3-{3-[(3,5,5-trimethylhexanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propoxy}-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate

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:

10 June 2013-15 January 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An amount of test item (250 mg) was added to the surface of 2.5 lites of culture medium to give the 100 mg/L loading rate. 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 mixture was 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 100 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
An aliquot (1 litre) of te 100 mg/L loading rate WAF was inoculated with algal suspension (6.2 mL) to give the required test concentration of 100 mg/L loading rate WAF.
- Eluate: Reconstituted water
- Differential loading: N/A
- Controls: Reconstituted water
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchnerella 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 house
- Age of inoculum (at test initiation): NDA
- Method of cultivation: In-house cultures were maintained in the laboratory by the periodic replenishment of culture medium


ACCLIMATION
- Acclimation period: until the algal cell density was approximately 10E04 – 10E05 cells/mL
- Culturing media and conditions (same as test or not):Same as test
- Any deformed or abnormal cells observed: NDA

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

N/A

Hardness:

NDA

Test temperature:

23-25 °C

pH:

pH of culture medium = 7.6 at 0 h; 8.2 at 72 h

Dissolved oxygen:

NDA

Salinity:

NDA

Nominal and measured concentrations:

nominal concentrations: 0 or 100 mg/L

Details on test conditions:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionised water Elga Optima 15+ or Elga Purelab Option R-15 BP
- Total organic carbon:NDA
- Particulate matter: NDA
- Metals: NDA
- Pesticides: NDA
- Chlorine: NDA
- Alkalinity: NDA
- Ca/mg ratio: NDA
- Conductivity: NDA
- Culture medium different from test medium: Same medium used
- Intervals of water quality measurement: NDA


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 24h
- Light intensity and quality: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm)
- Salinity (for marine algae):N/A


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter ® Multisizer Particle Counter
- Chlorophyll measurement: None


TEST CONCENTRATIONS
- Spacing factor for test concentrations: N/A
- Justification for using less concentrations than requested by guideline: Limit test based on results of range finding study
- Range finding study
- Test concentrations: Nominal loading rates: 0, 10 or 100 mg/L

The results showed no significant effect on growth rate at 10 or 100 mg/L nominal loading rate.

Based on this information a concentration of 100 mg/L nominal loading rate WAF was selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (non concurrent)

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth and yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: ErC50 (0-72h) = 1.2 mg/L; 95% confidence limits 1.0-1.3 mg/L

Reported statistics and error estimates:

Statistical analysis of the growth rate data and the yield data was carried out for the control and 100 mg/L loading rate WAF test group using a Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There was no statistically significant differences (p>=0.05) between the control and the 100 mg/L loading rate WAF test group and therefore the NOEL based on either growth rate or yield was 100 mg/L loading rate WAF.

See attached document for tables and figures.

Validity criteria fulfilled:

yes

Conclusions:

The 72-h EL50 was > 100 mg/L loading rate WAF. The NOELR was 100 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) 761/2009.

Methods:

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).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) 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 EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Chemical analysis of the 100 mg/L loading rate WAF at 0 and 72 hours showed measured test concentrations of 0.00068 and 0.0038 mg/L respectively. Analysis of the control preparation at 72 hours showed a peak was present, the profile of which was consistent with that of the test item. Given that this was not observed at 0 hours it was considered that possible contamination had occurred during the test. As such it was considered appropriate to perform a further confirmatory test. Analysis of the control preparations from this confirmatory test also showed a peak profile similar to that of the test item or a possible trace contaminant which had a characteristic profile similar to that of the test item. Given that no adverse effects were observed in the control group, this was considered to have had no impact on the outcome of the test.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 to 28 October 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

PHYSICO-CHEMICAL PROPERTIES
- Water solubility (under test conditions): Insoluble

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accommodated Fraction (WAF): A nominal 1000 mg/L stock solution was prepared in culture medium by the WAF method. The stock solution was prepared by stirring the test material in dilution water for 20 hours, followed by settling for 4 hours. The aqueous layer was then removed for testing. The test solutions were prepared by adding the 1000 mg/L stock solution to culture medium water in appropriate quantities to obtain the desired concentrations for the test.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, Windermere Laboratory
- Age of inoculum (at test initiation): Pre-culture growing in exponential phase
- Method of cultivation: Temperature: 23 ± 2 °C, Illumination: 6000 - 10000 lux continuous white light, shaking: 200 rpm, culture medium: according to OECD giudelines. Sterile nutrient stock solution were prepared and added to the dilution water to obtain the culture medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

test start: 22.5
test end: 21.5

pH:

test start: 7.4 - 7.6
test end: 7.2 - 7.3

Nominal and measured concentrations:

