Phosphoric acid, 2-ethylhexyl ester

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 6 June 2011 and 25 June 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a 1-Hour settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg/l . The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours - Sampling method:Samples were taken from the control (replicates R1 – R6 pooled) and each loading rate WAF test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis. - Sample storage conditions before analysis:All 0-Hour samples were stored at approximately -20°C prior to analysis. Duplicate samples were taken at each occasion and stored at approximately 20ºC for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)- Method:Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).Definitive test: Amounts of test item (12.5, 25, 50, 100 and 200 mg) were each separately added to the surface of 2 litres of culture medium to give the 6.25, 12.5, 25, 50 and 100 mg/l loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 6.25, 12.5, 25, 50 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.An aliquot (500 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (4.1 ml) to give the required test concentrations of 6.25, 12.5, 25, 50 and 100 mg/l loading rate WAF.- Controls:A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/l.Vortex depth measurements:Vortex depth measurement - the vortex depth was recorded at the start and end of the mixing period.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1°C.Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E3 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 10E4 – 10E5 cells/ml.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Samples were taken at 0, 24, 48 and 72 hours.

Hardness:

Not applicable.

Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test.

pH:

The pH value of the control cultures was observed to increase from pH 8.0 at 0 hours to pH 8.3 at 72 hours.

Dissolved oxygen:

Not applicable.

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg/l.Measured: Analysis of the test preparations at 0 hours showed measured concentrations to range from 8.6 to 98 mg/l whilst measured concentrations in the range of 8.8 to 95 mg/l were obtained at 72 hours.Given that the 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.

Details on test conditions:

TEST SYSTEMAs in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and three flasks each containing 100 ml were used for each treatment group.The control group was maintained under identical conditions but not exposed to the test item. Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 6.03 x 10E5 cells per ml. Inoculation of 500 ml of test medium with 4.1 ml of this algal suspension gave an initial nominal cell density of 5 x 10E3 cells per ml and had no significant dilution effect on the final test concentration.The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.GROWTH MEDIUM- Standard medium used: yes; The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.The culture medium is defined below:NaNO325.5mg/lMgCl2.6H2O12.164mg/lCaCl2.2H2O4.41mg/lMgSO4.7H2O14.7mg/lK2HPO41.044mg/lNaHCO315.0mg/lH3BO30.1855mg/lMnCl2.4H2O0.415mg/lZnCl20.00327mg/lFeCl3.6H2O0.159mg/lCoCl2.6H2O0.00143mg/lNa2MoO4.2H2O0.00726mg/lCuCl2.2H2O0.000012mg/lNa2EDTA.2H2O0.30mg/lNa2SeO3.5H2O0.000010mg/lThe culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl. EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Correction for the background count of the culture medium was made by counting a vessel containing culture medium alone. This value was then subtracted from all subsequent counts.TEST CONCENTRATIONS- Range finding study: Yes; The loading rates to be used in the definitive test were determined by a preliminary range-finding test. - Test concentrations: The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal loading rates of 10 and 100 mg/l for a period of 72 hours.- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/l.Samples of each loading rate WAF were taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration.

Reference substance (positive control):

yes

Remarks:

A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/l.

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

49 other: mg/l Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 46 - 52 mg/l Loading Rate WAF

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

25 other: mg/l Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

50 other: mg/l Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

33 other: mg/l Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL 30 - 35 mg/l Loading Rate WAF

