Toxicity of perfluorinated carboxylic acids for aquatic organisms

Toxicity of perfluorinated carboxylic acids with carbon chain C8 to C12 were tested with oligochaeta Tubifex tubifex. Toxicity was evaluated as the exposure time ET50 from onset of damage of the oligochaeta in saturated aqueous solutions. The ET50 fluctuated between 25 and 257 minutes. No statistically significant difference was found among the C8, C9 and C12 acids (ET50 between 143 and 257 minutes with large standard deviation). The acids with carbon chain C10 and C11 induced the effect significantly quicker (25 to 47 minutes). No acute toxicity measured in the three-minute test was observed in any case.


Introduction
It is impossible to mention all the fluorinated products used nowadays in miscellaneous human activities, whereas naturally occurring ones are more than rare. Moreover, fluorinated derivatives of organic compounds belong to the most stable compounds, badly degradable, easily distributed in various media and matrices over the world. Their unique chemical properties make them important ingredients in various products. .The compounds are found as widespread environmental contaminants. The global-scale multispecies mass balance model was used for simulation of the long-term transport of polyfluorinated carboxylic acids containing 8 to 13 carbons (Armitage et al., 2009). Model scenarios estimated their direct emission and indicated that the mass fluxes to the Arctic marine environment associated with oceanic transport were in excess of mass fluxes from indirect sources (atmospheric transport of precursors such as Miloň Tichý, Radka Valigurová, Radomír Čabala, Rut Uzlová, Marián Rucki  beneficial. Several antitumor agents of this type can be named, such as fluorouracil, floxuridin, tegafur, getitinib or camptothecin (Alloatti et al., 2008).
And all these compounds eventually end up in our environment.
Generally, acute toxicity of polyfluorinated compounds is low (Kovarova and Svobodova, 2008), yet there is a lack of toxicological data. There is also a lack of data on their physico-chemical properties. Due to discrepancies in measurements performed by different methods, confidence in the existing data is low. Data on physicochemical properties should therefore be used as estimates only (Hertzke et al., 2007). It can be concluded that QA/ QC should be carefully considered when generating and interpreting the results of analyses on both toxicological and physicochemical data. Often, uncommon properties of polyfluorinated organic compounds are a result of their structure, which resembles a rigid rod.
The aim of this contribution is a proposal how to estimate the relative toxicity of polyfluorinated compounds. Acute toxicity determination using oligochaeta Tubifex tubifex and the relevant mode of approach have been thoroughly described (Tichy & Rucki, 1996, Tichy et al., 20072008). The duration of action of polyfluorinated organic compounds in oligochaeta Tubifex tubifex was taken as the basis for this consideration.
Solutions of C 8 to C 12 linear perfluorinated acids were prepared and tested at the concentration of 400 μg/ml (from 0.44 × 10 -3 mol/l for C 12 to 0.58 × 10 -3 mol/l for C 8 ), which was close to saturated concentrations. The amount of exactly 0.01 g of acid was weighed and dissolved by 15 ml water in a 25 ml plastic volumetric flask and sonicated 10 min at room temperature (22-24 °C). The resulting aqueous acidic solution (pH 3 to 4) was neutralized by titration with a solution of sodium hydroxide (0.1 mol/l) to about pH 7 (checked by pH-meter) and filled up to 25 ml. Neutralization of the solutions was necessary because the low acidity itself could affect the oligochaeta.
ET n is the effective time when n % of individuals from the population exposed shows a recognizable reaction. In our case, ET 5 , ET 50 and ET 95 were determined by counting the number of immobilized Tubifex tubifex during the exposure time. Immobilized worms were counted each half hour for five hours of exposure at room temperature (22-24 °C). Deformation of bodies of the oligochaeta was observed: they wrinkled, bled and finally the worms were destroyed and broke up into pieces. Thus immobilization could be noticed only with difficulties. Each solution was tested three times in various seasons and always in a triplicate. The experimental data were processed with special computer software (Logistic Method for Determination of LD 50 by National Institute of Public Health, Prague) modified for calculation of ET 50 .

Results
At the concentration of 400 μg/ml, aqueous solutions of perfluorinated aliphatic carboxylic acid are not toxic if the acute toxicity is measured in three minutes with Tubifex tubifex (Tichý et al., 1996;. The three-minute exposure caused no apparent changes of Tubifex tubifex. Thus the solutions of the acids were not acutely toxic for the oligochaeta and the common testing procedure could not be applied.
However, the effect begins to be noticeable after a longer time, in chronic exposure, and the time necessary for induction of observable changes could be taken as a measure of toxicity of the acids. The start of the effect depends on the chain length of the given acid, however with the minimum at C 11 acid.
The results are summarized in Table 1, which contains values and confidence intervals (n = 3, α = 0.05) of ET 5 , ET 50 and ET 95 for the time necessary to immobilize 5%, 50% and 95%, respectively, of the oligochaeta from the whole population under exposure. The minimum ET 50 value appeared with perfluoroundecanoic acid (C 11 ) being comparable with perfluorodecanoic acid (C 10 ).

Discussion
We applied an uncommon measure of toxicity, ET 50 , the time necessary for the onset of destruction of the oligochaeta body. The shorter the ET 50 , the more toxic is a compound considered. We used this measure because no acute toxicity was observed, a fact that corresponded with the generally accepted opinion that perfluorinated Table 1. Effective time ET 50 , ET 5 and ET 95 (mins.). Tubifex tubifex was exposed in neutral aqueous saturated solutions of perfluorinated carboxylic acids (C 8 -C 12 ). Using the ET 50 measure, the most toxic of the set of the compounds tested, i.e. linear polyfluorinated carboxylic acids, were perfluorodecanoic (C 10 ) and perfluoroundecanoic (C 11 ) acids. No statistically significant differences between the toxic indices exist among C 8 , C 9 and C 12 acids. The C 10 and C 11 acids form a clear minimum, statistically significantly distinguished, and thus considered the most toxic ones in chronic exposures. Naturally, this conclusion applies only for our laboratory conditions in a closed system (on Petri dishes).
The question arises how does logP (n-octanol-water) of these acids or their salts behave. Unfortunately, even with simpler alcohols and fluorotelomers, it is still an unsolved problem because of the unique behavior of the fluorine substituents in the compounds. As far as the image of perfluorinated acids as rigid rods is true, a bending or a break may exist at C 10 and C 11 . We can thus just speculate that there is some non-linearity at the C 10 to C 11 carbon chain. The ability to penetrate membranes by this special form of compounds could then be influenced by this fact, although a better penetrating Tubifex surface with a rod is more probable than with anything else. The effect in the oligochaeta starts much sooner with C 10 and C 11 perfluorinated acids than with shorter or longer carbon chains.