Santicizer P1400

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:

21st September - 14th December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, effects on Biotic Systems, 201 Alga Growth inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Dangerous Chemicals Administration. GB/T 21805-2008 Chemicals- Alga Growth Inhibition Test [S]. Beijing: Standards Press of China, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: State Environmental Protection Administration of China. HJ/T 153-2004, The Guidelines for the test of chemical [S]. Beijing: China Environmental Science Press. 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: DI1045325
- Expiration date of the lot/batch: 21 October 2017
- Purity test date: 98.549%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: store in original tightly closed containers and away from incompatile materials
- Stability: stable in container after opening.in water/in light/in soil

TEST SUBSTANCE PREPARATION
- 0.101g test substance was obtained in a beaker and dissolved in some OECD medium and then ultrasonicated for 10 minutes.
- The mixture was transferred to a 1L-flask and diluted with OECD medium to 1000 mL to obtain the nominal concentration of 101 mg/L test suspension.
- The test suspension was stirred with a magnetic stirrer for about 48h and then filtered with a 0.45 µm polyether sulfone filter membrane to obtain the nominal concentration of 101 mg/L saturated solution (100% LS). The solution was used directly in the test.

Analytical monitoring:

not specified

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

TEST ALGA SOLUTION
- Alga cells were pre-cultured and was examined.
- The alga biomass was 2.58 x10^6 cells/mL calculated with the average optical density (650) of 1.031 measured by spectrophotometer.
- It was diluted to approximately 1 x10^6 cells/mL

TEST SOLUTION PREPARATION
- According to the design concentration,100 mL of the test solution was added to the test flask, and then flled by the 1.00 mL alga solution to prepare a test solution as 100% LS test solution group.
- The blank control comprised of 1.00 mL alga solution and 100 mL test medium.
- 100 mL of the test solution was added to the test flask and then filled by the 1.00 mL test medium to prepare 100%LS test solution group without alga.
- The final alga concentration of each test group was about 10^4 cells/mL.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: FACHB-271
- Source (laboratory, culture collection): bought from FACHB-Collection and cultured in the laboratory
- Batch number: APs20151113-1; APsU20170922-1 (pre-culture)
- Selection reason: the test organism species is recommended by 'The Guidelines for the Testing of Chemicals'
- Age of inoculum (at test initiation): logarithmic growth phase alga cells
- Method of cultivation: the pre-culture was incubated under the same conditions as the test cultures when still in exponential growth, after growing 2-3 times transit culture. The logarithmic growth phase Alga used for was selected were Alga cultures containing no deformed or abnormal cells after checks were made.

Test type:

static

Water media type:

other: Sterile deionised water

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Hardness:

Not specified

Test temperature:

Incubator: 22.6-22.9°C

pH:

Not specified

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Not specified

Details on test conditions:

TEST CONDITIONS:
- Initial cell concentration: 10^4 cells/ mL (±25%)
- Volume of test solution: 101 mL/test vessel
- Temperature in incubator: 22.6-22.9°C
- Light condition: continuous light, 5833 lx - 5993 lx
- Measurement of cell growth: cell counting method
- Analysis of concentrations: the actual concentrations of test substance in the blank control, 100% LS test group and 100% LS test group (without alga) were measured at 0h, 24h, 48h and 72h by Rapid Resolution Liquid Chromatography (RRLC)

PREPARATION OF STANDARD STOCK SOLUTION
- 250 mg of the test substances were dissolved and diluted with acetonitrile to 50 mL.
- A stock solution with a concentration of 404.1 mg/L was obtained.

PREPARATION OF CALIBRATION SOLUTIONS
- Defined volumes of standard stock solution were further diluted with 70% acetonitrile to obtain the set of calibration standards (Table 1).

BLANK SOLUTION
- The blank of water samples: OECD medium
- The blank of degradation samples: 6.2 mL inoculum were diluted with test medium to 250.0 mL and mixed, then 5 mL of the mixed solution was passed through a 0.45 µm filter.