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

25 other: mg/l Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

50 other: mg/l Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

RANGE-FINDING TEST:The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1.The results showed no effect on growth at 10 mg/l loading rate WAF. However, growth was observed to be reduced at 100 mg/l loading rate WAF.Based on this information loading rates of 6.25, 12.5, 25, 50 and 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, were selected for the definitive test.Chemical analysis of the test preparations at 0 and 72 hours (see Appendix 5 - attached background material) showed measured concentrations to range from 10 to 78 mg/l.DEFINITIVE TEST:Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.The mean cell densities versus time for the definitive test are presented in Figure 1 (see attached background information).GROWTH DATA:From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.Accordingly the following results were determined from the data:INHIBITION OF GROWTH RATE:ErL*10 (0 - 72 h): 37 mg/l loading rate WAFErL*20 (0 - 72 h): 41 mg/l loading rate WAFErL*50 (0 - 72 h): 49 mg/l loading rate WAF; 95% confidence limits 46 - 52 mg/l loading rate WAFwhere ErL*x is the loading rate that reduced growth rate by x%.Statistical analysis of the growth rate data was carried out for the control and all loading rates using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 6.25, 12.5 and 25 mg/l loading rate WAFs (P≥0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 25 mg/l loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 50 mg/l loading rate WAF.INHIBITION OF YIELD:EyL*10 (0 - 72 h): 23 mg/l loading rate WAFEyL*20 (0 - 72 h): 26 mg/l loading rate WAFEyL*50 (0 - 72 h): 33 mg/l loading rate WAF; 95% confidence limits 30 - 35 mg/l loading rate WAFwhere EyL*x is the loading rate that reduced yield by x%..Statistical analysis of the yield data was carried out as for growth rate. There were no statistically significant differences between the control, 6.25, 12.5 and 25 mg/l loading rate WAFs (P>0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 25 mg/l loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 50 mg/l loading rate WAF.OBSERVATIONS ON CULTURESAll test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 6.25, 12.5 and 25 mg/l loading rate WAF, however cell debris was observed to be present in the test cultures at 50 and 100 mg/l loading rate WAF. OBSERVATIONS ON TEST ITEM SOLUBILITYObservations on the test media were carried out during the mixing and testing of the WAFs. At the start of mixing the 6.25 and 12.5 mg/l loading rate WAFs were observed to have formed clear colourless media columns with a slick of test item at the surface. The 25 mg/l loading rate WAF was observed to have formed a clear colourless media column with a slick of test item at the surface and globules dispersed throughout whilst both the 50 and 100 mg/l loading rate WAFs were observed to have formed clear colourless media column with an extensive slick of test item at the surface and globules dispersed throughout. Following both the 23-Hour stirring period and 1-Hour settlement period the 6.25 mg/l loading rate WAF was observed to have formed a clear colourless solution. The 12.5, 25 and 50 mg/l loading rate WAFs were observed to have formed clear colourless solutions with a slight slick of test item at the surface whilst the 100 mg/l loading rate WAF was observed to have formed a clear colourless media column with a slick of test item at the surface.  Microscopic examination of the WAFs showed there to be no globules or micro-dispersions of test item present. At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-hour test period all control, 6.25, 12.5 and 25 mg/l loading rate WAF test cultures were observed to be pale green dispersions whilst the 50 and 100 mg/l loading rate WAF test cultures were observed to be clear colourless solutions. PHYSICO-CHEMICAL MEASUREMENTSThe pH values of the control and each test concentration are given in Table 2. Temperature was maintained at 24 ± 1ºC throughout the test.The pH value of the control cultures was observed to increase from pH 8.0 at 0 hours to pH 8.3 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.A concentration dependant decrease in pH in the range of pH 7.8 at 6.25 mg/l loading rate WAF to pH 3.7 at 100 mg/l loading rate WAF was observed at 0 hours. Given that this acidic nature was considered to be due to an intrinsic property of the test item no attempt was made to adjust the pH of the test preparations prior to exposure.VORTEX DEPTH MEASUREMENTSThe vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface (see Table 5). CHEMICAL ANALYSIS OF TEST LOADING RATESInformation 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 hours (see Appendix 5 - attached background material) showed measured concentrationsto range from 8.6 to 98 mg/l whilst measured concentrations in the range of 8.8 to 95 mg/l were obtained at 72 hours.Given that the 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.VALIDATION OF MIXING PERIODAlthough 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 sample 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.VALIDATION CRITERIA:The following data show that the cell concentration of the control cultures increased by a factor of 109 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.Mean cell density of control at 0 hours:5.78 x 10E3 cells per mlMean cell density of control at 72 hours:6.28 x 10E5 cells per mlThe mean coefficient of variation for section by section specific growth rate for the control cultures was 15% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/l.Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:ErC50 (0 – 72 h):1.2 mg/l, 95% confidence limits 0.98 – 1.4 mg/lEyC50 (0 – 72 h):0.48 mg/l, 95% confidence limits 0.43 – 0.54 mg/lNo Observed Effect Concentration (NOEC) based on growth rate:0.25 mg/lNo Observed Effect Concentration (NOEC) based on yield:0.25 mg/lLowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/lLowest Observed Effect Concentration (LOEC) based on yield:0.50 mg/lThe results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

See details on results.

