Phosphoric acid, decyl octyl ester

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From July 29, 2011 to September 21, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, 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 substances, 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 substance in cases where the test substance 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 substance with water for a prolonged period. Pre-study work showed that a preparation period of 24 h was sufficient to ensure equilibration between the test substance and water phase. At the completion of mixing and following a 1 h settlement period, the test substance phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test substance and/or leachates from the test substance). Exposures are expressed in terms of the original concentration of test substance in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test substance in the WAF.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L. The concentration and stability of the test substance in the test preparations were verified by chemical analysis at 0 and 72 h

- 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 h for quantitative analysis. Duplicate samples were taken at each occasion and stored at approximately 20ºC for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Validation of mixing period:
Pre-study investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances. A WAF of nominal loading rate of 100 mg/L was prepared, in duplicate, in reconstituted water. One loading rate was stirred for a period of 23 h and the other for a period of 95 h. After a 1-H standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis (See appendix 4 - attached background material).

Range-finding tests:
1st Test:
Due to the low aqueous solubility and complex nature of the test substance for the purposes of the test the test substance was prepared as a Water Accommodated Fraction (WAF). The initial 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 h. The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration. Amounts of test substance (20 and 200 mg) were each separately added to the surface of 2 litres of culture medium to give the 10 and 100 mg/L loading rates respectively. After the addition of the test substance, 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 h and the mixtures allowed to stand for 1 h. 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 10 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test substance to be present. An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (5.5 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF. The control group was maintained under identical conditions but not exposed to the test substance. At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 h. After 72 h the cell density of each flask was determined using a Coulter® Multisizer Particle Counter. Samples of each loading rate WAF were taken for chemical analysis at 0 and 72 h in order for determine the stability of the test substance over the test duration. All samples were stored at approximately -20°C prior to analysis.

2nd Test:
Based on the results obtained from the initial range-finding test, and following discussion with the Sponsor, it was considered appropriate to conduct a second range-finding test using a modified culture medium (see details on test conditions for details on culture medium). The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration. An amount of test substance (200 mg) was added to the surface of 2 litres of culture medium to give the 100 mg/L loading rate. After the addition of the test substance, 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 h and the mixture allowed to stand for 1 h. 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 100 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test substance to be present. An aliquot (200 mL) of the WAF was inoculated with algal suspension (2.3 mL) to give the required test concentration of 100 mg/L loading rate WAF. The control group was maintained under identical conditions but not exposed to the test substance. Exposure conditions in the second range-finding test were the same as those in the initial test.

Definitive test:
Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L. Following discussion with the Sponsor, it was considered appropriate, for the purposes of the definitive test to use a modified culture medium. Furthermore, it was considered appropriate, based on the acidic nature of the test substance, to adjust the pH of the test preparations to that of the control preparation prior to the addition of the algal inoculum.

Experimental Preparation:
Amounts of test substance (10, 32, 20, 64 and 200 mg) were each separately added to the surface of 10, 10, 2, 2 and 2 litres of culture medium to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. After the addition of the test substance, 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 h and the mixtures allowed to stand for 1 h. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test substance was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). 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. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed there to be no particles of test substance present. The pH of each test preparation was measured and adjusted, where necessary to pH 7.8.An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (6.6 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF. The concentration and stability of the test substance in the test preparations were verified by chemical analysis at 0 and 72 h.

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

Hardness:

For the purposes of the second range-finding test and the definitive test additional CaCl2.2H2O (198.4 mg/L) was added to the culture medium increasing the hardness from 15 mg/L to approximately 150 mg/L as CaCO3 in order to overcome possible chelation effects.

Test temperature:

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

pH:

The pH of each test preparation was measured and adjusted, where necessary to pH 7.8.

Dissolved oxygen:

Not applicable.

Salinity:

Not applicable.

Nominal and measured concentrations:

Definitive Test:
Nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L.
Chemical analysis of the test preparations at 0 h showed measured test concentrations to range from 0.30 mg/L at 1.0 mg/L loading rate WAF through to 2.4 mg/L at 100 mg/L loading rate WAF. A decline in measured test concentration was observed at 72 h in the range of less than the limit of quantitation (LOQ) of the analytical method employed (which was determined to be 0.031 mg/L at 1.0 mg/L loading rate WAF through to 1.1 mg/L at 100 mg/L loading rate WAF.

