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Toxicity to aquatic algae and cyanobacteria

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Description of key information

The effect of the test material on the growth of Scenedesmus subspicatus has been investigated and gave EC50 values of greater than 0.10 mg/l.  Correspondingly the No Observed Effect Concentration was 0.10 mg/l.
The test material was shown to degrade in culture medium. In order to give a worst case analysis of the data the results of the test were also calculated based on the geometric mean measured concentration of the parent test material, AS 100, in the centrifuged test samples. In terms of the geometric mean measured test concentrations the EC50 values were estimated to be greater than 0.00084 mg/l. Correspondingly the No Observed Effect Concentration was 0.00084 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.1 mg/L
EC10 or NOEC for freshwater algae:
0.1 mg/L

Additional information

Introduction.

A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods.

Preliminary work showed that on addition to culture medium there was an initial rapid loss of test material, however the rate of degradation slowed resulting in a low but relatively stable parent test material concentration and complete degradation of the test material did not occur. A previous study conducted on the test material (Determination of Hydrolysis Half-Life at Environmentally Relevant pH and Temperature) showed that the test material hydrolysed to form Di-isopropyl xanthogen disulphide (DIXD). In the preliminary work conducted for the Algal Inhibition Test the concentration of DIXD did not increase as the concentration of parent test material (AS 100) declined. These results suggested that degradation of the test material in culture medium may not be due to hydrolysis alone and may include other processes such as oxidation and the formation of further unidentified degradation products.

The preliminary analytical work conducted indicated that under experimental conditions the water solubility of AS100 was approximately 0.10 mg/l. Therefore, following a preliminary range-finding test, Scenedesmus subspicatus was exposed to an aqueous dispersion of the test material at a nominal concentration of 0.10 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

The test concentration of 0.10 mg/l was prepared using a solvent spike method of preparation followed by a 24-Hour stirring period in order to allow degradation of the test material to occur thus ensuring that the test organisms were exposed to a relatively stable concentration of both parent test material and degradation product(s).

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.

Results.

Exposure of Scenedesmus subspicatus to the test material gave EC50 values of greater than 0.10 mg/l and correspondingly the No Observed Effect Concentration was 0.10 mg/l.

The test material was shown to degrade in culture medium. Analysis of the test preparations therefore included analysis for the parent test material (AS 100) and one of its known degradants, DIXD. Preliminary work conducted at a nominal test concentration of 0.75 mg/l suggested that a significant proportion of both AS 100 and DIXD may be present as dispersed rather than dissolved material. Therefore samples taken for chemical analysis were analysed untreated and after centrifugation (40000g, 30 minutes) in order to confirm the dissolved and hence bioavailable test material concentration that the test organisms are exposed to.

The recovery analyses conducted showed that the recovery of both AS 100 and DIXD from culture medium was relatively low, 84% and 72% respectively, and so the untreated test sample results were corrected for procedural recoveries analysed alongside the test sample analyses. Recovery analyses conducted in centrifuge tubes showed that both AS 100 and DIXD adsorbed to the centrifuge tubes and hence the centrifuged test sample results were corrected for the centrifuge tube only recovery rate.

Analysis of the test preparations at 0 hours showed measured concentrations of AS 100 to be 0.00813 mg/l and 0.00969 mg/l in the untreated samples, and 0.0134 mg/l and 0.0119 mg/l in the centrifuged test samples. The centrifuged test sample results were slightly higher than the untreated test sample results. This was considered to be due to the centrifuged test sample results having been corrected for the relatively low centrifuge tube only recovery rate where small variations in the measured concentration lead to relatively large variations in the final calculated result.

Overall the measured concentrations of AS 100 at 0 hours were approximately 8% to 13% of the nominal test concentration. The relatively low measured concentrations were considered to be due to degradation of the test material in culture medium and were in line with the results obtained from the media preparation trials conducted.

Analysis for the degradation product DIXD was also performed. At 0 hours measured concentrations of DIXD were 0.0428 mg/l and 0.0445 mg/l in the untreated samples, and 0.0456 mg/l and 0.0381 mg/l in the centrifuged test samples.

Given that there were no significant differences in the measured concentrations between the untreated and centrifuged test samples it was considered that all the AS 100 and DIXD present in the test system was dissolved and hence bioavailable. The difference between the results obtained in the definitive test and those obtained from the preliminary work conducted which suggested that dispersed material may be present was considered to be due to the definitive test being conducted at a lower nominal test concentration (0.10 mg/l) which was equivalent to the water solubility level of the test material under experimental conditions.

The sum of the concentrations of AS 100 and DIXD determined at 0 hours did not result in a mass balance. This was in line with the preliminary work conducted to determine the rate of hydrolysis in culture medium which suggested that degradation of the test material in culture medium was not due to hydrolysis to DIXD alone and may include other processes such as oxidation. No other degradation products were detected using the analytical method employed in the study.

After 72 hours there was a marked decline in the measured concentrations of AS 100 to 0.00744 mg/l and 0.00228 mg/l in the untreated samples and to less than the Limit of Quantitation (LOQ) in the centrifuged samples. A similar decline was observed in the measured concentrations of DIXD to 0.00148 mg/l and 0.000946 mg/l in the untreated samples, and to 0.00407 mg/l and 0.00463 mg/l in the centrifuged samples. These results were in line with the stability analyses conducted which showed both AS 100 and DIXD to be unstable in culture medium over a 72-Hour period.

Given that toxicity cannot be attributed to the parent test material or its degradation product(s) but to the parent test material/degradation product(s) mixture as a whole, EC50values would not normally be calculated in terms of measured concentrations. However in order to give a worst case analysis of the data it was considered justifiable in this case to express the results in terms of the geometric mean measured test concentrations of the parent test material AS 100. 

The EC50values based on the geometric mean measured test concentrations of the centrifuged test media were greater than 0.00084 mg/l and correspondingly the No Observed Effect Concentration was 0.00084 mg/l.