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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-09 - 2018-02-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
28th July 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Regulation (EU) 2017/735 of 14 February 2017
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA Ecological Effects Test Guidelines, OCSPP 850.5400, Algal Toxicity, Tiers I and II, EPA 712-C-96-164
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch/Lot Number: 7703200
Description: clear, pale yellow liquid
Purity: considered as 100%
Manufacture date: 04 February 2016
Expiry date: 28 February 2019
Storage condition: Controlled room temperature (15-25°C, below 70 RH%)
Analytical monitoring:
yes
Details on sampling:
Analytical measurement was performed at the control and at the applied test concentration levels at the beginning of the test and in 24-hour intervals thereafter during the experiment.
Determination of test substance concentrations was performed at each test concentration level and from the additional samples without algae in order to distinguish degradation and adsorption of the test item to algae.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Because the test item is very poorly soluble in water, test solutions were prepared using a saturated solution method (water accommodated fraction, WAF) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
Saturated test item solutions (nominal loading rates of 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L) were prepared individually by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two days before the start of the study. These solutions were shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C.
The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give test solutions at the appropriate nominal loading rates.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of Citoxlab Hungary Ltd
- Initial cell number: The initial cell number in the test cultures was 104 cells/mL.
- Pre-culturing: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
not applicable
Hardness:
Not reported
Test temperature:
22.6 – 23.0 °C
pH:
7.29 – 8.60
Dissolved oxygen:
Not reported
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rates WAFs
The corresponding measured geometric mean test item concentrations were: 1.78; 2.02; 8.95; 18.33 and 25.38 mg/L in presence of algae, while the measured geometric mean test item concentrations were: 3.46; 6.15; 11.99; 18.18 and 24.79 mg/L in absence of algae
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: fitted with air-permeable stoppers
- Material, size, headspace, fill volume: glass
- Initial cells density: 10E04 algal cells per mL test medium
- Control end cells density: 69.33E04 cell/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 24 h light
- Light intensity and quality: Approx 7813 lux (equivalent to 106 μE/m2/s). Fluorescent lamps (with a spectral range of 400-700 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
- Other: Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x2
- Range finding study
- Test concentrations: Nominal concentrations (% saturated solution) - 0.1, 1, 10, 100
- Results used to determine the conditions for the definitive study:
Nominal concentration (% saturated solution)/ Average cell number at 72 hours (x10E04 cell/mL): untreated control/72.00; 0.1/69.50; 1/67.00; 10/45.00; 100/4.00
Reference substance (positive control):
yes
Remarks:
Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 28.92 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
15.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
15.29 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.53 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.71 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.71 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: none reported
- Colour differences: none reported
- Flocculation: none reported
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no (NOEC)
Results with reference substance (positive control):
The date of the last study (Study Code: 17/367-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 04 – 07 December 2017.
The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EbC 50: 0.63 mg/L, (95 % confidence limits: 0.58 – 0.69 mg/L)
The 72h EyC 50: 0.53 mg/L, (95 % confidence limits: 0.49 – 0.58 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).
Reported statistics and error estimates:
Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.
The ErL50/ErC50 values of the test item could not be calculated (due to the slight effect observed) and therefore determined directly from the raw data. The EbL50/EbC50 and EyL50/EyC50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software.
With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates were significantly different from that of the control group in the
initial nominal loading rate range of 25.0 - 100.0 mg/L (WAFs). The overall NOELR was determined as 12.5 mg/L nominal loading rate (WAF); the overall LOELR was determined as 25.0 mg/L nominal loading rate (WAF). The 0-72 h areas and yield were significantly different from that of the control group in the examined nominal concentration range of 12.5 - 100.0 mg/L (nominal loading rates, WAFs). The overall NOELR was determined as 6.25 mg/L nominal loading rate WAF; the overall LOELR was determined as 12.5 mg/L nominal loading rate WAF.
Validity criteria fulfilled:
yes
Executive summary:

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours (OECD TG 201).

A significant toxic response was observed during the preliminary range-finding test, therefore five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment.

The concentrations of test item used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rates WAFs. Test concentrations were analytically determined at the start of the test and at 24 hour intervals thereafter in order to better define loss of the test substance during the exposure period.

The test design included three replicates at each test concentration and six replicates for the untreated controls. There was an additional replicate without algae at each test concentration level for further analytical measurements (0, 24, 48 and 72 hours) in order to distinguish degradation and adsorption of the test item to algae.