Details on test conditions:

Test system
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 3.80 x 10E5 cells per mL. Inoculation of 500 mL of test medium with 6.6 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 h.

Growth medium
Culture Medium:
The culture medium used was the same as that used to maintain the stock culture. However, as the test item was known to contain phosphonates, it was considered possible that complexation may have affected the toxicity of the test item by reducing the availability of salts such as magnesium and calcium and other key nutrients in the test medium which are essential for supporting healthy algal growth. Inhibition of algal growth by metal complexing agents can be eliminated by compensating for the deficit in the concentration of the essential ion(s). Therefore, for the purposes of the second range-finding test and the definitive test, in accordance with the OECD Guidance Document on Aquatic Testing of Difficult Substances and Mixtures additional CaCl2.2H2O (198.4 mg/L) was added to the culture medium increasing the hardness from 15 mg/L to approximately 150 mg/L as CaCO3 in order to overcome possible chelation effects.

Culture MediumNaNO325.5mg/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/L

Effect parameters measured (with observation intervals if applicable):
Samples were taken at 0, 23, 43 and 72 h and the cell densities determined using a Coulter® Multisizer Particle Counter.

Physico-chemical measurements:
The pH of the control and each test concentration was determined at initiation of the test and after 72 h exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily

Vortex depth measurements:
The vortex depth was recorded at the start and end of the mixing period

Test concentrations
Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: mg/L Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 1.1 other: mg/L Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

< 1 other: mg/L Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Growth rate and biomass

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

ca. 1 other: mg/L Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Growth arte and biomass

Details on results:

Validation of mixing period:
Pre-study investigational work (see Appendix 4- attached background material) indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 h. Therefore, for the purpose of testing the test substance was prepared using a stirring period of 23 h followed by a 1 h settlement period.

Range-finding tests:
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test substance during the range-finding tests are given in Table 1 and Table 2.Growth was observed to be reduced at 10 and 100 mg/L loading rate WAF. Based on this information loading rates of 1.0, 3.2, 10, 32 and 100 mg/L, using a stirring period of 23 h followed by a 1 h standing period, were selected for the definitive test. Chemical analysis of the 10 and 100 mg/L loading rate WAF preparations taken for the initial range-finding test at 0 h (see Appendix 5) showed measured test concentrations of 2.7 and 8.2 mg/L respectively were obtained. A slight decline in measured test concentration was observed at 72 h with measured concentrations of 1.7 and 6.0 mg/L being obtained for the 10 and 100 mg/L loading rate WAF preparations respectively.

Definitive test:
Cell density values determined at each sampling time are given in Table 3. Daily specific growth rates for the control cultures are given in Table 4. Growth rate and yield values for the control and test cultures after 72 h and percentage inhibition values are given in Table 5.The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage inhibition values are plotted against loading rate in Figure 2 and Figure 3 (see attached background material).

Growth data:
From the data given in Tables 3 and 5, 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 substance over the 72 h exposure period.
Accordingly the following results were determined from the data: Inhibition of growth rate:
ErL*10 (0 - 72 h) :< 1.0 mg/L loading rate WAF
ErL*20 (0 - 72 h) : >100 mg/L loading rate WAF
ErL*50 (0 - 72 h) : >100 mg/L loading rate WAF

Where,
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 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). Statistically significant differences were observed between the control and all loading rate WAFs (P≥0.05) and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was less than 1.0 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 1.0 mg/L loading rate WAF.

Inhibition of yield:
EyL*10 (0 - 72 h): <1.0 mg/L loading rate WAF
EyL*20 (0 - 72 h): <1.0 mg/L loading rate WAF
EyL*50 (0 - 72 h): 1.1 mg/L loading rate WAF

Where,
EyL*x is the loading rate that reduced yield by x%.

Statistically significant differences were observed between the control and all loading rate WAFs (P≥0.05) and therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was less than 1.0 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 1.0 mg/L loading rate WAF.

Observations on cultures:
All test and control cultures were inspected microscopically at 72 h. There were no abnormalities detected in any of the control or test cultures.

