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EC number: 947-969-4 | CAS number: -
Table 1 Cumulative Mortality Data in the Range-finding Test
(Initial Population = 3)
Table 2 Cumulative Mortality Data in the Definitive Test
Cumulative Mortality(Initial Population =7)
R1and R2= Replicates 1 and 2
Table 3 Vortex Depth Measurements at the Start and End of Each Mixing Period
FIRST MIXING PERIOD
Nominal Loading Rate (mg/l)
Height of Water Column (cm)
Depth of Vortex (cm)
Observation of Vortex
SECOND MIXING PERIOD
THIRD MIXING PERIOD
FOURTH MIXING PERIOD
R1- R2= Replicates 1 and 2
*= Start of mixing period
+= End of mixing period
Appendix 3: Validation of Mixing Period
Pre-study investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher measured levels of test item, in the WAF. A WAF of a nominal loading rate of 100 mg/l was prepared in duplicate in deionised reverse osmosis purified water and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 71 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for chemical analysis.
The results are summarised as follows:
Test Item Loading Rate
Measured Concentration (mg/l)
Although the results from this pre-study investigational work showed that the measured concentration increased from 24 hours to 72 hours, chemical analysis of the 100 mg/l Loading rate WAF test samples from the range-finding test showed that 24 hours was a sufficient preparation period to ensure the maximum amount of the dissolved water soluble fraction of the test item. The preparation of the WAF was maintained at 24 hours.
LOQ = Limit of Quantitation (assessed down to 0.03 mg/l)
Appendix 5 Physico-Chemical Measurements
0 Hours (Fresh Media)
24 Hours (Old Media)
24 Hours (Fresh Media)
48 Hours (Old Media)
48 Hours (Fresh Media)
72 Hours (Old Media)
72 Hours (Fresh Media)
96 Hours (Old Media)
*ASV= Dissolved oxygen concentration expressed as a percentage of Air Saturation Value
A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.
Following a preliminary range-finding test, fish were exposed, in groups of seven, to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l for a period of 96 hours at a temperature of approximately 14ºC under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.
The 96-Hour LL50based on nominal loading rates was greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.
Information supplied by the Sponsor, indicated that the test item contained approximately 42% 2-ethylhexyl dihydrogen phosphate (MW = 210 g/mol) and 49% bis(2-ethylhexyl) hydrogen phosphate (MW = 322 g/mol)). Under the acidic conditions of the analysis, both compounds could be observed, but the lower acidity and greater response of the bis(2-ethylhexyl) hydrogen phosphate meant that it appeared as the main peak in the chromatogram.
Analysis of the test preparations at 0 (fresh media), 24 and 96 hours (old media) showed measured concentrations to range from 94.9 to 98.0 mg/l with the exception of the 100 mg/l loading rate WAF replicate R2 at 0 and 24 hours which showed measured concentrations of 60.7 and 66.0 mg/l respectively. Analysis of the duplicate samples, stored at approximately -20°C prior to analysis, showed measured concentrations of 69.7 and 68.9 mg/l respectively. The results were lower than expected as results from the remainder of the samples showed measured concentrations in excess of 90 mg/l. A review of the data could not reveal a reason for this, however, this anomalous result was considered to have no adverse effect on the outcome of the test.
Given that toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole the results were based on nominal loading rates only.
Oncorhynchus mykiss; OECD TG 203; read-across to EC 235-741-0.
96-h LL50 value of greater than 100 mg/L loading rate WAF
No study is available for Phosphoric Acid, Esters with Alcohols, C11-14 iso, C13-rich. Results are read across from an OECD TG 203 study performed on the 2-ethylhexyl- analogue. In this study, groups of 7 rainbow trout (Oncorhynchus mykiss) were exposed, in groups of seven, to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l for a period of 96 hours under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours. The 96-h LL50 based on nominal loading rates was > 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF.
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