According to the relevant guideline (OECD 201) disappearance of the test substance from solution by absorption to the increasing algal biomass does not mean that it is lost from the test system. When the result of the test was analysed, it was checked whether a decrease in concentration of the test substance in the course of the test was accompanied by a decrease in growth inhibition.

The corresponding measured geometric mean test item concentrations were: 1.78; 2.02; 8.95; 18.33 and 25.38 mg/L in presence of algae (more details are given in 4.2.). In addition, results of the analytical measurements of samples in absence of algae showed measured geometric mean test item concentrations of 3.46; 6.15; 11.99; 18.18 and 24.79 mg/L for the nominal concentrations of 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rate WAFs respectively (more details are given in 4.2.).

However based on the section-by-section average specific growth rates, it is indicating that a decrease of concentration of the test item was not clearly accompanied by a decrease in growth inhibition. Based on these results most probably the test item is indeed absorbed to the increasing algal biomass, and should not be considered as lost from the test system.

Therefore it is recommended to express the EL50/EC50 and NOELR/NOEC//LOELR/LOEC values in the initial test item concentrations (nominal or measured) and not in the mean measured concentrations from the algae containing samples, thus biological results are related to the initial nominal loading rates (WAFs) and initial measured test item concentrations.

Results based on initial nominal loading rates (WAFs):

72 -h ErL50 > 100 mg/L; 72 -h EγL50 = 37.8 mg/L; 72 -h EbL50 = 36.51 mg/L

72 -h NOErLR = 12.5 mg/L; 72 -h NOEγLR = 6.25 mg/L; 72 -h NOEbLR = 6.25 mg/L

Results based on initial measured concentrations:

72 -h ErC50 > 28.92 mg/L; 72 -h EγC50 = 15.70 mg/L; 72 -h EbC50 = 15.29 mg/L

72 -h NOErC = 6.53 mg/L; 72 -h NOEγC = 3.71 mg/L; 72 -h NOEbC = 3.71 mg/L

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting 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:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
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. certificate)
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):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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.


Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis proposed is that the organism is not exposed to common compounds but rather, because of structural similarity, that different compounds have similar ecotoxicological and fate properties. In this case the ECHA Read-Across Assessment Framework (RAAF) Scenario 2 is used.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Primary Source: Phosphoric acid, C8-12 (even-numbered) alkyl esters [EC not yet assigned; CAS not assigned]
Supporting Source: Phosphoric acid, 2-ethylhexyl ester [EC 235-741-0; CAS 12645-31-7]
Target: Phosphoric Acid, linear and branched Octyl Esters [EC not yet assigned; CAS not assigned]
3. ANALOGUE APPROACH JUSTIFICATION
Available data for the Primary Source substance suggest that algae are the most sensitive species. This is confirmed by data available for Daphnia and algae from studies performed on the Supporting Source substance. In the available studies the effects levels are similar in both species. In this case, it is acceptable to read-across for the toxicity to aquatic algae endpoint from the Primary Source to the Target.
4. DATA MATRIX
Short-term toxicity to fish:
Primary Source: No data
Supporting Source: 96-h LL50 value of greater than 100 mg/L loading rate WAF
Target: No data
Short-term toxicity to aquatic invertebrates:
Primary Source : Daphnia magna; OECD TG 202.
48-h EC50 > 5.62 mg/L (measured) (100 mg/L nominal loading rate WAF)
48-h NOEC = 5.62 mg/L (measured) (100 mg/L nominal loading rate WAF)
Supporting Source: Daphnia magna; OECD TG 202.
48-h EL50 >100 mg/L loading rate WAF
48-h NOELR = 100 mg/L
Target: No data
Toxicity to aquatic algae:
Primary source: Pseudokirchneriella subcapitata; OECD TG 201
Results based on initial nominal loading rates (WAFs):
72 -h ErL50 > 100 mg/L; 72 -h EγL50 = 37.8 mg/L; 72 -h EbL50 = 36.51 mg/L
72 -h NOErLR = 12.5 mg/L; 72 -h NOEγLR = 6.25 mg/L; 72 -h NOEbLR = 6.25 mg/L
Results based on initial measured concentrations:
72 -h ErC50 > 28.92 mg/L; 72 -h EγC50 = 15.70 mg/L; 72 -h EbC50 = 15.29 mg/L
72 -h NOErC = 6.53 mg/L; 72 -h NOEγC = 3.71 mg/L; 72 -h NOEbC = 3.71 mg/L
Supporting source:
72-h ErL50 = 49 mg/L loading rate WAF
72-h NOErLR = 25 mg/L
Target: No data
Reason / purpose:
read-across source
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 28.92 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
15.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
15.29 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.53 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.71 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.71 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass

