Reaction product of 2,4-bis(2-methylbutan-2-yl)phenol and phosphorous pentoxide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27th April 2017 - 7th May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD, 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

impurity 1

Reference substance name:

2,4-di-tert-pentylphenol

EC Number:

204-439-0

EC Name:

2,4-di-tert-pentylphenol

Cas Number:

120-95-6

Molecular formula:

C16H26O

IUPAC Name:

2,4-bis(1,1-dimethylpropyl)phenol

Specific details on test material used for the study:

The test material is a UVCB

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Approximately 15 mL of all test solutions (exposure and control) were collected into 20 mL vials (pre-cleaned glass with Teflon lined caps) provided by Investigative Science Inc.(ISI) (no preservative). For the range-finding test single samples of all test solutions were collected and submitted for analysis, while for the definitive test, duplicate samples were submitted. The vials were filled, leaving some headspace. Samples were collected immediately prior to test commencement (0 hour) and at test termination (72 hour, from pooled replicates) for analysis. All samples were stored at 4 ± 2°C in the dark prior to pick up and delivery to ISI’s laboratory for analysis. The samples were kept cool (packed in a cooler with an ice pack) during transit. All samples were analyzed the same day that they were received by ISI, and within 24 hours of sample collection.

Test solutions

Vehicle:

no

Details on test solutions:

Individual test solutions of the test item for both range-finding and definitive tests were prepared from stock solutions (1,000 mg/L nominal concentration) prepared in algal nutrient media without the use of any solubilizing agent. All stock solutions were prepared in 1-L glass aspirator bottles, stirred for approximately 23 hours at a rate sufficient to maintain a vortex between approximately 10 – 35% of the solution depth using a stir bar. The solutions were then settled for approximately 1 hour. The first ~ 100 mL of solution removed from of each glass aspirator was disposed of. The 1,000 mg/L stock solution used for the range-finding test was prepared by adding 1.0001g of the test item to 1 L of algal nutrient media in a 1- L glass with aspirator bottle and treated as noted above. The individual range-finding test solutions were prepared by adding an appropriate amount of the stock solution into a 200 mL volumetric flask making this up to volume in algal nutrient media. The 1,000 mg/L stock solution used for the definitive test was prepared by adding 1.0009 g of the test item to 1 L of algal nutrient media in a 1- L glass with aspirator bottle and treated as noted above. The individual definitive test solutions were prepared by adding an appropriate amount of the stock solution into a 500 mL volumetric flask making this up to volume in algal nutrient media. All test solutions were thoroughly mixed prior to being dispensed into the individual test vessels (i.e., 250 mL Erlenmeyer flasks covered with Jaece® non-toxic foam plugs).

Test organisms

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata used in testing were obtained from an in-house culture. The starter culture was purchased from the University of Waterloo Culture Collection (CPCC 37). Cultures of P. subcapitata were maintained according to AquaTox Standard Operating Procedure No. 206. Cultures were aseptically transferred twice weekly (typically from 3 – 7 day old donors) and maintained in temperature and light controlled environments isolated from all testing. The axenic nature of the stock culture was verified by plating on Trypticase Soy Agar (TSA) and Plate Count Agar (PCA). Algal growth curves conducted semi-annually ensured that algae were in an exponential growth phase, suitable for testing.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23 ± 1 °C

pH:

7.5 ± 0.1

Nominal and measured concentrations:

Range-finding test
Nominal concentrations: 0.1; 1; 10; 100 & 1000 mg/L
TWM (time-weighted mean) concentrations: < MDL; < MDL; 3.5; 21 & 203 mg/L, respectively

Definitive test
Nominal concentrations: 0 (negative control); 1.562; 3.125; 6.25; 12.5; 25; 50 & 100 mg/L
TWM concentrations: < MDL; 1.6; 3.1; 7; 11.7; 22.6 & 49 mg/L, respectively

MDL = 9 mg/L for nominal test solutions of 100 and 50 mg/L, 2.25 mg/L for nominal test solutions of 25
and 12.5 mg/L, and 1.13 mg/L for nominal concentrations of 6.25 and below

Details on test conditions:

Table 3. Summary of Test Conditions for Determining Toxicity of X-19933
to P. subcapitata
Parameter Conditions

Test type Static algal flask test

Test species Uni-algal cultures of P. subcapitata (Korschikov) Hindák (current sourceCPCC 37)

