1,4-phenylenebis(nitrilo-2,2-dimethylprop-1-yl-3-ylidene) didodecanoate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-06-07 to 2016-06-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

23 March 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

01 March 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

April 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

TOCI-COOP Toxicological Research Center Zrt.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: In order to prepare the test solutions, a saturated stock solution was first prepared as follows: A supersaturated solution (nominal loading: 100 mg/L) was prepared by adding an excess of test item in OECD medium. An amount of 0.08 g test item was diluted in 800 mL OECD medium. Subsequently, this solution was shaken for at least 24 hours (~26 hours in this test) and thereafter the non-dissolved test material was removed by filtration through a 0.22 μm membrane filter to obtain the saturated stock solution (i.e. 100 % v/v saturated solution). The test solutions of subsequent lower concentrations were prepared by appropriate dilution of this stock solution. The test solutions were prepared immediately before introduction of algae (start of the experiment).
- Controls:
Untreated Control: Algal Mineral Salts Test Medium (without test item) was inoculated and examined in parallel in the study.
Toxic Reference Control: For the evaluation of the quality of the algae and the experimental conditions, potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Test organisms (species):

other: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapita and Selenastrum capricornutum) (Printz-Starr)

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Method of cultivation: The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, after an incubation period of four days.

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.4 – 22.8 °C

pH:

7.64 – 8.34

Nominal and measured concentrations:

Nominal concentration: 0.5, 1.4, 4.1, 11.9, 34.5 and 100 mg/L
No measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel (Material, size, fill volume): Erlenmeyer flasks of ~250 mL volume with 100 mL test medium.
- Type: closed, the flasks were covered with air-permeable stoppers
- Initial cells density: about 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- The composition of test medium according to OECD 201was used.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: The algal culture flasks were continuously illuminated
- Light intensity and quality: The average light instensity was 7826 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell Number: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber.
- Morphological Changes of Algal Cells: The morphological changes of algal cells compared to the control were examined at 24, 48 and 72 hours after starting the test using a microscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.9 (Main test)
- Range finding study:
- Results used to determine the conditions for the definitive study:
Preliminary Algal Growth Inhibition Test:
The algal cells were exposed to test item concentrations of 0, 0.1, 1, 10 and 100 mg/L. Please find the results of the preliminary Range-Finding Test in the "any other information on materials and methods incl. tables" section (Table 1).

Preliminary analytical investigations:
Based on the measured data obtained during the preliminary analytical investigations (Study Number: 644-100-0596) the test item undergoes a fast hydrolysis reaction in the presence of 20% water. Its concentration decreased below 10% of the nominal within 13 minutes. Stability of the hydrolysis product, 1,4-Phenylenediamine was investigated under the same conditions. This compound also decomposed rapidly, less than 20% of the nominal concentration was measured within 13 minutes.
Consequently it was not possible to validate a suitable analytical method for the verification of test item concentration in aqueous test media. Analytical determination of the test item concentration was not feasible due to instability of the test item. Therefore, biological endpoints of the test are based on the nominal test item concentrations (loading rate).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 3.5 - 5.6

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 14.3 - 19.5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CL: 1.2 - 1.9

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CL: 4.9 - 6.7

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Growth rate and Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Growth rate and Yield

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50 (72h): 0.70 mg/L, (95 % confidence limits: 0.64 – 0.78 mg/L)
- EyC50 (72h): 0.39 mg/L, (95 % confidence limits: 0.36 – 0.43 mg/L)

Reported statistics and error estimates:

Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, after 0 h, 24 h and 48 h and at the end of the test (72 hours after the start of the test) using Excel for Windows software.
Percentage inhibition of growth rate (μ) and yield (y) were calculated using EXCEL for Windows software.
The ErCx, and EyCx values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software (based on the measured geometric mean concentrations).
For the determination of the LOEC and NOEC, the calculated mean growth rate (μ) and yield (y) at the test concentrations were tested on significant differences to control values by Bonferroni t-Test using TOXSTAT software.

