2,4,6-triisopropyl-m-phenylene diisocyanate

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:

2020-06-23 - 2020-06-26 (experimental part)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 'Freshwater Alga and Cyanobacteria, Growth inhibition test' (2009)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

OECD Guideline for Testing of Chemicals No. 201 (2006)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document on aqueous-phase Aquatic Toxicity testing of Difficult Test Chemicals No.23 (2019)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from the only test concentration of 100 mg/L, the test concentration of 100 mg/L without algal inoculum and the control.
- Sampling method: Samples were taken at test start, after 24 hours, 48 hours and 72 hours of exposure.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: To produce the only test item concentration 399.7 mg of the test item were added to 4 litres of dilution water. The emulsion was treated for 60 seconds at 8000 rpm with an ultra turrax and was gentle stirred for 20 h on a magnetic stirrer. The emulsion was allowed to settle for one hour. Afterwards, 1500 mL of the solution were removed from the middle water column with a vacuum pump and a silicone tube. Undissolved particles of the test item were not removed with sponsors agreement. The pH was measured to be pH 7.9.
- Control: Inoculum in growth medium / dilution water

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Source (laboratory, culture collection): stock culture bred at CURRENTA Analytik
- Age of inoculum (at test initiation): Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium (OECD medium of OECD TG 201)
- Method of cultivation: Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21 - 24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 µE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a particle counter, Z2, Beckmann Coulter.

ACCLIMATION
- Acclimation period: Reinoculation of stock culture once a week.
- Culturing media and conditions (same as test or not): same (OECD medium of OECD TG 201)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Water hardness of the final nutrient medium was 1.3 °dH, corresponding to 22.5 mg/L CaCO3.

Test temperature:

21 °C - 24 °C (+/- 2 °C)

pH:

Control: 8.0 - 8.9
100 mg/L: 7.8 - 8.5
100 mg/L (without algae): 7.9 - 8.0

Nominal and measured concentrations:

nominal: 100 mg/L
geometric mean measured: 2.467 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks with cotton stoppers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: test volume: 100 mL
- Initial cells density: 5000 cells/mL
- Control end cells density: 1107000 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- Other: Additionally, test item concentration 100 mg/L without algae (1 replicate).

GROWTH MEDIUM
- Standard medium used: yes (OECD medium of OECD TG 201)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium of OECD TG 201
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous uniform illumination
- Light intensity and quality: 60 to 120 µE x m-2 x s-1 equivalent to 4000 to 8000 lux
- Other: Cultivation of green algae over a period of three days in a light chamber including a shaking device.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Daily measurements of growth of unicellular green algae over a period of three days.
- Determination of cell concentrations: particle counter Z2 (Beckman Coulter)

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: The selection of the test concentration depends on the result of the preliminary test.
- Range finding study
- Test concentrations: 100, 10, 1.0 and 0.1 mg/L

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 2.467 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 2.467 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.467 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): not specified
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:The solution of the test item concentration 100 mg/L was not clear. It was a cloudy emulsion.

Reported statistics and error estimates:

The measured values were reduced by the number of cells that were measured in the replicates without algae. These values formed the data basis for the evaluation in the statistic program ToxRat.

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to EU Method C.3, which is equivalent to OECD TG 201 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxoicity of the test substance towards algae.
Exponentially growing cultures of the freshwater green algal species Desmodesmus subspicatus were exposed to the test subsantce at the limit concentration of nominal 100 mg/L, representing the limit of water solubility, for a period of 72 hours under defined conditions. At this concentration no inhibition of algal yield and growth rate was determined at the end of the 72 hour study period, relative to control cultures grown under identical conditions. The 72h ErC50 and the 72h EyC50, as well as the 72h ErC10 and the 72h EyC10 were determined to be greater than the limit concentration of nominal 100 mg/L, corresponding to a geometric mean measured concentration of 2.467mg/L. Accordingly, no toxic effects against algae were observed at the limit of water solubility under test conditions.
Based on the obtained results, the test substance does not need to be classified as toxic to freshwater algae following the provisions laid down in Council Directive 67/548/EEC and Regulation (EC) No. 1272/2008.

Executive summary:

The acute toxicity of the test substance to aquatic algae was tested according to EU Method C.3, which is equivalent to OECD TG 201, in a static freshwater test with Desmodesmus subspicatus (former name: Scenedesmus subspicatus) as test organism. The study was conducted under certificated GLP compliance.

Due to the test substance properties, a limit test was performed at the limit of water solubility under test conditions in order to demonstrate that the EC₅₀values (based on yield as well as on growth rate) are greater than this concentration. Pre-experiments were conducted, which provided the concentration, as well as the biological and analytical design to be used in the main test. The final test concentration was chosen as nominal 100 mg/L, whereby the test medium was prepared by direct weighing, prolonged stirring and a subsequent settling period before withdrawal of the solved fraction in the middle.

For the test item concentration as well as for the control 6 replicates were prepared. The algal inocula for the experiment were taken from an exponentially growing pre-culture and were mixed with the nutrient medium to make up to a final cell density of about 5000 cells per milliliter in the test medium. The algae were exposed for a period of 72 hours and the cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate and yield, relative to control cultures grown under identical conditions. All calculations were carried out using a statistical software programme. The maintenance of test item concentrations was proven by analytical measurements (HPLC MS/MS). Based on the analytical measurements in all test medium samples a geometric mean measured concentration of 2.647 mg/L was determined for the limit concentration of nominal 100 mg/L.