Nominal: 0, 100, 180, 320, 560, 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 250 mL conical flasks filled with 100 mL test medium
- Initial cells density: 10000 cells/ml
- Control end cells density: 153.89 x 10 exp 4 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water sterilised by autoclaving at 120°C for 30 minutes
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH and temperature were measured on the pooled replicates for each test and control solution
immediately prior to initiating the test and at the end of the full 72 hour test period.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes, to 8.0 ± 0.2
- Photoperiod: Continuous light
- Light intensity and quality: 6000 - 10000 lux white light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density was determined at 24, 48 and 72 hours on a small volume removed from each replicate
flask. The cell counts were made using a haemocytometer and microscope.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1000 mg/l
- Results used to determine the conditions for the definitive study: EC50 as being approx. 1000 mg/l.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: WAF loading rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: WAF loading rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: WAF loading rate

Details on results:

- Exponential growth in the control (for algal test): Yes

Reported statistics and error estimates:

The inhibition of growth was calculated using both the biomass integral (area under the growth curve) and the growth rate method.
The EL50 values were estimated where possible using a logarithm-linear or logarithm-probit plot of concentration and percent growth inhibition.

The test substance showed to be of low toxicity to Pseudokirchnerella subcapitata. The inhibiton for the WAF loading rate of 1000 mg/l after 72h by biomass integral was 18% and by growth rate 2%. Therefore, the 72 hour EL50 was estimated as > 1000 mg/l using the biomass integral and also by growth rate calculation. Because of the serial dilution from the highest concentration (1000 mg/L), only the 1000 mg/L test concentration was valid and could be used for evaluation of toxicity of the test substance. Without chemical analytics of the diluted concentrations, no information about the exact test concentration could be stated.

Table 1: Cell density measurements (Mean initial cell density: 1 x 10 exp 4 cells/ml)

Concentration [mg/l]	Replicate No.	Cell density measurements [cells/ml x 10 exp 4]
		24 hours	48 hours	72 hours
0	1 2 3 4 5 6	3.00 2.67 2.67 2.33 1.67 2.00	37.00 30.33 27.33 41.33 28.00 33.67	149.33 157.33 150.67 148.67 168.00 149.33
	Mean	2.39	32.94	153.89
100	1 2 3	2.00 3.33 3.67	42.67 47.00 38.33	166.67 165.33 140.00
	Mean	3.00	42.67	157.33
180	1 2 3	2.67 3.33 2.67	34.33 30.67 28.67	164.00 182.67 158.67
	Mean	2.89	31.22	168.45
320	1 2 3	1.33 2.67 1.67	37.33 30.00 25.33	168.00 172.33 164.33
	Mean	1.89	30.89	168.22
560	1 2 3	2.67 3.00 1.67	30.67 24.67 20.00	129.33 144.00 168.67
	Mean	2.45	25.11	147.33
1000	1 2 3	2.33 1.33 1.67	22.33 24.00 19.67	140.00 136.00 136.67
	Mean	1.78	22.00	137.56

 

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2007-02-29 to 2007-03-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accomodated Fraction (WAF): A concentration of 100 mg/L of the test substance was moderately stirred in algal medium for 24 h at
room temperature. After this incubation time, the undissolved materials were removed by sterile filtration (0.45 µm) to obtain a sterile test substance solution for the algal growth test. The resulting water accommodated fraction was used in the test.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures, Institute of Freshwater Ecology, University of Göttingen, D-37073 Göttingen,
Germany
Age of inoculum (at test initiation): Exponentially growing liquid preculture were used to inoculate sterile test flasks.
- Method of cultivation: 250 mL flask containing 100 mL of sterile OECD medium inoculated with cell material from axenic slope culture, continuous
illumination from warm white fluorescent tubes (8500 - 9000 lux), temperature: 22 ± 0.2 °C thermocontrolled room, periodically conducted
reference test with potassium dichromate.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22 ± 0.2 °C

pH:

8.0 ± 0.2

Nominal and measured concentrations:

Nominal: 0, 100 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 250 m flaks, all-glass, with 100 mL of test medium
- Initial cells density: 10000- 20000 / mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 5