Observations on test substance solubility:
Observations on the test media were carried out during the mixing and testing of the WAFs. At the start of stirring all loading rate WAFs were observed to have formed clear colourless media columns with a slick of test substance at the media surface and globules of test substance dispersed throughout. After the 23 h stirring period all loading rate WAFs were observed to have formed clear colourless media columns with white crystalline flakes at the surface and dispersed throughout, the quantity of which increased with increased loading rate. After the 1 h standing period the 1.0, 3.2, 10 and 32 mg/L loading rate WAFs were observed to have formed clear colourless media columns with white crystalline flakes settled on the bottom of the mixing vessel and floating on the surface. The 100 mg/L loading rate WAF was observed to have flakes of test substance dispersed throughout the media column. Microscopic observations made on the WAFs indicated that dispersed particles of test substance were present and therefore it was considered appropriate to filter the WAFs through a glass wool plug. Microscopic observations made on the WAFs after filtering showed there to be no particles of test substance present. At the start of the test all control, 1.0, 3.2, 10 and 32 mg/L loading rate WAF cultures were observed to be clear colourless solutions, the 100 mg/L loading rate WAF cultures were observed to be cloudy dispersions. After the 72 h test period all control, 1.0, 3.2, 10 and 32 mg/L loading rate WAF test cultures were observed to be pale green dispersions whilst the 100 mg/L loading WAF test cultures were observed to be very pale green dispersions with coarse white particles present.

Physico-chemical measurements
The pH values of the control and each test concentration prior to and post adjustment at 0 h and following 72 h exposure are given in Table 6. Temperature was maintained at 24 ± 1ºC throughout the test. The pH value of the control cultures (see Table 6) was observed to increase from pH 7.8 at 0 h to pH 8.0 at 72 h. The pH deviation in the control cultures was less than 1.5 pH units after 72 h and therefore was within the limits given in the Test Guidelines.

Vortex depth measurements
The 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 7).

Chemical analysis of test loading rates:
Chemical analysis of the test preparations at 0 h (see Appendix 5) showed measured test concentrations to range from 0.30 mg/L at 1.0 mg/L loading rate WAF through to 2.4 mg/L at 100 mg/L loading rate WAF. A decline in measured test concentration was observed at 72 h in the range of less than the limit of quantitation (LOQ) of the analytical method employed (which was determined to be 0.031 mg/L) at 1.0 mg/L loading rate WAF through to 1.1 mg/L at 100 mg/L loading rate WAF. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test substance as a whole the results were based on nominal loading rates only.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference substance 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 substance gave the following results:
ErC50 (0 – 72 h):1.2 mg/L, 95% confidence limits 0.98 – 1.4 mg/L
EyC50 (0 – 72 h):0.48 mg/L, 95% confidence limits 0.43 – 0.54 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference substance.

Reported statistics and error estimates:

Refer to details on results.

Table1 Cell densities and percentage inhibition of growth from the initial range-finding test

Nominal Loading Rate (mg/L)		Cell Densities*(cells per mL)		Inhibition Values (%)
		0 H	72 H	Growth Rate	Yield
Control	R1	5.04E+03	9.32E+05	-	-
	R2	5.13E+03	9.56E+05
	Mean	5.08E+03	9.44E+05
10	R1	5.05E+03	4.53E+05	15	53
	R2	5.10E+03	4.38E+05
	Mean	5.08E+03	4.46E+05
100	R1	5.06E+03	1.28E+04	85	99
	R2	5.06E+03	9.46E+03
	Mean	5.06E+03	1.12E+04

 *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

Table 2 Cell densities and percentage inhibition of growth from the second range-finding test

Nominal Loading Rate (mg/L)		Cell Densities*(cells per mL)		Inhibition Values (%)
		0 H	72 H	Growth Rate	Yield
Control	R1	5.92E+03	1.04E+06	-	-
	R2	6.27E+03	1.04E+06
	Mean	6.10E+03	1.04E+06
100	R1	5.54E+03	1.21E+04	93	100
	R2	5.60E+03	3.58E+03
	Mean	5.57E+03	7.86E+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