Table 1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate (mg/l)		Cell Densities*(cells per ml)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	5.14E+03	4.67E+05	-	-
	R2	5.05E+03	4.57E+05
	Mean	5.10E+03	4.62E+05
10	R1	5.43E+03	7.67E+05	[10]	[68]
	R2	5.18E+03	7.80E+05
	Mean	5.30E+03	7.74E+05
100	R1	5.11E+03	9.89E+03	95	100
	R2	5.15E+03	2.45E+03
	Mean	5.13E+03	6.17E+03

*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁and R₂= Replicates 1 and 2

[Increase in growth compared to controls]

Table 2              Cell Densities and pH Values in the DefinitiveTest

Nominal Loading Rate (mg/l)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	8.0	6.10E+03	2.82E+04	1.33E+05	8.04E+05	8.3
	R2		5.49E+03	1.88E+04	1.01E+05	5.96E+05
	R3		6.02E+03	1.88E+04	9.46E+04	4.87E+05
	R4		6.15E+03	2.21E+04	1.26E+05	7.94E+05
	R5		5.70E+03	1.87E+04	1.09E+05	6.21E+05
	R6		5.19E+03	1.30E+04	7.68E+04	4.64E+05
	Mean		5.78E+03	1.99E+04	1.07E+05	6.28E+05
6.25	R1	7.8	5.33E+03	2.57E+04	1.09E+05	6.13E+05	8.2
	R2		4.60E+03	2.64E+04	1.40E+05	6.59E+05
	R3		4.52E+03	2.15E+04	1.17E+05	3.78E+05
	Mean		4.82E+03	2.45E+04	1.22E+05	5.50E+05
12.5	R1	7.5	5.58E+03	3.39E+04	1.40E+05	6.64E+05	8.1
	R2		6.00E+03	3.33E+04	1.70E+05	7.37E+05
	R3		5.52E+03	3.28E+04	1.65E+05	7.37E+05
	Mean		5.70E+03	3.33E+04	1.58E+05	7.12E+05
25	R1	7.2	5.55E+03	2.72E+04	1.30E+05	4.05E+05	8.0
	R2		5.06E+03	3.49E+04	1.45E+05	5.27E+05
	R3		5.28E+03	3.32E+04	1.49E+05	6.48E+05
	Mean		5.30E+03	3.18E+04	1.41E+05	5.26E+05
50	R1	6.2	5.11E+03	9.93E+03	2.07E+04	5.36E+04	7.3
	R2		4.52E+03	1.07E+04	1.95E+04	4.53E+04
	R3		4.18E+03	9.23E+03	1.62E+04	3.75E+04
	Mean		4.61E+03	9.97E+03	1.88E+04	4.55E+04
100	R1	3.7	5.46E+03	3.26E+03	4.22E+03	6.52E+03	3.7
	R2		5.25E+03	3.26E+03	4.01E+03	4.94E+03
	R3		6.39E+03	2.69E+03	2.18E+03	1.90E+03
	Mean		5.70E+03	3.07E+03	3.47E+03	4.45E+03

 *Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁- R₆= Replicates 1 to 6

Table 3              Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/ml/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.072	0.065	0.075
	R2	0.055	0.070	0.074
	R3	0.055	0.067	0.068
	R4	0.062	0.072	0.077
	R5	0.055	0.073	0.073
	R6	0.040	0.074	0.075
	Mean	0.057	0.070	0.074

R₁- R₆= Replicates 1 to 6

Table 4              Inhibition of Growth RateYield in the Definitive Test

Nominal Loading Rate (mg/l)		Growth Rate (cells/ml/hour)		Yield (cells/ml)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.071		7.98E+05
	R2	0.066		5.90E+05
	R3	0.064		4.81E+05
	R4	0.070	-	7.88E+05	-
	R5	0.067		6.15E+05
	R6	0.063		4.59E+05
	Mean	0.067		6.22E+05
	SD	0.003		1.46E+05
6.25	R1	0.067	0	6.07E+05
	R2	0.068	[1]	6.54E+05
	R3	0.060	10	3.73E+05
	Mean	0.065	3	5.45E+05	12
	SD	0.004		1.50E+05
12.5	R1	0.068	[1]	6.58E+05
	R2	0.069	[3]	7.31E+05
	R3	0.069	[3]	7.31E+05
	Mean	0.069	[2]	7.07E+05	[14]
	SD	0.001		4.20E+04
25	R1	0.061	9	3.99E+05
	R2	0.065	3	5.22E+05
	R3	0.068	[1]	6.42E+05
	Mean	0.065	4	5.21E+05	16
	SD	0.004		1.21E+05
50	R1	0.033	51	4.85E+04
	R2	0.031	54	4.08E+04
	R3	0.028	58	3.34E+04
	Mean	0.031	54	4.09E+04	93
	SD	0.003		7.56E+03
100	R1	0.004	94	1.06E+03
	R2	0.000	100	-3.04E+02
	R3	-0.013	119	-4.50E+03
	Mean	-0.003	104	-1.25E+03	100
	SD	0.009		2.89E+03

