fish, plants), the toxicities of the acids to further end points should now be determined.

’ INTRODUCTION The toxicity of the so-called naphthenic acids from commercial (petroleum) sources, from offshore oil platforms and from oil sands, to a variety of organisms, has been widely studied, particularly recently.1 5 However, most experimental evidence for the toxicity has been gathered from assays of incompletely characterized extracts2 5 and it has proven difficult to ascribe the toxicities to individual components or even to compound classes in such mixtures, except in one or two cases.1 Such mixtures may contain toxic components other than acids.2,6,7 Therefore, recent advances in the characterization of both oil sands and commercial petroleum-derived naphthenic acids,8 10 which have allowed some of the individual carboxylic acids to be identified, have assumed considerable importance. Toxicity assays can now, in principle, be carried out on acids which are actually known to be present in the mixtures. Such an approach may help to define which of the acids are most toxic, and/or whether components other than acids should also be studied. However, pure samples of individual acids in at least milligram quantities are required before such assays are possible. We synthesized numerous examples of individual acids recently11 and now report use of a well-accepted toxicity screening assay to determine the toxicity of r 2011 American Chemical Society

over thirty-five of these acids. We also show the relationship between the measured toxicity of the acids to the bioluminescent bacterium, Vibrio fischeri, and the toxicity to the water flea, Daphnia magna, predicted by the US EPA computer modeling program ECOSAR.12

’ MATERIALS AND METHODS Reference Acids. Alkylphenylalkanoate and cis/trans alkylcyclohexylalkanoate (alkyl = methyl to hexyl, nonyl; alkanoate = ethanoate and butanoate) acids were synthesized by a route based on the Kindler modification of the Willgerodt reaction.11 Other acids were obtained by Freidel-Crafts13 or Willgerodt chemistry.11 Straight chain and methyl branched, phytanic, citronellic, adamantane-1-carboxylic, adamantane-1-ethanoic, and 3,5-dimethyl-adamantane-1-carboxylic acids were purchased from Sigma (U.K.) with stated purities g97%. Decalin carboxylic, decalin Received: June 8, 2011 Accepted: September 26, 2011 Revised: August 26, 2011 Published: September 26, 2011 9776

dx.doi.org/10.1021/es201948j | Environ. Sci. Technol. 2011, 45, 9776–9782

Environmental Science & Technology

ARTICLE

Table 1. Measured EC50 Values (( Standard Error, n = 3) for the Toxicity of Individual Carboxylic Acids to Vibrio fischeri (Microtox Assay) with Log Kow Values Used to Generate ECOSAR Predictionsa

9777

dx.doi.org/10.1021/es201948j |Environ. Sci. Technol. 2011, 45, 9776–9782


ARTICLE

Table 1. Continued

9778



ARTICLE

Table 1. Continued

a

Cn = carbon number. S = synthetic, C = commercial.

ethanoic, and decalin propanoic acids were synthesized from the aromatic analogues by hydrogenation.11,13 The 2,6-dimethylheptanoic and 2,6,10-trimethylundecanoic acids were synthesized previously and 3,7-dimethyloctanoic acid was obtained by hydrogenation of citronellic acid (PtO2, 8 psi H2). Purity of methyl esters of synthetic acids was assayed by gas chromatography mass spectrometry and was generally g99%. Microtox (Vibrio fischeri) Assay. Synthesized acids were carefully weighed on an Oxford five figure balance and placed into preweighed and prerinsed 7 mL vials. The acids (maximum 20 mg) were subsequently dissolved in 1 or 2 mL of 1 M NaOH and mixed on an autovortex mixer (Stuart Scientific) until completely dissolved. These solutions were then pH adjusted by dropwise titration with a Pasteur pipet with 1, 0.1, and 0.01 M HCL until a pH of between 6 and 8 was achieved. The amount of HCL/NaOH added to the solution by the titration was then calculated (on average 0.04 mL per drop) and a calculated amount was added to 10.0 mL of Microtox diluent which was then agitated by an autovortex mixer and titrated using 0.01 M HCL/NaOH until a pH of 7.5 ( 0.1 was achieved. The concentrations of these solutions ranged between 5 and 200 mg L 1 ((2%) depending on predicted solubility and toxicity. The acids tested were initially screened on the Microtox M500 analyzer (SDI Europe) using the 45% basic (15 min) test as a toxicity screening method. Briefly: Microtox glass cuvettes were placed in the M500 analyzer and an amount of diluent was added (either 1000 or 500 μL depending on position in analyzer). 2500 μL of acid solution was then added and osmotically adjusted, this was mixed and an amount was discarded before a 2 serial dilution was carried out. The subsequent test was performed (in triplicate) according to Microtox protocols. Phenol was tested as a positive control and found to be within the parameters set by Microtox (viz: EC50 between 13 and 26 mg L 1).