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was determined in a control flask at the start and at the end of the test. PH was determined in 1 control flask after sterilization before the test and in all test vessels at the end of the test. Observations of mis-shapen cells and cell-debris were
made after 24, 48 and 72 h of exposure.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuous
- Light intensity and quality: From warm white fluorescent tubes (8500-9000 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Every 24h using a spectrophotometer (680 nm wavelength). Aliquots of 5 ml were removed from each test
substance served as controls. The starting cell density was determined using a counting chamber.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: EL50 (24-72h)

Duration:

72 h

Dose descriptor:

EL0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: EL0 (24-72h)

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: EL50 (24-72h)

Duration:

72 h

Dose descriptor:

EL0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: EL0 (24-72h)

At the WAF loading rate of 100 mg/L no inhibition with respect to algal growth rate and algal cell growth (area under growth curve) compared to the untreated controls were observed. The no-observed-effect concentration for growth rates (ErL0) and biomass production (EbL0) with respect to the loading rate was ≥ 100 mg/l. The EL50 -value was conducted as EL50 (24 -72h). But the EL50 can also be stated as EL50 (72h) > 100 mg/l for the algal growth rate and algal cell growth.

Table 1: Cell concentration (A680) in individual test cultures after 24, 48 and 72 h of exposure to the test material. ((b) = Mean absorbance (A680))

Nominal concentration [mg/l]	Code	Cell concentration (A680)
		0 h (b)	24 h	48 h	72 h
Control	A B C D E	0.008 0.008 0.008 0.008 0.008	0.033 0.038 0.038 0.036 0.040	0.228 0.222 0.208 0.212 0.233	0.685 0.733 0.676 0.624 0.694
100	A B C	0.008 0.008 0.008	0.040 0.038 0.044	0.224 0.207 0.207	0.700 0.631 0.675

 

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1999/06/22 - 1999/06/25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(1984)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, date of inspection 23 March 1998

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Water samples were taken from the control (replicates R1-R2 pooled) and the 100 mg/L loading rate WSF test group (replicates R1-R3 and R4-R6 pooled) at 0 and 72 h.
- Sample storage conditions before analysis: Duplicate samples were taken at 0 h and stored frozen (approx. -20°C) for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An amount of test material (2000 mg) was added to the vortex created in 20 L of culture medium to give a 100 mg/L loading rate. The mixture was stirred for 23 h with a vortex depth of approx. 25%. After stirring the mixture was allowed to stand for 1 h prior to the removal of the WSF by filtration through 0.45 µm filters. An aliquot (2000 mL) of the WSF was inoculated with algal suspension (10 mL) to give the final test concentration of 100 mg/L loading rate WSF.
- Controls: reverse osmosis purified deionised water (Elga Optima 15+) without test item

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CCAP 276/20
- Source: Culture Centre for Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria
- Method of cultivation: The culture was maintained in the laboratory at a temperature of 21 ± 1 °C under continuous light (approx. 7000 lux) and constant aeration. The culture medium was prepared according to the OECD guideline (AAP medium).

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

7.3-7.6 (0 h), 8.0-8.8 (control, 72 h), 8.8 - 9.7 (100 mg/L, 72 h)

Nominal and measured concentrations:

Nominal concentrations: control - 100 mg/L
Measured concentrations: control (< detection limit), 100 mg/L (12.6 and 17.9 mg/L, 0 h; 5.89 and 2.07 mg/L, 72 h)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks each containing 100 mL of solution
- Type: covered with aluminium foil
- Aeration: constantly shaken at 100 rpm
- Initial cells density: 1.26 - 1.66x10^4 cells/mL
- Control end cells density: 3.64x10^5 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes according to OECD guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionised water (Elga Optima 15+)


OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to 7.5 +/- 0.1 with 0.1 N NaOH or HCl
- Photoperiod: continuous light
- Light intensity and quality: approx. 7000 lux


EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: samples were taken at 0, 24, 48 and 72 h and the cell densities were determined by using a Coulter Multisizer II Particle Counter


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: control - 100 mg/L (WSF)
- Results used to determine the conditions for the definitive study: No effect on growth at 100 mg/L loading rate WSF.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: yes
- Any stimulation of growth found in any treatment: an increase of growth was observed in the highest test concentration after 72 h.
- Effect concentrations exceeding solubility of substance in test medium: The nominal effect concentration of 100 mg/L exceeded the water solubility of the test substance.

Reported statistics and error estimates:

Statistical analysis of the area under the growth curve data was carried out for the control and 100 mg/L loading rate WSF test group using a Students t-test.