PREPARATION OF SPIKED RECOVERY SAMPLES (Table 2)
- Defined volumes (A) of the standard stock solution and calibration solutions were further diluted with OECD medium to defined volumes (B) and mixed.
- Defined volumes (C) of the mixed solution were further filuted with acetonitrile to defined volumes (D) as the spiked recovery samples.

PREPARATION OF SPIKED RECOVERY DEGRADATION SAMPLES
- 7.64 mg, 7.58 mg and 7.59 mg of test substance were weighed into triangular flask 1, 2 and 3 respectively, and then dissolved in 250 mL of deionised water.
- Stock solutions (1, 2, 3) of the non-biological degradation contrast solution with the theoretical concentration 30 mg/L were obtained.
- 7.56 mg, 7.65 mg, 7.60 mg of the test substance were weighted into triangular flasks 4, 5 and 6 respectively, and then dissolved in 243.8 mL of test medium and 6.2 mL inoculum.
- Stock solutions (1, 2, 3) of the biodegradation solution with the theoretical concentration 30 mg/mL were obtained.
- The solution in bottles 1-6 were diluted with 250 mL acetonitrile and then mixed as spiked recovery samples.
- All recovered samples were passed through 0.45 µm filteres before testing.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

ErC50

Effect conc.:

ca. 101 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Key result

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

LOErC

Effect conc.:

ca. 101 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

NOErC

Effect conc.:

ca. 101 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

1. Alga growth
- Unusual cell shape: No. There was no significant difference on shape and size of cells between the test group and the blank control under the microscope.
- Observation of abnormalities: The condition of cells in the blank control was not abnormal.
- The mean coefficient of variation for section-by-section specific growth rate in the blank control was 26%.
- The coefficient of variation of average specific growth rates during the whole test period in control cultures was 1%.
- A t-test performed showed that there was no significant difference (p>0.05) on the specific growth rate of 100% LS test group compared with the blank control, therefore the 72h-LOErC was greater than the nominal concentration of 101 mg/L saturated concentration (the geometric mean of the measured concentrations of the test substance was 2.66 mg/L). The 72h-NOErC was the nominal concentration of 101 mg/L saturated concentration (the geometric mean of the measured concentrations of the test substance was 2.66 mg/L).

2. Water quality and environmental conditions
- pH values: blank control = 7.67, 100% LS test group = 7.90
- Incubator temperature = 22.6-22.9 °C
- Mean light intensity = 5833-5993 lx

3. Actual concentration of the test substance in the test solution
- The limit of detection (LOD) = 0.101 mg/L and the limit of quantitation (LOQ) = 0.335 mg/L
- Blank control: the measured concentrations of the test substance in the blank control at 0h, 24h, 28h and 72h after the start of the test were all lower than the LOD (0.101 mg/L).
- 100% LS test group: - the measured concentrations of test substance at 0h, 24, 48h and 72h after the start of the test were 2.76, 2.80, 2.68 and 2.30 mg/L.
- the measured concentration at the end of the test corresponded to 83.3% of the initial concentration, which could be maintained within ± 20% of the initial concentration.
- the geometric mean of the measured concentrations of test substance was 2.66 mg/L during the test.
- 100% LS test group (without alga): - the measured concentrations of test substance at 0h, 24, 48h and 72h after the start of the test were 2.76, 2.73, 2.58 and 2.58 mg/L.
- the measured concentration at the end of the test corresponded to 93.5% of the initial concentration, which could be maintained within ± 20% of the initial concentration.
- this showed that the alga cells had no influence on the stability of the test substance.

Results with reference substance (positive control):

- The 72h-ErC50 of reference substance, K2Cr2O7, was determined to be 1.34 mg/L and the 95% confidence limit was 1.32-1.37 mg/L, which was within the range of 1.19 mg/L ± 0.27 mg/L.
- The item number of the sensitivity test was 2017GST0006.