’ RESULTS AND DISCUSSION We measured the concentrations required to produce a 50% decrease (EC50) in the bioluminescence of the bacterium, Vibrio fischeri, in triplicate experiments at pH 7.5 (Table 1). Given

the pKa of such acids, at this pH the EC50 values therefore relate to those of free acids and/or carboxylate ions. EC50 values ranged from 0.004 to about 0.7 mM, depending on both the structures and the carbon numbers of the acids (Table 1). The mechanism of action of this toxicity is widely accepted to be due to nonspecific narcosis12 and the decreases in EC50, representing increasing toxicity, with increased carbon number of the acids (Figures 1 and 2), were consistent with this. The toxicities of most of the acid classes approached an effective limit at the higher carbon numbers assayed (Figures 1 and 2) and above these, the acids were insoluble at the pH of the assay. The maximum average toxicity was ca. 0.016 mM. So-called “straight-chain” or normal (n- or fatty) acids in oil sands process water extracts range from about C9 (nonanoic) to C15 (pentadecanoic) and in petroleum-derived acids from about C8 (octanoic) to C18 (octadecanoic).10,14 However, the proportions in the oil sands extracts appear to be low and are dominated by even numbered acids (Rowland et al., unpublished data and reference 14; earlier low resolution data may have been in error14), likely of biological origin. Our results (Table 1; Figure 1) show that n-C12 (dodecanoic) acid had the highest toxicity (EC50 0.019 ( 0.002 mM) of those n-acids tested and that acids larger than this were too insoluble to be assayed. For hexanoic acid and decanoic acid, Frank et al12 reported V. fischeri EC50s of 19.1 and 0.33 mM respectively; a D. magna LC50 of 10.04 mM and ECOSAR predicted LC50 of 7.15 mM were also reported. Oil sands acids from Alberta, Canada, also include monomethyl branched acids over a similar carbon number range, (but again in low proportions).14 A more complex distribution was present in a sample of petroleum-derived acids.10 Our measurements indicated (Table 1; Figure 1) that C11 13 acids were most toxic, maximizing at 0.012 ( 0.001 mM, before a solubility limit was reached (Figure 1). Polymethyl branched, acyclic isoprenoid acids have yet to be reported in oil sands acids mixtures, but are common in biodegraded petroleum due to microbial oxidation of the related hydrocarbons which are virtually ubiquitous in petroleum. Unsurprisingly therefore, they were abundant in a sample of petroleum-derived naphthenic acids10 and are likely present in at 9779



ARTICLE

Figure 1. Measured EC50 values ((standard error, n = 3) for the toxicity of individual carboxylic acids to Vibrio fischeri (Microtox assay). Where error bars are not apparent the error was smaller than the symbol. The identities of the individual acids are shown in Table 1 along with the numerical EC50 values. R2 and P values represent the goodness of fit of the polynomial trend lines.

least some oil sands acids, which are well-known to vary in composition with age. C10 14 acids were relatively toxic (Table 1: up to 0.015 ( 0.007 mM) but by C20 (phytanic acid) a solubility limit had been reached. Monocyclic acids are apparently not abundant in oil sands acids,14 but were present in petroleum-derived acids.10 Again, C12 14 acids were most toxic (Table 1; up to 0.012 mM).

Bicyclic acids are abundant (ca. 30%) in both oil sands and petroleum-derived naphthenic acids mixtures,10,14 but to date none have been firmly identified in the former. Bicyclic acids in a petroleum-derived naphthenic acids mixture however, included numerous decalin-type compounds10 and we therefore determined the toxicity of such species as the best models currently available. No doubt, more relevant acids can be synthesized, once 9780



Figure 2. Summarized measured EC50 values ((standard error, n = 3) for the toxicity of all tested carboxylic acids to Vibrio fischeri (Microtox assay). Depending on the individual acid class (e.g., n-acids, C13) there was a solubility limit beyond which the acids were too hydrophobic to dissolve at pH 7.5.