No effects on the growth rate was observed in the range of water solubility of the test substance with an EL50 of > 100 mg/L based on the growth rate. The measured concentrations of the test item in the test vessels were much lower than the nominal loading rate (Table 3).

Table 1: Cell densities in the definitive study

	Cell densities [cells/mL]
Nominal loading rate [mg/L]	0 h	24 h	48 h	72 h
Control R1	1.34x10^4	2.29 x10^4	7.69 x10^4	3.79 x10^5
R2	1.34 x10^4	2.16 x10^4	8.80 x10^4	3.53 x10^5
R3	1.10 x10^4	2.22 x10^4	9.83 x10^4	3.60 x10^5
Mean	1.26 x10^4	2.22 x10^4	8.78 x10^4	3.64 x10^5
100 R1	1.67 x10^4	2.38 x10^4	6.14 x10^4	5.19 x10^5
R2	1.80 x10^4	2.42 x10^4	8.11 x10^4	5.23 x10^5
R3	1.37 x10^4	2.44 x10^4	6.29 x10^4	3.50 x10^5
R4	1.54 x10^4	2.49 x10^4	7.26 x10^4	4.01 x10^5
R5	1.93 x10^4	2.41 x10^4	7.64 x10^4	6.62 x10^5
R6	1.61 x10^4	2.41 x10^4	6.60 x10^4	6.65 x10^5
Mean	1.66 x10^4	2.42 x10^4	7.01 x10^4	5.20 x10^5

Table 2: Inhibition of growth rate and biomass

Nominal loading rate [mg/L]	Area under curve at 72 h	% inhibition	Growth rate (0-72 h)	% inhibition
control	6.25 x10^6	-	0.047
100	7.51 x10^6	[20]	0.048	[2]

[increase in growth as compared to the controls]

Table 3: Chemical analysis of test loading rates

Samples	Nominal loading rate [mg/L]	Concentration found [mg/L]	Expressed as a % of the nominal loading rate
0 h	control	< LOQ	-
	100 R1-R3	12.6	13
	100 R4-R6	17.9	18
72 h	control	< LOQ	-
	100 R1-R3	5.89	6
	100 R4-R6	2.07	2

Reference 5

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:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth and yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: ErC50 (0-72h) = 1.2 mg/L; 95% confidence limits 1.0-1.3 mg/L

Reported statistics and error estimates:

Statistical analysis of the growth rate data and the yield data was carried out for the control and 100 mg/L loading rate WAF test group using a Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There was no statistically significant differences (p>=0.05) between the control and the 100 mg/L loading rate WAF test group and therefore the NOEL based on either growth rate or yield was 100 mg/L loading rate WAF.

See attached document for tables and figures.

Validity criteria fulfilled:

yes

Conclusions:

The 72-h EL50 was > 100 mg/L loading rate WAF. The NOELR was 100 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) 761/2009.

Methods:

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).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) 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 EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Chemical analysis of the 100 mg/L loading rate WAF at 0 and 72 hours showed measured test concentrations of 0.00068 and 0.0038 mg/L respectively. Analysis of the control preparation at 72 hours showed a peak was present, the profile of which was consistent with that of the test item. Given that this was not observed at 0 hours it was considered that possible contamination had occurred during the test. As such it was considered appropriate to perform a further confirmatory test. Analysis of the control preparations from this confirmatory test also showed a peak profile similar to that of the test item or a possible trace contaminant which had a characteristic profile similar to that of the test item. Given that no adverse effects were observed in the control group, this was considered to have had no impact on the outcome of the test.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Reference 6

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: WAF loading rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: WAF loading rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: WAF loading rate

Details on results:

- Exponential growth in the control (for algal test): Yes

Reported statistics and error estimates:

The inhibition of growth was calculated using both the biomass integral (area under the growth curve) and the growth rate method.
The EL50 values were estimated where possible using a logarithm-linear or logarithm-probit plot of concentration and percent growth inhibition.

The test substance showed to be of low toxicity to Pseudokirchnerella subcapitata. The inhibiton for the WAF loading rate of 1000 mg/l after 72h by biomass integral was 18% and by growth rate 2%. Therefore, the 72 hour EL50 was estimated as > 1000 mg/l using the biomass integral and also by growth rate calculation. Because of the serial dilution from the highest concentration (1000 mg/L), only the 1000 mg/L test concentration was valid and could be used for evaluation of toxicity of the test substance. Without chemical analytics of the diluted concentrations, no information about the exact test concentration could be stated.

