Poly[oxy(methyl-1,2-ethanediyl)], .alpha.-hydro-.omega.-[2-[[2,2-dimethyl-3-[(1-oxododecyl)oxy]propylidene]amino]methylethoxy]-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1)

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:

2016-09-01 to 2016-11-28

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:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

No sampling

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions were performed using the WAF method (according to OECD Series on Testing and Assessment No. 23) as follows: Each test concentration was prepared individually by adding an amount of test item (appropriate for the concentration) into the dilution water (OECD medium) and mixing for a period of one hour to achieve an equilibrated concentration. Each solution was then filtrated through a membrane filter (0.22 μm) to separate the possible non-dissolved test material. The test solutions were prepared immediately before introduction of algae (start of the experiment).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapita and Selenastrum capricornutum) (Printz-Starr).
- 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 stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines.
The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The pre-culture was incubated for three days at this test.)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.5 and 24.0°C

pH:

7.73 – 9.31

Nominal and measured concentrations:

The following nominal concentrations were tested: 0.1, 0.4, 1.6, 6.3, 25, 100 mg/L (loading rate)
No measured concentrations as no analytical determination possible.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: ~250 mL volume with 100 mL test medium.
- Aeration: No
- Initial cells density: 1 × 10^4 cells/mL
- Control end cells density: 84.00 × 10^4 cells/mL (mean value) after 72 hours in the Untreated control.
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to OECD guideline

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: 24 h
- Light intensity and quality: 7980 lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: 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
- Other: 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: 4
- Range finding study: Each test concentration was prepared individually by adding an amount of test item (appropriate for the concentration) into the dilution water (OECD medium) and mixing for a period of one hour to achieve an equilibrated concentration. Each solution was then filtrated through a membrane filter (0.22 μm) to separate the possible non-dissolved test material. Untreated control ran parallel in the test.
- Results used to determine the conditions for the definitive study: Based on the results of the non-GLP Preliminary Range-Finding Test (see above) six test concentrations in a geometric series (with a spacing factor of 4) were used in the main test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.58 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL: 0.46 - 0.73 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.84 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL: 0.69 - 1.01 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL: 1.45 - 1.99 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.27 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL: 0.60 - 0.81 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.38 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL: 0.31 - 0.45 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL: 0.60 - 0.81 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: No
- Any observations that might cause a difference between measured and nominal values: No morphological abnormalities were observed in the control and in the lowest test item concentration (0.1 mg/L) during the experiment. No precipitation was observed.

Validity criteria fulfilled:

yes

Conclusions:

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 results are based on nominal test item concentrations (loading rate). The calculated biological endpoints are EC10 (growth rate) 0.58 mg/L, EC50 (growth rate) 1.70 mg/L, EC10 (yield) 0.27 mg/L, EC50 (yield) 0.70 mg/L, NOEC (growth rate) 0.4 mg/L and NOEC (yield) 0.1 mg/L.

Executive summary:

The purpose of this study was to determine the effect of the test item SIKA Hardener LTJ on the growth of a unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). Exponentially growing cultures of Raphidocelis subcapitata were exposed to various concentrations of the test item over several generations under defined conditions. 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 method of application and the test species Raphidocelis subcapitata are recommended by the test guidelines. Based on the results of non-GLP Preliminary Range-Finding Test the following six test concentrations in a geometric series (with a spacing factor of 4) were tested: 0.1, 0.4, 1.6, 6.3, 25, 100 mg/L (loading rate). The test concentrations were prepared using the WAF method (by preparing individual WAFs). Due to the rapid hydrolysis of the test item (SIKA Hardener LTJ ) 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). Biological results are based on the nominal test item concentrations (loading rate).For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test using TOXSTAT software. 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 in concentrations of 1.6, 6.3, 25 and 100 mg/L and based on yield in concentrations of 0.4, 1.6, 6.3, 25 and 100 mg/L (Bonferroni t-Test, 1 tailed, α=0.05) when compared to control. The ErCx and EyCx values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software. The calculated biological endpoints are EC10 (growth rate) 0.58 mg/L, EC50 (growth rate) 1.70 mg/L, EC10 (yield) 0.27 mg/L, EC50 (yield) 0.70 mg/L, NOEC (growth rate) 0.4 mg/L and NOEC (yield) 0.1 mg/L.

Description of key information

The purpose of this study was to determine the effect of the test item SIKA Hardener LTJ on the growth of a unicellular green algal species Raphidocelis subcapitata according to OECD guideline 201. The calculated biological endpoints are EC10 (growth rate) 0.58 mg/L, EC50 (growth rate) 1.70 mg/L, EC10 (yield) 0.27 mg/L, EC50 (yield) 0.70 mg/L, NOEC (growth rate) 0.4 mg/L and NOEC (yield) 0.1 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.7 mg/L

EC10 or NOEC for freshwater algae:

0.58 mg/L

Additional information

Growth Inhibition Test of SIKA Hardener LTJ on Algae (Raphidocelis subcapitata)

The purpose of this study was to determine the effect of the test item SIKA Hardener LTJ on the growth of a unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). Exponentially growing cultures of Raphidocelis subcapitata were exposed to various concentrations of the test item over several generations under defined conditions. 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 method of application and the test species Raphidocelis subcapitata are recommended by the test guidelines. Based on the results of non-GLP Preliminary Range-Finding Test the following six test concentrations in a geometric series (with a spacing factor of 4) were tested: 0.1, 0.4, 1.6, 6.3, 25, 100 mg/L (loading rate). The test concentrations were prepared using the WAF method (by preparing individual WAFs). Due to the rapid hydrolysis of the test item (SIKA Hardener LTJ ) 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). Biological results are based on the nominal test item concentrations (loading rate).For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test using TOXSTAT software. 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 in concentrations of 1.6, 6.3, 25 and 100 mg/L and based on yield in concentrations of 0.4, 1.6, 6.3, 25 and 100 mg/L (Bonferroni t-Test, 1 tailed, α=0.05) when compared to control. The ErCx and EyCx values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software. The calculated biological endpoints are EC10 (growth rate) 0.58 mg/L, EC50 (growth rate) 1.70 mg/L, EC10 (yield) 0.27 mg/L, EC50 (yield) 0.70 mg/L, NOEC (growth rate) 0.4 mg/L and NOEC (yield) 0.1 mg/L.