Test duration 72 hour

Agitation during testing Continuous shaking of test vessels on a rotary shaker set at 100 rpm

Incubation chamber Incubated in a growth chamber

Temperature 23 ± 1 °C, as recorded daily with a maximum/minimum thermometer

Light quality Cool-white fluorescent

Light intensity Measured at the surface of the liquid in the flasks. 4440 – 8880 Lux for testing

Photoperiod Continuous (24 hours)

Test vessel Clear glass 250-mL Erlenmeyer flasks covered with Jaece® non-toxic foam plugs

Nutrient medium Filter-sterilized

Nutrient medium/test
solution volume 50 mL

pH of the nutrient medium 7.5 ± 0.1

pH of the test solutions pH is measured (but not adjusted) in all concentrations at the beginning and end of the test (pooled replicates)

Age of algal cells used for
testing 3 – 7 days old and in exponential growth

Number of definitive test
Concentrations 7 plus a nutrient medium (negative) control

Number of replicate test
vessels/concentration in
definitive test 4 per treatment/4 per control

Initial concentration of
cells per test vessel 5 x 10^3 to 1 x 10^4 cells/mL

Measured endpoints Cell number counted daily, at approximately 24, 48, and 72 hours

Cell count methodology Conducted using a haemocytometer and a phase-contrast microscope (at 100 – 200 times magnification)

Observations Changes in cell development or appearance, such as cell clumping, cell morphology, cell colour, cell shape, cell size, etc.
Additional observations, such as sedimentation of the test solution, precipitation of cells, solution appearance / colouration, or other abnormalities

Calculated endpoints Average specific growth rate, cell yield, section-by-section growth rate for controls

Statistical endpoints Average Specific Growth Rate = EC10, EC20, and EC50 (0 – 3 days) and NOEC and LOEC; Cell Yield = EC10, EC20, and EC50; NOEC and LOEC at 72 hours

Reference substance (positive control):

yes

Remarks:

Reference toxicant testing (under non-GLP conditions) is performed in AquaTox's Ecotoxicity Laboratory on a regular basis to demonstrate consistency in laboratory test performance.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

37 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

47.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

69.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

log (72-h EC25): 2.8738; 95% warning limit: 2.6646 - 3.1960

Reported statistics and error estimates:

Calibration Verification Standard percent recovery between 100.4 and 101.8, n=4).
Calibration Verification Standard percent recovery between 100.4 and 101.2, n=2).
Results of duplicate analysis. RPD = 6.09.
MDL = 9 mg/L for nominal test solutions of 100 and 50 mg/L, 2.25 mg/L for nominal test solutions of 25
and 12.5 mg/L, and 1.13 mg/L for nominal concentrations of 6.25 and below

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Biomass in control vessels increased by a factor > 16x Mean CV for section-by-section specific growth rates in the control cultures did not exceed 35%. CV of average specific growth rates during test period in replicate control cultures did not exceed 7%.

Conclusions:

From the definitive test and based on the nominal concentrations, the NOEC and LOEC for average specific growth rate and cell yield were estimated to be 12.5 and 25.0 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for average specific growth rate based on the nominal concentrations were estimated to be 37.0, 47.6 and 69.6 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 19.1, 24.1, and 37.3 mg/L, respectively.

Executive summary:

The toxicity of the test substance to the freshwater green alga,Pseudokirchneriella subcapitatawas investigated under static test conditions, according to OECD Guideline 201,“Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (OECD, 2011). 

 

With the following exceptions, all components of this study were conducted in accordance with the requirements of OECD Principles of Good Laboratory Practice (GLP) (OECD, 1998):

 

•P. subcapitataculture;

• Reference toxicant testing with P. subcapitata; and,

• Ecotoxicological and analytical method development.

 

Analytical concentrations were verified, however, due to the nature of the test item (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results of the range-finding and definitive tests are reported here in terms of nominal concentrations only.

 

Results of the range-finding test indicated that algal growth expressed in terms of average specific growth rate and cell yield was adversely affected at nominal concentrations above 10 mg/L.

From the definitive test and based on the nominal concentrations, the NOEC and LOEC for average specific growth rate and cell yield were estimated to be 12.5 and 25.0 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for average specific growth rate based on the nominal concentrations were estimated to be 37.0, 47.6 and 69.6 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 19.1, 24.1, and 37.3 mg/L, respectively.