Reported statistics and error estimates:

The area A of growth curves and the growth rate r for each parallel was calculated and the arithmetic mean growth

Validity of test

- During the 72h test period, the blank control group, the number of Alga cells from the test was increased from 1.02 x10^4 cells/mL in at least 9.02 x10^5 cells/mL, signifying an increas of 85.1, which met the 16 -fold increase (specific growth rate ≥ 0.92/d) requirements.

- The mean coefficient of variation for section-by-section specific growth rates (days 0 -1, 1 -2 and 2 -3 , for 72 -hour tests) in the control cultures was 26%, met with the requirements of not exceeding 35%.

- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 1%, which met with the requirements of not exceeding 7% in tests.

- The pH drift was 0.94, which met with the requirements of not being more than 1.5 during the test.

- The 72h-EC50 of potassium dichromate on Alga growth inhibition was 1.34 mg/L, which met with the requirements, falling in the 1.19mg/L ± 0.27 mg/L range.

Toxicity effect of test organism

- The average specific growth rate of the blank control and 100% LS test froup was 1.50/d and 1.49/d respectively.

- A t-test was performed, whcih showed that there were no signficant differences (p > 0.05) in the specific growth rate of 100% LS test group compared to the blank control.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the median effective concentration (ErC50), the lowest observable effect coecnentration (LOErC) and the no observable effect concentration (NOErC) for the test substance, Santicizer Platinum P1400, was 101 mg/L.

Executive summary:

The aim of this study was to determine the effects of the test substances, Santicizer Platinum P1400 and 1 -butyl 2 -(phenylmethyl) ester, on the growth of the unicellular green alga species, Pseudokirchneriella subcapitata. The study was carried out in accordance with the following guidelines: OECD 201; Chemical Registration Center of MEP, The Guidelines for the Testing of Chemicals, Effects on Biotic Systems, 201 Alga Growth Inhibition Test; Dangerous Chemicals Administration, GB/T 21805 -2008 Chemicals - Alga Growth Inhibition Test; ISO8692 Water Quality - Freshwater algal growth inhibition with unicellular green algae; OECD series on testing and assessment number 23; and State Environmental Protection Administration of China, HJ/T 153 -2004, The Guidelines for the test of chemical [S].

Exponentially growing freshwater microalgae were exposed to various concentrations of the test substances in batch cultures over 72 hours. Alga biomass was recorded at 24h, 48h and 72h after the start of the test, in order to determine the inhibition rate (compared with the control), EC50 with 95% confidence interval, the lowest observable effect concentration (LOEC) and no observable effect concentration (NOEC).

The test substance was prepared in a solution with OECD medium until a nominal concentration of 101 mg/L test suspension was obstained. Alga cells were also pre-cultured and examined. The alga biomass and average optical density was determined via spectrophotometry and then was diluted to approximately 1 x10^6 cells/mL. 100 mL of test solution was added to the test flask with 1 mL alga solution for the 100% LS test solution group. The blank control consisted of 1 mL alga solution and 100 mL test medium and the 100% LS test solution without alga was made up from 100 mL test solution and 1 mL test medium. The final alga concentration of each test group was about 10^4 cells/mL.

The test solutions of each group were placed at a constant temperature in a light incubator for 72 hours and shaken at least 3 -4 times to ensure a good carbon dioxide exchange between the air and test solution. There were 6 replicates/blank control and 100% LS test group, and 1 replicate/100% LS test group without alga cell. The alga growth, water quality and environmental conditions, analysis of the actual concentration of the test substance in the test solutions and growth rate were measured.