the individual bicyclic compounds in oil sands process water have been firmly identified. The bicyclic acids included the most toxic of the diverse ranges of acids tested (3-decalin-1-yl propanoic acid; Table 1; 0.004 mM). Tricyclic acids, along with the bicyclic compounds are often the most abundant species present in oil sands process-affected water.8,14 Numerous structures have been identified as “nanodiamond” adamantane acids (Table 1; Figure 1) and some of these are also present in petroleum-derived acids.8,10 The maximum EC50 was 0.337 ((0.022 mM). Interestingly, the extrapolated trend was somewhat different to those of the other acids (Figure 1). The trends of EC50 versus carbon number for the other acids approached asymptotes (Figure 2), whereas that of the diamondoid acids did not. Thus, C15 adamantane acids (if soluble) might exhibit toxicity at least comparable with the other classes of acids studied herein. Finally, we measured the toxicities of a range of monoaromatic acids found in petroleum-derived naphthenic acids from a commercial suppier,11 but not, to date, in oil sands acids from Alberta, Canada8 (Table 1; Figure 1), though some aromatics are likely present. These too followed a clear relationship with carbon number with a maximum toxicity of 0.023 ( 0.005 mM; C14). The effective solubility limit observed in our assay with several acid classes (Figure 2) likely also applies to many of the constituents of the complex naphthenic acids mixtures in oil sands and petroleum, though overall lower concentrations of individual acids in such mixtures and possibly some synergistic or antagonistic cosolubilization, may extend or limit the effective toxicity ranges of these somewhat. This solubility limit correlation with carbon number suggests that the gC15 pentacyclic diamondoid acids identified in oil sands process-affected water,9 would probably be too insoluble to exhibit a toxic effect to V. fischeri. Unfortunately insufficient amounts or diversities of such diamantane acids were available for assay, so we decided to estimate the toxicity using the U.S. Environmental Protection Agency ECOSAR program.12 This model is also based on action by narcotic activity12 and allows calculation of octanol water coefficients, solubility and thereby toxicity, to various biological

ARTICLE

end points. We chose to compute the toxicities of the acids to the water flea, D. magna (lethal response in 50% of a population of daphnids after 48 h (LC50)), since a previous study12 established a relationship between this widely used end-point and three acids. We noted (Figure 1S of the Supporting Information) that there were indeed, clear relationships between the measured (V. fischeri, x axis) and modeled (D. magna, y-axis) end points for each acid class measured, though the responses were not always linear, nor 1:1, nor consistent between classes (R2 = 0.9095 to 0.9865, p < 0.001 to >0.1). Of course, there is no reason ipso facto, why the toxicities measured or predicted to these two end points, should be the same. Concentrations predicted to produce a LC50 ranged from 4 mM for the least toxic n-hexanoic acid to 0.004 mM for the most toxic, 3-decalin-1-yl propanoic acid. When the model was used to compute the toxicities of the pentacyclic diamantane acids, however, the results suggested the acids would be too insoluble to be reliably predicted. This data set may however be useful for future QSAR studies. Since the high concentrations of the acids in oil sands processaffected water have led others to conclude that such components are responsible for the measured toxicities to a range of biota,2 5,12 it would be useful to try to gauge what proportion of the observed toxicity might be assignable to the individual acids we have studied. However, the assays of oil sands or petroleum naphthenic acids published previously have all been carried out on mixtures in which, not only the individual acid constituents were unknown, but in which many toxic nonacids may have been present. For example, the acid extracts of oil sands are known to contain so-called O3, O4 and heteroatomic compounds15 and phenols and polycyclic aromatic hydrocarbons may also be present,16,17 as the emulsifying behavior of acids can make it difficult to achieve clear separations of the acids from other toxic constituents. Similarly, some petroleum-derived naphthenic acids contain phenols and PAH.7,10 It is thus inappropriate to compare the measured toxicities with those of the acids measured here, in most instances. However, Frank et al.,18 reacted the acid extractables from oil sands process water from Alberta, Canada with diazomethane to convert those compounds containing esterifiable carboxylic acid groups to the corresponding methyl esters, separated these by distillation and resaponified the distilled ester fractions back to the carboxylic acids. The toxicities of the distilled, saponified fractions to V. fischeri were then assayed. While compounds other than acids could still be present in such fractions, none were detected by comprehensive twodimensional gas chromatography mass spectrometrycf.8 Conversion of the toxicities of these distilled oil sands acids to mM concentrations by dividing concentrations by the measured18 median molecular weights of each distilled fraction, produced EC50 values of 0.14 to 0.28 mM. These values compare to our measured values of 0.004 to 0.7 mM for the individual acids. Thus, mixtures of various classes of the acids tested, especially in the range C10 14 (typically with EC50 values of

Toxicity of Individual Naphthenic Acids to Vibrio fischeri

Recommend Documents