Validity criteria fulfilled:

yes

Conclusions:

In this 72 hour algal growth inhibition test with Raphidocelis subcapitata, an EC10 and EC50 based on growth rate of 4.4 and 16.7 mg/Lwere observed, respectively. The EC10 and EC50 values based on Yield were 1.5 and 5.7mg/L, respectively.The determined NOEC was 1.4 mg/L based on growth rate and Yield and a LOEC of 4.1 mg/L was observed based on growth rate and Yield.

Executive summary:

A Growth Inhibition Test with the algal Raphidocelis subcapitata was performed according to OECD Guideline 201. Exponentially growing cultures of Raphidocelis subcapitata were exposed to test item concentrations of 0.5, 1.4, 4.1, 11.9, 34.5 and 100 mg/L. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and thus, over several algal generations. The test concentrations were obtained by serial dilution of a saturated stock solution (100 mg/L loading rate). Due to the rapid hydrolysis of the test item and its hydrolysis product (1,4-Phenylenediamine), validation of suitable analytical method was not possible for the verification of test item concentration in aqueous test media. Analytical determination of the test item concentrations was not feasible, therefore biological endpoints of the test are based on the nominal test item concentrations (loading rate).

In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item had significant toxic effects on the growth of Raphidocelis subcapitata. All validity criteria were met. The test item had a statistically significant inhibitory effect on the growth of Raphidocelis subcapitata after the exposure period of 72 hours based on the average specific growth rate and yield in concentrations of 4.1, 11.9, 34.5 and 100 mg/L when compared to control. Accordingly, the 72-hour NOEC based on growth rate and yield was determined to be 1.4 mg/L and the LOEC as 4.1 mg/L. An EC10 and EC50 based on growth rate of 4.4 and 16.7 mg/L were observed, respectively. The EC10 and EC50 values based on Yield were 1.5 and 5.7 mg/L, respectively.

 

 

Description of key information

In a Growth Inhibition test with the algal Raphidocelis subcapitata a 72-hour NOEC based on growth rate and yield was determined to be 1.4 mg/L and the LOEC as 4.1 mg/L. An EC10 and EC50 based on growth rate of 4.4 and 16.7 mg/L were observed, respectively. The EC10 and EC50 values based on Yield were 1.5 and 5.7 mg/L, respectively.

 

 

Key value for chemical safety assessment

EC50 for freshwater algae:

16.7 mg/L

EC10 or NOEC for freshwater algae:

4.4 mg/L

Additional information

A Growth Inhibition Test with the algal Raphidocelis subcapitata was performed according to OECD Guideline 201. Exponentially growing cultures of Raphidocelis subcapitata were exposed to test item concentrations of 0.5, 1.4, 4.1, 11.9, 34.5 and 100 mg/L. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and thus, over several algal generations. The test concentrations were obtained by serial dilution of a saturated stock solution (100 mg/L loading rate). Due to the rapid hydrolysis of the test item and its hydrolysis product (1,4-Phenylenediamine), validation of suitable analytical method was not possible for the verification of test item concentration in aqueous test media. Analytical determination of the test item concentrations was not feasible, therefore biological endpoints of the test are based on the nominal test item concentrations (loading rate).

In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item had significant toxic effects on the growth of Raphidocelis subcapitata. All validity criteria were met. The test item had a statistically significant inhibitory effect on the growth of Raphidocelis subcapitata after the exposure period of 72 hours based on the average specific growth rate and yield in concentrations of 4.1, 11.9, 34.5 and 100 mg/L when compared to control. Accordingly, the 72-hour NOEC based on growth rate and yield was determined to be 1.4 mg/L and the LOEC as 4.1 mg/L. An EC10 and EC50 based on growth rate of 4.4 and 16.7 mg/L were observed, respectively. The EC10 and EC50 values based on Yield were 1.5 and 5.7 mg/L, respectively.