Table 3 Cell densities in the definitive test

Nominal Loading Rate (mg/L)		Cell Densities*(cells per mL)
		0 h	23 h	43 h	72 h
Control	R1	5.13E+03	3.54E+04	1.41E+05	9.34E+05
	R2	6.05E+03	3.73E+04	1.21E+05	8.01E+05
	R3	6.38E+03	3.45E+04	1.33E+05	9.22E+05
	R4	5.60E+03	3.24E+04	1.25E+05	8.60E+05
	R5	5.79E+03	3.02E+04	1.36E+05	1.05E+06
	R6	5.62E+03	3.38E+04	1.23E+05	6.98E+05
	Mean	5.76E+03	3.39E+04	1.30E+05	8.78E+05
1.0	R1	5.36E+03	2.66E+04	6.34E+04	4.71E+05
	R2	5.28E+03	3.20E+04	6.38E+04	4.66E+05
	R3	5.06E+03	3.07E+04	6.52E+04	5.08E+05
	Mean	5.23E+03	2.98E+04	6.41E+04	4.82E+05
3.2	R1	5.18E+03	2.54E+04	6.82E+04	3.33E+05
	R2	5.09E+03	2.13E+04	7.44E+04	3.63E+05
	R3	5.13E+03	2.70E+04	7.75E+04	3.84E+05
	Mean	5.13E+03	2.45E+04	7.34E+04	3.60E+05
10	R1	5.00E+03	2.80E+04	7.35E+04	2.60E+05
	R2	5.13E+03	3.37E+04	7.07E+04	3.31E+05
	R3	5.17E+03	3.33E+04	6.75E+04	2.37E+05
	Mean	5.10E+03	3.17E+04	7.05E+04	2.76E+05
32	R1	5.42E+03	3.86E+04	1.17E+05	3.98E+05
	R2	5.70E+03	3.87E+04	1.06E+05	5.08E+05
	R3	5.60E+03	3.58E+04	1.11E+05	3.60E+05
	Mean	5.57E+03	3.77E+04	1.11E+05	4.22E+05
100	R1	5.42E+03	3.22E+04	7.75E+04	5.56E+05
	R2	5.22E+03	2.90E+04	7.56E+04	5.54E+05
	R3	5.27E+03	2.54E+04	5.01E+04	3.30E+05
	Mean	5.30E+03	2.89E+04	6.77E+04	4.80E+05

 *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- R₃= Replicates 1 to 3

Table 4 Daily specific growth rates for the control cultures in the definitive test

		Daily Specific Growth Rate (cells/mL/h)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.085	0.069	0.065
	R2	0.087	0.059	0.065
	R3	0.084	0.067	0.067
	R4	0.081	0.068	0.066
	R5	0.078	0.075	0.071
	R6	0.083	0.065	0.060
	Mean	0.083	0.067	0.066

 R₁- R₆= Replicates 1 to 6

Table 5 Inhibition of growth rate yield in the definitive test

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/h)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.073		9.29E+05
	R2	0.071		7.95E+05
	R3	0.072		9.16E+05
	R4	0.071	-	8.54E+05	-
	R5	0.074		1.05E+06
	R6	0.069		6.93E+05
	Mean	0.072		8.72E+05
	SD	0.002		1.22E+05
1.0	R1	0.063	13	4.66E+05
	R2	0.063	13	4.60E+05
	R3	0.064	11	5.03E+05
	Mean	0.063	12	4.76E+05	45
	SD	0.001		2.31E+04
3.2	R1	0.058	19	3.27E+05
	R2	0.060	17	3.58E+05
	R3	0.060	17	3.79E+05
	Mean	0.059	18	3.55E+05	59
	SD	0.001		2.59E+04
10	R1	0.055	24	2.55E+05
	R2	0.058	19	3.26E+05
	R3	0.054	25	2.32E+05
	Mean	0.056	23	2.71E+05	69
	SD	0.002		4.91E+04
32	R1	0.061	15	3.93E+05
	R2	0.064	11	5.03E+05
	R3	0.059	18	3.55E+05
	Mean	0.061	15	4.17E+05	52
	SD	0.003		7.68E+04
100	R1	0.065	10	5.50E+05
	R2	0.065	10	5.49E+05
	R3	0.058	19	3.25E+05
	Mean	0.063	13	4.75E+05	46
	SD	0.004		1.30E+05

 * 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

Table 6 pH measurements taken in the definitive test

Nominal Loading Rate (mg/L)	pH
	0 H			72 H
	Pre-adjustment		Post-adjustment
Control		7.8	-		8.0
1.0		7.7	7.8		8.0
3.2		7.6	7.8		7.9
10		7.6	7.8		7.9
32		7.2	7.8		7.9
100		4.0	7.8		7.9