There were no significant differences in the alga cell size or shapes between the test control and blank control when observed under the microscope. The average specific growth rate of the blank control and 100% LS test group were 1.50/d and 1.49/d respectively. A t-test revealed that there was no significant difference (p>0.05) on the specific growth rate of 100% LS test group compared to the blank control. The median effective concentration (ErC50), the lowest observable effect concentration (LOErC) and the no observable effect concentration (NOErC) for the test substance, Santicizer Platinum P1400, was 101 mg/L. HPLC was also performed to determine whether the analytical method is scientific and reasonable and to ensure accuracy of the concentration results, which was validated. The validity criteria was also fulfilled. In terms of the actual concentration of the test substance in the test solution, the limit of detection (LOD) was 0.101 mg/L and the limit of quantitation (LOQ) was 0.335 mg/L. The measured concentrations of the test substance in the blank control at 0h, 24h, 28h and 72h after the start of the test were all lower than the LOD. In terms of the 100% LS test group, the test substance concentrations at 0h, 24, 48h and 72h were 2.76, 2.80, 2.68 and 2.30 mg/L. The measured concentration at the end of the test corresponded to 83.3% of the initial concentration, which could be maintained within ± 20% of the initial concentration, and the geometric mean of the measured concentrations of test substance was 2.66 mgL. Regarding the 100% LS test group (without alga), the concentrations of the test substance at 0h, 24, 48h and 72h were 2.76, 2.73, 2.58 and 2.58 mg/L, and at the end of the test, the concentration corresponded to 93.5% of the initial concentration, which could be maintained within ± 20% of the initial concentration. Therefore, this showed that the alga cells had no influence on the stability of the test substance.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-01-07 to 2016-02-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Polyadd Limited; Batch no. 0900
- Expiration date of the lot/batch: 2016-10-22
- Purity test date: 2015-10-22
- Purity: 98.8%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

FORM AS APPLIED IN THE TEST (if different from that of starting material): Clear colorless liquid

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Range-finding Test: nominal test concentrations of 1.0, 10 and 100% v/v saturated solution
Definitive Test: 1.0, 3.2, 10, 32 and 100% v/v saturated solution
- Sampling method:

Range-finding Test:
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10 and 1.0% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.8 mL) to give the required test concentrations of 1.0, 10 and 100% v/v saturated solution.

Definitive Test:
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 32, 10, 3.2 and 1.0% v/v saturated solution. An aliquot (700 mL) of each of the stock solutions was separately inoculated with 4.5 mL of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution.The stock solutions and each of the prepared concentrations were inverted several times to
ensure adequate mixing and homogeneity

- Sample storage conditions before analysis: All 0-Hour samples were stored frozen prior to analysis

Vehicle:

yes

Remarks:

culture medium prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Information provided by the Sponsor indicated the water solubility of the test item to be 5 mg/L. Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions

Culture medium:
NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L

The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green Alga (Pseudokirchneriella subcapitata)
- Strain: CCAP 278/4
- Source (laboratory, culture collection): 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 constant 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 10^3 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 10^4-10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1 ºC

pH:

8.1 to 8.2

Nominal and measured concentrations:

Nominal concentrations: Range-finding Test: 0, 1.0, 10, 100 (% v/v)
Measure concentrations: Definitive Test: 0, 0.032, 0.11, 0.35, 0.75, 4.0 (mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 250 mL
- Aeration: not specified
- Initial cells density: 10^3 cells/mL
- Control end cells density: 10^4 - 10^5 cells/mL
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

OTHER TEST CONDITIONS
- Light intensity and quality: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell densities; appearance: shape and size

TEST CONCENTRATIONS
- Range finding study: nominal test concentrations of 1.0, 10 and 100% v/v saturated solution
- Test concentrations: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

Yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.75 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

Yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.75 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: No
- Colour differences: No
- Any stimulation of growth found in any treatment: yes
- 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: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.032, 0.11, 0.35 and 0.75 mg/L test cultures were
observed to be green dispersions whilst the 4.0 mg/L test cultures were observed to be clear colorless solutions.