*In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

 

Table 5              Vortex Depth Measurements at the Start and End of the Mixing Period

	Nominal Loading Rate (mg/l)
	Control		6.25		12.5
	*	+	*	+	*	+
Height of Media Column (cm)	12	12	12	12	12	12
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

 

	Nominal Loading Rate (mg/l)
	25		50		100
	*	+	*	+	*	+
Height of Media Column (cm)	12	12	12	12	12	12
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

 

*= Start of mixing period

+= End of mixing period

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results:Growth rate:EL50: 49 mg/l Loading Rate WAFYield:EL50: 33 mg/l Loading Rate WAF

Executive summary:

Introduction.

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No. 440/2008.

Methods.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

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 Pseudokirchneriella subcapitata to the test item gave the following results:

Response Variable	EL*50 (mg/l Loading Rate WAF)	95% Confidence Limits (mg/l Loading Rate WAF)			No Observed Effect Loading Rate (NOEL) (mg/l)	Lowest Observed Effect Loading Rate (LOEL) (mg/l)
Growth Rate	49	46	-	52	25	50
Yield	33	30	-	35	25	50

*EL = Effective Loading Rate

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 hours showed measured concentrations to range from 8.6 to 98 mg/l whilst measured concentrations in the range of 8.8 to 95 mg/l were obtained at 72 hours.

Given that the 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.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 to 11 January 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The test was conducted according to the principles of GLP and a recognised ISO marine test guideline. Also, the validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

GLP compliance:

yes

Analytical monitoring:

no

Details on test solutions:

The test solutions were prepared as water accomodated fractions as follows: The test substance was mixed in culture medium in a 1 L glass flask with stopcock on a magnetic stirrer (-100 rpm) for -20 hours (overnight). The flasks were wrapped in aluminum foil to avoid light exposure.After mixing, the solution was separated for 1-4 hours.The undissolved substances distributed in the top (or the bottom) layers were not used. The first 50 mL was discarded and the middle phase was collected for testing

Test organisms (species):

Skeletonema costatum

Details on test organisms:

The strain NIVA BAC 1 was used. Algal medium 1/2 T from Guillard and Ryther (1962) was used.

Test type:

not specified

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20.5-21.7°C

pH:

8.24-8.38

Nominal and measured concentrations:

Nominal test concentrations: 10, 50, 250 and 1000 mg/L

Details on test conditions:

A range-finding test was conducted followed by a definitive test including three test concentrations replicated three times and an untreated control replicated six times. The growth rate of the algal culture at the start of the test was 1.72/day. The light intensity during the test varied from 7150 to 9100 lux.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

The growth rate inhibition of 1.5 mg/L 3,5-dichlorophenol was 39% after 72 h.

Validity criteria fulfilled:

yes

Conclusions:

The 72 h EC50 for Skeletonema exposed to Servo RM 93700 was found to be 15 mg/L WAF.

Executive summary:

A study was conducted in accordance with IOS 10253 in which the marine algal species Skeletonema costatum was exposed to Servo RM 93700 for 72 h. The 72 h EC50 for Skeletonema exposed to Servo RM 93700 was found to be 15 mg/L WAF.

Description of key information

72h EL50 (growth rate) = 49 mg/L, 72h NOELr (growth rate) = 25 mg/L (Pseudokirchnerella subcapitata); Vryenhoef & Mullee (2011); OECD 201
72h EL50 (growth rate) = 15 mg/L, 72h NOELr (growth rate) = 10 mg/L (Skeletonema costatum); Furst (2002); ISO 10253

Key value for chemical safety assessment

EC50 for freshwater algae:

49 mg/L

EC50 for marine water algae:

15 mg/L

EC10 or NOEC for freshwater algae:

25 mg/L

EC10 or NOEC for marine water algae:

10 mg/L

Additional information

Two key studies are submitted for the toxicity to aquatic algae and cyanobacteria endpoint, one for a freshwater species and one for marine species. Each study was conducted using a standard approach for testing poorly soluble, multi-component substances, the preparation of a water accommodated fraction (WAF). As such, the effect values are presented as loading rates (e.g. EL₅₀or NOELr), and not measured aqueous concentrations.