As analytical recoveries in the test media samples were not within 80 -120 % of nominal, all endpoints were determined referring to the geometric mean measured concentration of the test material. Accordingly, the following results were determined for yield (y) and growth rate (r) of the algal population within 72 h exposure period:

E_rC₅₀(72h) > 2.467mg/L

E_rC₁₀(72h) > 2.467 mg/L

NOE_rC (72h) ≥ 2.467 mg/L

E_yC₅₀(72h) > 2.467mg/L

E_yC₁₀(72h) > 2.467 mg/L

NOE_yC (72h) ≥ 2.467 mg/L

Description of key information

1) Key_Toxicity to aquatic algae and cyanobacteria: E_rC₅₀ (72h) > 2.467 mg/L (geometric mean measured), E_rC₁₀(72h) > 2.467 mg/L (geometric mean measured) for Desmodesmus subspicatus (static, freshwater, OECD 201, GLP)

2) Supporting_Toxicity to aquatic algae and cyanobacteria: EC₅₀ (72h) = 4300 mg/L for Chlorella vulgaris (static, freshwater, non-guideline study, non-GLP)

3) Supporting_Toxicity to aquatic algae and cyanobacteria: EC₅₀(72h) = 3230 mg/L for Skeletonema costatum (static, freshwater, non-guideline study, non-GLP)

Key value for chemical safety assessment

EC50 for freshwater algae:

2.467 mg/L

EC10 or NOEC for freshwater algae:

2.467 mg/L

Additional information

The acute toxicity of the test substance to aquatic algae was tested according to EU Method C.3, which is equivalent to OECD TG 201, in a static freshwater test with Desmodesmus subspicatus (former name:Scenedesmus subspicatus) as test organism. The study was conducted under certificated GLP compliance.

Due to the test substance properties, a limit test was performed at the limit of water solubility under test conditions in order to demonstrate that the EC₅₀values (based on yield as well as on growth rate) are greater than this concentration. Pre-experiments were conducted, which provided the concentration, as well as the biological and analytical design to be used in the main test. The final test concentration was chosen as nominal 100 mg/L, whereby the test medium was prepared by direct weighing, prolonged stirring and a subsequent settling period before withdrawal of the solved fraction in the middle.

For the test item concentration as well as for the control 6 replicates were prepared. The algal inocula for the experiment were taken from an exponentially growing pre-culture and were mixed with the nutrient medium to make up to a final cell density of about 5000 cells per milliliter in the test medium. The algae were exposed for a period of 72 hours and the cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate and yield, relative to control cultures grown under identical conditions. All calculations were carried out using a statistical software programme. The maintenance of test item concentrations was proven by analytical measurements (HPLC MS/MS). Based on the analytical measurements in all test medium samples a geometric mean measured concentration of 2.647 mg/L was determined for the limit concentration of nominal 100 mg/L.

As analytical recoveries in the test media samples were not within 80 -120 % of nominal, all endpoints were determined referring to the geometric mean measured concentration of the test material. Accordingly, thefollowing results were determined for yield (y) and growth rate (r) of the algal population within 72 h exposure period:

E_rC₅₀(72h) > 2.467mg/L

E_rC₁₀(72h) > 2.467 mg/L

NOE_rC (72h)≥2.467 mg/L

E_yC₅₀(72h) > 2.467mg/L

E_yC₁₀(72h) > 2.467 mg/L

NOE_yC (72h) ≥ 2.467 mg/L

These results are supported by two experiments on the toxicity of the read-across substance m-tolylidene diisocyanate (TDI) to aquatic algae.

The acute toxicity of m-tolylidene diisocyanate to the freshwater algae Chlorella vulgaris was investigated according to OECD Guideline 201 for a test duration of 96 hours (Tadokoro et.al., 1997). 15g/L of T-80 was added to alga medium and the medium was stirred for 24 hours. The solution was allowed to settle for 1 hour. The stock solution was diluted to desired concentrations. The nominal concentration range was 938 mg/L - 15000 mg/L. An initial cell number of 1x10E7 cells/mL was used and the temperature was held constant at 20 °C. At a test concentration of 1 g/L, only 2.8 % biomass inhibition was observed. This value can be considered as the No Observed Effect Concentration (NOEC; EC2.8). The EC50 was determined as 4300 mg/L, whereby the rapid hydrolysis rate of the test substance was taken into consideration.

Also the acute toxicity of m-tolylidene diisocyanate to the marine algae species Skeletonema costatum was investigated according to OECD Guideline 201 for a test duration of 96 hours (Tadokoro et.al., 1997). 10g/L of T-80 was added to enriched sea water medium and the medium was stirred for 24 hours. The solution was allowed to settle for 1 hour and filtered with no. 5C filter paper and 0.22 µm millipore filter to remove undissolved component and to sterilize the medium. Then the stock solution was diluted to desired concentration with sterilized enriched seawater to prepare test solutions (highest concentration was 5000 mg/L with 5 others diluted by a factor of 1.8). The initial number of cells was 10E3 cells/mL and the test temperature 20 °C. The EC₅₀ was determined as 3230 mg/L, taken the rapid hydrolysis rate of the substance into consideration.