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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9th of May 2017 to 20th May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

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:

The range-finding test

Nominal test solutions of 0.1, 1.0, 10, 100 and 1000 mg/L were prepared by adding 0.025, 0.25, 2.5, 25, and 250 mL of a nominal 1,000 mg/L stock solution, respectively into a 250 mL graduated cylinder and making this up to volume in dilution water. The nominal 1,000 mg/L solution was prepared by adding 1.0009 g of X-19933 into a 1-L glass aspirator bottle with a 1-L volume of dilution water following the procedures described in Section 4.3.1. All test solutions were hand mixed by inverting the cylinder several times, prior to being dispensed into each of 2 replicate 100 mL test vessels (75 mL per replicate). A total of ten daphnids (i.e., 2 replicates with 5 daphnids each) were exposed to each test and control solution under static-test conditions. Test organisms were transferred to the test and the
control solutions using a pipette and bulb. Test results were based on immobility over a 48-hour exposure period.


Definitive Test

Results of the range-finding test indicated that the 48-hour EC50 for daphnid immobilization was in the range of 1 to 10 mg/l (nominal concentration) of the test item. The nominal 1,000 mg/L solution was prepared by adding 1.0002 g of X-19933 into a 1-L glass aspirator bottle with a 1-L volume of dilution water. Nominal concentrations of 0.625, 1.25, 2.5, 5, 10, 20 and 40 mg/L were prepared by adding 0.313, 0.625, 1.25, 2.5, 5, 10, and 20 mL of a nominal 1,000 mg/L stock solution, respectively into a 500 mL graduated cylinder and making this up to volume in dilution water. All test solutions hand mixed by inverting the cylinder several times, prior to being dispensed into each of 4 replicate 100 mL test vessels (75 mL per replicate). A total of 20 daphnids (i.e., 4 replicates with 5 daphnids each) were exposed to the test and control solution under static test conditions. All test organisms were transferred to the control and test solutions using a pipette and bulb. Test results were based on immobility over a 48- hour exposure period.

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 dilution water without the use of any solubilizing agent. Stock solutions were prepared in 1-L glass aspirator bottles, stirred for approximately 23 hours (or longer if needed) at a rate sufficient to maintain a vortex between approximately 10 – 35% of the solution depth using a stir bar and stir plate. The solutions were then settled for approximately 1 hour. The first ~ 100 mL of solution removed from each glass aspirator was disposed of. Individual test solutions were prepared by addition of the appropriate amount of stock solution to dilution water to achieve the desired concentrations for testing. All tests (range-finding and definitive) included a negative control consisting of dilution water with no test item added.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna were cultured continuously from a population that has been activelyreproducing in AquaTox’s laboratory since 1988. The organisms were originally obtained from the Ministry of the Environment and Climate Change (Toronto, Ontario, Canada). Certification of the taxonomic identity of the D. magna is provided in Appendix C. Culturing and holding of the test species prior to testing was conducted according to AquaTox SOP # 230 (AquaTox,2016a).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

200 – 210 mg/L as CaCO3;

Test temperature:

20 ± 2 °C

pH:

pH 8.0 – 8.5

Dissolved oxygen:

The concentration of dissolved oxygen in the water was maintained at >80% of the air saturation value

Nominal and measured concentrations:

Range-finding test
Nominal concentrations: 0, 0.1; 1; 10; 100 & 1000 mg/L

Definitive test
Nominal concentration (mg/L) 40, 20, 10, 5, 2.5, 1.25, 0.625, 0 (negative control)

Details on test conditions:

Parameter Condition
Test type Static
Test duration 48 hour
Temperature 20 ± 2 °C, as recorded daily with a maximum/minimum thermometer (constant within ± 1 °C)
Light quality Ambient laboratory illumination
Light intensity 400 to 800 lux
Photoperiod 16-hour light, 8-hour dark (with 30 minute transition period)
Feeding prior to test Once/day: 1) yeast, Cerophyll broth, Trout Chow (YCT) and 2) Pseudokirchneriella subcapitata algae
Feeding regime None during test
Test vessel Glass beaker, 100 mL
Loading rate 15 mL/daphnid
Test volume 75 mL (min)
Source of test organisms In-house culture
Age of test organisms First instar, neonate (<24-hours old)
Number of concentrations Range-finding test: 5 + a negative control; Definitive multiple concentration test: 7 + a negative control;
Number of Replicates 2 (Range-finding test) 4 (Definitive test)
Number of organisms per replicate 5
Total number of organisms 10 (Range-finding test) 20 (Definitive multiple concentration test)
per test level

Aeration None
Dilution water Groundwater diluted with Reverse Osmosis water (water hardness 200 – 210 mg/L as CaCO3; pH 8.0 – 8.5)
Monitoring parameters and Temperature daily; dissolved oxygen, pH, and conductivity on all solutions at start and end of test; dilution water
observations made during the test hardness at 0-hours; and visual observations of immobilisation of D. magna at 24 and 48 hours.

Measured endpoints Immobility, stressed behaviour
Calculated endpoints 24 and 48-hour EC50s; NOEC and LOEC at 48 hours.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

11.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.25 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All of the validity criteria for the definitive tests were met as listed below • Immobility of the controls did not exceed 10%. • Dissolved oxygen concentrations at the end of the test were ≥ 3 mg/L in the control and test solutions.

Conclusions:

Results of the range-finding test indicated that the 48-h EC50 for immobility based on the nominal concentrations was in the range of 1 to 10 mg/L. Results of the definitive test showed that there was no immobility of daphnids at or below 1.25 mg/L. The 24- and 48-hour EC50s for immobility of D. magna determined from the definitive test were estimated to be 11.7 and 1.8 mg/L, respectively. The NOEC and LOEC for immobility of daphnids were estimated to be 1.25 and 2.50 mg/L, respectively.

Executive summary:

The toxicity of the test substance to the Daphnia Magna was investigated under static test conditions, according to OECD Guideline 202,“ Daphnia sp., Acute Immobilisation Test” (OECD, 2004). This study were conducted in accordance with the requirements of OECD Principles of Good Laboratory Practice (GLP).

 

Both the range-finding and definitive tests met all the validity criteria and the test results were based on measured concentrations at the beginning and end of the test, or more specifically, the Time-Weighted Mean (TWM) concentrations. Results of the range-finding test indicated that the 48-hour EC50 for daphnid immobilization was in the range of 1 to 10 mg/l(nominal concentration) of the test item.

Results of the definitive test showed that there was no immobility of daphnids at or below 1.25 mg/L. The 24- and 48-hour EC50s for immobility of D. magna determined from the definitive test were estimated to be 11.7 and 1.8 mg/L, respectively. The NOEC and LOEC for immobility of daphnids were estimated to be 1.25 and 2.50 mg/L, respectively.