Table 7 Vortex depth measurements at the start and end of the mixing period

	Nominal Loading Rate (mg/L)
	Control		1.0		3.2
	*	+	*	+	*	+
Height of Media Column (cm)	12.0	12.0	26.0	26.0	26.0	26.0
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)
	10		32		100
	*	+	*	+	*	+
Height of Media Column (cm)	12.0	12.0	12.0	12.0	12.0	12.0
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:

Based on the result of the read across study, 72 h ELy50, ELr50, NOELy, NOELr, LOELy, LOELr for test substance, mono- and di- C8-10 PSE were determined to be 1.1, >100, <1, <1, 1 and 1 mg/L (nominal loading rate WAF), respectively.

Executive summary:

 A study was conducted to determine the effect of the read across substance, mono- and di C6-10 PSE, on the growth of the green algae, Pseudokirchneriella subcapitata, according to the OECD Guideline 201 and EU C3 Method, in compliance with GLP. Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the read across substance over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 h, 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. Chemical analysis of the test preparations at 0 h showed measured test concentrations to range from 0.30 mg/L at 1.0 mg/L loading rate WAF through to 2.4 mg/L at 100 mg/L loading rate WAF. A decline in measured test concentration was observed at 72 h in the range of less than the limit of quantitation (LOQ) of the analytical method employed (which was determined to be 0.031 mg/L) at 1.0 mg/L loading rate WAF through to 1.1 mg/L at 100 mg/L loading rate WAF. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the read across substance as a whole the results were based on nominal loading rates only. Based on exposure of Pseudokirchneriella subcapitata to the read across substance the study results, 72 h ELy50 (Effective loading rate for biomass), ELr50 (Effective loading rate for growth rate), NOELy (No observed effect loading rate for biomass), NOELr (No observed effect loading rate for growth rate), LOELy (Lowest observed effect loading rate for biomass), LOELr (Lowest observed effect loading rate for growth rate) were determined to be 1.1, >100, <1, <1, 1 and 1 mg/L loading rate WAF. Under the study conditions, 72 h ELy50, ELr50, NOELy, NOELr, LOELy, LOELr were determined to be 1.1, >100, <1, <1, 1 and 1 mg/L (nominal loading rate WAF), respectively (XXXX, 2012). Based on the result of the read across study, similar values can be expected for the test substance, mono- and di- C8-10 PSE.

Description of key information

Based on the result of the read across study, 72 h ELy50, ELr50, NOELy, NOELr, LOELy, LOELr for test substance, mono- and di- C8-10 PSE were determined to be 1.1, >100, <1, <1, 1 and 1 mg/L (nominal loading rate WAF), respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

1 mg/L

Additional information

 A study was conducted to determine the effect of the read across substance, mono- and di C6-10 PSE, on the growth of the green algae, Pseudokirchneriella subcapitata, according to the OECD Guideline 201 and EU C3 Method, in compliance with GLP. Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the read across substance over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 h, 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. Chemical analysis of the test preparations at 0 h showed measured test concentrations to range from 0.30 mg/L at 1.0 mg/L loading rate WAF through to 2.4 mg/L at 100 mg/L loading rate WAF. A decline in measured test concentration was observed at 72 h in the range of less than the limit of quantitation (LOQ) of the analytical method employed (which was determined to be 0.031 mg/L) at 1.0 mg/L loading rate WAF through to 1.1 mg/L at 100 mg/L loading rate WAF. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the read across substance as a whole the results were based on nominal loading rates only. Based on exposure of Pseudokirchneriella subcapitata to the read across substance the study results, 72 h ELy50 (Effective loading rate for biomass), ELr50 (Effective loading rate for growth rate), NOELy (No observed effect loading rate for biomass), NOELr (No observed effect loading rate for growth rate), LOELy (Lowest observed effect loading rate for biomass), LOELr (Lowest observed effect loading rate for growth rate) were determined to be 1.1, >100, <1, <1, 1 and 1 mg/L loading rate WAF. Under the study conditions, 72 h ELy50, ELr50, NOELy, NOELr, LOELy, LOELr were determined to be 1.1, >100, <1, <1, 1 and 1 mg/L (nominal loading rate WAF), respectively (XXXX, 2012). Based on the result of the read across study, similar values can be expected for the test substance, mono- and di- C8-10 PSE.