Table 1: Cell density measurements (Mean initial cell density: 1 x 10 exp 4 cells/ml)

Concentration [mg/l]	Replicate No.	Cell density measurements [cells/ml x 10 exp 4]
		24 hours	48 hours	72 hours
0	1 2 3 4 5 6	3.00 2.67 2.67 2.33 1.67 2.00	37.00 30.33 27.33 41.33 28.00 33.67	149.33 157.33 150.67 148.67 168.00 149.33
	Mean	2.39	32.94	153.89
100	1 2 3	2.00 3.33 3.67	42.67 47.00 38.33	166.67 165.33 140.00
	Mean	3.00	42.67	157.33
180	1 2 3	2.67 3.33 2.67	34.33 30.67 28.67	164.00 182.67 158.67
	Mean	2.89	31.22	168.45
320	1 2 3	1.33 2.67 1.67	37.33 30.00 25.33	168.00 172.33 164.33
	Mean	1.89	30.89	168.22
560	1 2 3	2.67 3.00 1.67	30.67 24.67 20.00	129.33 144.00 168.67
	Mean	2.45	25.11	147.33
1000	1 2 3	2.33 1.33 1.67	22.33 24.00 19.67	140.00 136.00 136.67
	Mean	1.78	22.00	137.56

 

Reference 7

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: EL50 (24-72h)

Duration:

72 h

Dose descriptor:

EL0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: EL0 (24-72h)

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: EL50 (24-72h)

Duration:

72 h

Dose descriptor:

EL0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: EL0 (24-72h)

At the WAF loading rate of 100 mg/L no inhibition with respect to algal growth rate and algal cell growth (area under growth curve) compared to the untreated controls were observed. The no-observed-effect concentration for growth rates (ErL0) and biomass production (EbL0) with respect to the loading rate was ≥ 100 mg/l. The EL50 -value was conducted as EL50 (24 -72h). But the EL50 can also be stated as EL50 (72h) > 100 mg/l for the algal growth rate and algal cell growth.

Table 1: Cell concentration (A680) in individual test cultures after 24, 48 and 72 h of exposure to the test material. ((b) = Mean absorbance (A680))

Nominal concentration [mg/l]	Code	Cell concentration (A680)
		0 h (b)	24 h	48 h	72 h
Control	A B C D E	0.008 0.008 0.008 0.008 0.008	0.033 0.038 0.038 0.036 0.040	0.228 0.222 0.208 0.212 0.233	0.685 0.733 0.676 0.624 0.694
100	A B C	0.008 0.008 0.008	0.040 0.038 0.044	0.224 0.207 0.207	0.700 0.631 0.675

 

Reference 8

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

To register EC453-460-3 under REACH program, a read-across approach for fulfillment of the endpoints has been adopted. EC453-460-3 is an aliphatic ester and fits the description of polyol esters category in HPV program (High Production Volume, US-EPA). The distinguishing feature of polyol esters category is that the fatty acids were linked to one or more of the multiple hydroxyl groups present in the polyol (alcohol portion of ester). The focus of this correspondence is to support read across to EC613-848-7 (target) which provided key studies to determine hazard profile of EC453-460-3 (target), published information on other structurally analogous polyol esters were served as supporting evidence.

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: yes
- Any stimulation of growth found in any treatment: an increase of growth was observed in the highest test concentration after 72 h.
- Effect concentrations exceeding solubility of substance in test medium: The nominal effect concentration of 100 mg/L exceeded the water solubility of the test substance.

Reported statistics and error estimates:

Statistical analysis of the area under the growth curve data was carried out for the control and 100 mg/L loading rate WSF test group using a Students t-test.

No effects on the growth rate was observed in the range of water solubility of the test substance with an EL50 of > 100 mg/L based on the growth rate. The measured concentrations of the test item in the test vessels were much lower than the nominal loading rate (Table 3).