Description of key information

Read-across to Phosphoric acid, C8 -12 (even-numbered) linear alkly esters (OECD 201):

Results based on initial nominal loading rates (WAFs):

72 -h ErL50 > 100 mg/L; 72 -h EγL50 = 37.8 mg/L; 72 -h EbL50 = 36.51 mg/L

72 -h NOErLR = 12.5 mg/L; 72 -h NOEγLR = 6.25 mg/L; 72 -h NOEbLR = 6.25 mg/L

Results based on initial measured concentrations:

72 -h ErC50 > 28.92 mg/L; 72 -h EγC50 = 15.70 mg/L; 72 -h EbC50 = 15.29 mg/L

72 -h NOErC = 6.53 mg/L; 72 -h NOEγC = 3.71 mg/L; 72 -h NOEbC = 3.71 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
28.92 mg/L
EC10 or NOEC for freshwater algae:
6.53 mg/L

Additional information

No data are available for Phosphoric acid, linear and branched Octyl Esters. A study is available for the analogue substance Phosphoric acid, C8 -12 (even-numbered) linear alkyl esters. In this study the effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours (OECD TG 201).

A significant toxic response was observed during the preliminary range-finding test, therefore five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment.

The concentrations of test item used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rates WAFs. Test concentrations were analytically determined at the start of the test and at 24 hour intervals thereafter in order to better define loss of the test substance during the exposure period.

The test design included three replicates at each test concentration and six replicates for the untreated controls. There was an additional replicate without algae at each test concentration level for further analytical measurements (0, 24, 48 and 72 hours) in order to distinguish degradation and adsorption of the test item to algae.

According to the relevant guideline (OECD 201) disappearance of the test substance from solution by absorption to the increasing algal biomass does not mean that it is lost from the test system. When the result of the test was analysed, it was checked whether a decrease in concentration of the test substance in the course of the test was accompanied by a decrease in growth inhibition.

The corresponding measured geometric mean test item concentrations were: 1.78; 2.02; 8.95; 18.33 and 25.38 mg/L in presence of algae (more details are given in 4.2.). In addition, results of the analytical measurements of samples in absence of algae showed measured geometric mean test item concentrations of 3.46; 6.15; 11.99; 18.18 and 24.79 mg/L for the nominal concentrations of 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rate WAFs respectively (more details are given in 4.2.).

However based on the section-by-section average specific growth rates, it is indicating that a decrease of concentration of the test item was not clearly accompanied by a decrease in growth inhibition. Based on these results most probably the test item is indeed absorbed to the increasing algal biomass, and should not be considered as lost from the test system.

Therefore it is recommended to express the EL50/EC50 and NOELR/NOEC//LOELR/LOEC values in the initial test item concentrations (nominal or measured) and not in the mean measured concentrations from the algae containing samples, thus biological results are related to the initial nominal loading rates (WAFs) and initial measured test item concentrations.

Results based on initial nominal loading rates (WAFs):

72 -h ErL50 > 100 mg/L; 72 -h EγL50 = 37.8 mg/L; 72 -h EbL50 = 36.51 mg/L

72 -h NOErLR = 12.5 mg/L; 72 -h NOEγLR = 6.25 mg/L; 72 -h NOEbLR = 6.25 mg/L

Results based on initial measured concentrations:

72 -h ErC50 > 28.92 mg/L; 72 -h EγC50 = 15.70 mg/L; 72 -h EbC50 = 15.29 mg/L

72 -h NOErC = 6.53 mg/L; 72 -h NOEγC = 3.71 mg/L; 72 -h NOEbC = 3.71 mg/L

A supprting study is available for the 2 -ethylhexyl analogue, performed according to EOCD TG 201 and EU Method C.3.

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.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

ErL50 = 49 mg/L loading rate WAF.

NOErL = 25 mg/L loading rate WAF.

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 torange 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.