The toxicity of investigated ketones, aldehydes, oximes, and oxime ethers on Daphnia magna, R. subcapitata, Spirodela polyrhiza, and A. fischeri is described below. The test results indicate that the compounds being tested range from non acute toxicity to very high acute toxicity, depending on the organism tested. For example, the most toxic compound for R. subcapitata was found to be cyclocitral oxime (Figs. 1 and 2, Table S1) with an EC50 of 0.034 mg/L, followed by citral (EC50 = 0.13 mg/L), which both fall into the V class of toxicity. Figure 2 represents growth inhibition in % vs. the logarithm of the concentration in mg/L. The more the curve is shifted to the left, the higher the toxicity. The slope of the curve is also important; for example, the oxime o-tolualdehyde curve has the lowest slope, and the tested range of the concentration logarithm does not reach the 50% inhibition value in the tested concentration, which means that this compound is the least toxic. Other compounds such as o-tolualdehyde oxime (EC50 = 1.04 mg/L) and p-tolualdehyde (EC50 = 1.27 mg/L) are classified as high acute hazards and fall into the IV class of toxicity. Most of the other compounds, with the exception of the p-tolualdehyde oxime O-methyl ether (EC50 = 92.35 mg/L) and dihydrocinamaldehyde (EC50 = 75.79 mg/L) which fall into the III class of toxicity, are classified as slight acute hazards or non/low-toxic and fall into the I or II class of toxicity. It should be noted that no EC50 values were given for the cyclocitral oxime O-methyl ether and p-tolualdehyde oxime as they significantly exceeded the test range. (Table S1). Toxicity values have been previously reported for only 2 compounds, commercially available oximes stemone and buccoxime. The difference from our results might come from the variability in the experimental procedure and in the strain used.
Evaluation of ecotoxic properties on D. magna (Table S2, Fig. 3) revealed that all compounds show only two levels of toxicity: high acute toxicity (IV class) and acute toxicity (III class). The least toxic among the compounds tested was propiophenone with an EC50 of 80.14 mg/L. Other compounds that belong to this type of toxicity included buccoxime, citral, citral oxime, cyclocitral oxime, dihydrocinnamaldehyde, perillaldehyde, perillaldehyde oxime, perillaldehyde oxime O-methyl ether, and stemone. The highest toxicity was found for cyclocitral oxime with an EC50 of 2.56 mg/L after 48 hours, which shows high acute toxicity, together with the remaining compounds. In almost all cases oxime ethers were comparable or less toxic than parental oximes or commercially available carbonyl compounds. In the series of two isomers (m- and p-) of tolualdehyde a clear pattern can be noticed where oximation and subsequent alkylation led to the significant decrease of toxicity.
In the case of the effect of flavour and fragrance carbonyl compounds, oximes and oxime ethers on the development of S. polyrhiza, the EC50 of all compounds were in the range of 5 · 10-5-0.15 mg/L (Fig. 4, Table S3), indicating very high acute toxicity (class V). Perillaldehyde oxime O-methyl ether (EC50 = 5 · 10− 5 mg/L) was the most toxic to S. polyrhiza and dihydrocinamaldehyde was the least toxic with EC50 0.15 mg/L after 72 h.
Microtox
Figure 5 shows the toxicity effect EC50(15) of the tested chemical compounds in relation to the luminescent bacteria Alivibrio fischeri. Detailed information on the tested parameters, i.e. EC50 values, toxicity units (TU) and estimating equation and 95% confidence range, are included in Table S4 in Supporting Information.
Alivibrio fischeri are naturally luminescent Gram-negative marine bacteria, also known as Photobacterium phosphorum NRRL-B-11177 and Vibrio fischeri. A. fischeri reacts quickly to bioavailable toxicants by decreasing its natural bioluminescence in seconds to minutes, depending on the chemical structure of the toxicant and its concentration (Kurvet et al. 2017). Biotests using Alivibrio fischeri bacteria are a very useful tool used to assess the degree of toxicity of a given compound (Kaiser &Devillers 1994, Kurvet et al. 2011, Parvez et al. 2006, Suppi et al. 2015). Under optimal conditions, these bacteria are able to use about 10% of the energy that comes from metabolism for glowing. In a polluted environment or in the presence of compounds that have an unfavorable effect on bacterial cells, their metabolic processes are disturbed and the luminescence disappears. Therefore, the reduction in light output reflects the inhibition of bacterial metabolic activity and is proportional to the toxicity of the chemical or test sample (Bulich 1982, Woodland Hastings et al. 1987).
EC50(15) values were obtained from the line of best fit through the four data points on a plot (usually log-log) of test solution concentration vs. Gamma light loss factor. Gamma tells us the ratio of the amount of light lost to the amount of light remaining and can be converted into %effect. The Gamma value cannot be negative. This would prove that the addition of a toxicant stimulates the bacteria to glow. Therefore, negative values were not taken into account for further calculations. The EC50(15) value, corresponding to the halving of light intensity after 15 minutes, is the (anti-logarithm) of the intercept where Gamma = 1, i.e. where log Gamma = 0 (Gellert 2000). The lower the EC50 value, the more toxic the sample is. In order to be able to present these values in a clearer way, toxic equivalents were used, where the higher the value of the TU factor, the more toxic the substance is (Ashworth &Symonds 2013, ISO Technical Committee 2018, Pogorzelec &Piekarska 2017, SDI 1992).
All chemical compounds tested by us should be classified as toxic class V (acute toxicity) based on the EC50 parameter (Table S4) and calculated values of the TU factor after 15 minutes of contact of luminescent bacteria with the tested substance. The highest value of this coefficient (347,056) was obtained for m-tolualdehyde oxime O-methyl ether, and the lowest value of 6,480 for propiophenone oxime O-ethyl ether.
Reducing the toxicity in relation to the starting compounds of the synthesis was observed only for cyclocitral oxime O-methyl ether and propiophenone oxime O-ethyl ether. Such high values of toxicity units obtained in this test compared to other tests could be due to the shortest contact time of the test organisms with volatile chemicals.