Table 1: Cell densities in the definitive study

	Cell densities [cells/mL]
Nominal loading rate [mg/L]	0 h	24 h	48 h	72 h
Control R1	1.34x10^4	2.29 x10^4	7.69 x10^4	3.79 x10^5
R2	1.34 x10^4	2.16 x10^4	8.80 x10^4	3.53 x10^5
R3	1.10 x10^4	2.22 x10^4	9.83 x10^4	3.60 x10^5
Mean	1.26 x10^4	2.22 x10^4	8.78 x10^4	3.64 x10^5
100 R1	1.67 x10^4	2.38 x10^4	6.14 x10^4	5.19 x10^5
R2	1.80 x10^4	2.42 x10^4	8.11 x10^4	5.23 x10^5
R3	1.37 x10^4	2.44 x10^4	6.29 x10^4	3.50 x10^5
R4	1.54 x10^4	2.49 x10^4	7.26 x10^4	4.01 x10^5
R5	1.93 x10^4	2.41 x10^4	7.64 x10^4	6.62 x10^5
R6	1.61 x10^4	2.41 x10^4	6.60 x10^4	6.65 x10^5
Mean	1.66 x10^4	2.42 x10^4	7.01 x10^4	5.20 x10^5

Table 2: Inhibition of growth rate and biomass

Nominal loading rate [mg/L]	Area under curve at 72 h	% inhibition	Growth rate (0-72 h)	% inhibition
control	6.25 x10^6	-	0.047
100	7.51 x10^6	[20]	0.048	[2]

[increase in growth as compared to the controls]

Table 3: Chemical analysis of test loading rates

Samples	Nominal loading rate [mg/L]	Concentration found [mg/L]	Expressed as a % of the nominal loading rate
0 h	control	< LOQ	-
	100 R1-R3	12.6	13
	100 R4-R6	17.9	18
72 h	control	< LOQ	-
	100 R1-R3	5.89	6
	100 R4-R6	2.07	2

Description of key information

One reliable study is available for an analogue of the registered substance. In this study Pseudokirchneriella subcapitata were exposed to a Water Accommodated Fraction of the substance at a nominal loading rate of 100 mg/L under static conditions for 72 hours. The ErL50(72 h) value was > 100 mg/L loading rate WAF. The NOELR was 100 mg/L loading rate WAF. The substance is not  harmful to algae.

Additional information

One reliable study is available for an analogue of the registered substance. In this study (Vryenhoef, 2014) performed under GLP according to OECD TG 201 and EC Method C.3, green alga Pseudokirchneriella subcapitata were exposed to the substance under static conditions for 72 hours at 24 °C. 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). Following a preliminary range-finding test, Pseudokirchneriella subcapitata (6 replicates) was exposed to a WAF of the test material at a nominal loading rate of 100 mg/L. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group using a Coulter Multisizer Particle Counter. ErL50 (72 h) value was > 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

The results of this study are supported by data available for the read-across substances fatty acid polyols (Fatty acids, C5-9, esters with pentaerythritol (EC 270-290-3, CAS 68424-30-6) and Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234-392-1, CAS 11138-60-6)) and their analogues is applicable based on the similarity in structure and physico-chemical properties. The substance with the CAS No. 131459-39-7 is a structural analogue of one of the read-across substances (CAS No. 68424-30-6) and can therefore be used for read-across.

The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

Two studies, investigating the toxicity to freshwater algae of Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234 -392 -1, CAS-No. 11138-60-6), were available.

During both static Algal Growth Inhibition tests no inhibiting effects on growth rate were observed within 72 hour test period at the tested concentrations. The first study (Häner, 2007) according to OECD 201 with Desmodesmus subspicatus determined a no-observed-effect concentration for growth rate (ErL0) of ≥ 100 mg/L and an ErL50 > 100 mg/L (WAF loading rate). In the second study (Craig, 2005) similar to OECD 201 with Pseudokirchnerella subcapitata an ErL50 and ErL10 > 1000 mg/L (WAF loading rate) was estimated. In addition, another algae toxicity test (Kuttler, 1998) with Desmodesmus subspicatus showed no toxicity of the test substance after 72 h at a WAF loading rate of 10000 mg/L. All results show no toxicities of Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS-No.11138-60-6) in the range of water solubility (< 0.3 mg/L). Therefore, the test substance is not harmful to algae.

In a static GLP study on 3,5,5-trimethylhexanoic acid mixed tetraesters with PE and valeric acid (CAS-No. 131459-39-7), (Mead, 1999) with Scenedesmus subspicatus according to OECD 201, no inhibition on growth rate and biomass was observed at the tested WAF loading rates (EL50 (72h) > 100 mg/L, NOELR (72h) ≥ 100 mg/L). An increase in growth rate and biomass (growth rate: 2 - 4%, biomass: 2 -20%) in comparison to the controls was determined. Chemical analysis showed measured test concentrations of 2 -18 mg/L. No toxic effects were observed within the water solubility under the conditions tested.