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Reported statistics and error estimates:

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) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Table 1. Inhibition of Growth Rate and Yield in the Definitive Test
0-Hour Measured Test Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0-72 h	% Inhibition	0-72 h	% Inhibition*
Control	R1	0.069		7.27E+05
	R2	0.070		7.72E+05
	R3	0.071		7.98E+05
	R4	0.071	-	8.47E+05	-
	R5	0.070		7.54E+05
	R6	0.068		6.77E+05
	Mean	0.070		7.63E+05
	SD	0.001		5.86E+04
0.032	R1	0.072	[3]	8.81E+05
	R2	0.073	[4]	9.37E+05
	R3	0.069	1	7.40E+05
	Mean	0.071	[2]	8.53E+05	[12]
	SD	0.002		1.02E+05
0.11	R1	0.073	[4]	9.87E+05
	R2	0.069	1	6.99E+05
	R3	0.063	10	4.65E+05
	Mean	0.068	2	7.17E+05	6
	SD	0.005		2.62E+05
0.35	R1	0.067	4	6.20E+05
	R2	0.064	9	4.92E+05
	R3	0.067	4	6.12E+05
	Mean	0.066	6	5.75E+05	25
	SD	0.002		7.15E+04
0.75	R1	0.065	7	5.31E+05
	R2	0.066	6	5.59E+05
	R3	0.065	7	5.19E+05
	Mean	0.065	7	5.36E+05	30
	SD	0.001		2.05E+04
4.0	R1	0.045	36	1.26E+05
	R2	0.047	33	1.44E+05
	R3	0.039	44	8.03E+04
	Mean	0.044	38	1.17E+05	85
	SD	0.004		3.28E+04

* 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]

Validity criteria fulfilled:

yes

Conclusions:

Growth rate and yield of Pseudokirchneriella subcapitata were affected by exposure to the test material. For inhibition of yield, the EC50 was determined to be 1.2 mg/L (0.92 – 1.5 mg/L; 95% C.I.); the NOEC was 0.75 mg/L and the LOEC was determined to be 4.0 mg/L. For inhibition of growth rate, the EC50was >4.0 mg/L while the NOEC and LOEC were determined to be 0.75 and 4.0 mg/L, respectively.

Executive summary:

In a key study, the algal growth inhibition potential of the test material (1,2 Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester, CAS# 1200806-67-2) was tested using the green alga Pseudokirchneriella subcapitata.

 

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (equivalent to 0.032, 0.11, 0.35, 0.75, 4.0 mg/L) (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

 

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

 

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.032 to 4.0 mg/L. A decline in measured test concentrations was observed at 72 hours in the range of 0.019 to 3.4 mg/L (58% to 100% of the 0-Hour measured test concentrations) typically following a concentration dependent decline with the lowest test concentrations exhibiting the greatest decline. Analysis of test samples at 72 hours prepared at 0 hours with the omission of algal cells showed measured test concentrations to range from 0.027 to 3.2 mg/L (86% to 153% of the 0-Hour measured concentrations) indicating that the decline in measured concentrations

observed in the test samples prepared with the addition of algal cells was due to adsorption of the test item to the algal cells present rather than instability.

 

Growth rate and yield of Pseudokirchneriella subcapitata were affected by exposure to the test material. For inhibition of yield, the EC₅₀ was determined to be 1.2 mg/L (0.92 – 1.5 mg/L; 95% C.I.); the NOEC was 0.75 mg/L and the LOEC was determined to be 4.0 mg/L. For inhibition of growth rate, the EC₅₀ was >4.0 mg/L while the NOEC and LOEC were determined to be 0.75 and 4.0 mg/L, respectively.

 

 

Description of key information

Growth rate and yield of Pseudokirchneriella subcapitata were affected by exposure to the test material. For inhibition of yield, the EC50 was determined to be 1.2 mg/L (0.92 – 1.5 mg/L; 95% C.I.); the NOEC was 0.75 mg/L and the LOEC was determined to be 4.0 mg/L. For inhibition of growth, the EC50was >4.0 mg/L while the NOEC and LOEC were determined to be 0.75 and 4.0 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.2 mg/L

EC10 or NOEC for freshwater algae:

0.75 mg/L