4,4'-methylenebis(2,6-diisopropylaniline)

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:

January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At start and end of the study (72 h).

Vehicle:

no

Details on test solutions:

The test solutions were prepared by mechanical dispersion, without the use of any solubilising agent. The test item is poorly water soluble material. In order to achieve maximum dissolved concentration, a supersaturated solution was prepared by dispersing/dissolving the test item amount of 0.76 g into 1000 mL test medium (OECD medium; see 5.4) one day before the start of the test. This test item solution was agitated by orbital shaker (~300 rpm) for a period of about 20 hours and then the non-dissolved test material was separated by filtration through a fine (0.22 μm) filter in order to obtain the saturated test solution (i.e. 100 % v/v saturated solution). The further test solutions were freshly prepared from this test item stock solution (TISS) in the testing laboratory just before introduction of algae (start of the experiment) by appropriate diluting of this stock solution.

Test organisms (species):

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

Details on test organisms:

- Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
- Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Origin: The algae were supplied by the SAG: Collection of Algal Cultures, D-37073 Göttingen, Germany
- Breeding Conditions: 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 standardized conditions according to the test guidelines.
- Pre-culturing: 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 preculture was incubated for four days at this test.) The algal cultures used in this study did not contain deformed or abnormal cells.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment.

Test temperature:

Temperature was in the range of 22.1 – 22.7 °C measured in the flasks.

pH:

Range of the pH was 7.39 – 9.11 during the experiment.

Dissolved oxygen:

Not measured

Salinity:

Freshwater used

Nominal and measured concentrations:

Based on the results of a preliminary tests, the test solutions were prepared from a test item stock solution (TISS) at the concentration equal to the water solubility (water accommodated fraction, i.e. WAF) and the following test item concentrations were used in the main study: TISS/39.0625; TISS/15.625; TISS/6.25; TISS/2.5 and TISS.
The analytically measured test item concentrations deviated more than ± 20 % of the initial test item concentrations over the test period of 72 hours, therefore the biological results are based on the measured concentrations.
The corresponding calculated geometric mean test item concentrations (based on the analytical measurements) were NA1*, NA2*, 0.03, 0.06 and 0.16 mg/L.

* remark: not applicable; the two lowest concentrations were below the analytical detection limit (LOD = 0.01 mg/L) at the end of the test and therefore excluded from the result evaluation

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.06 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.06 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Results with reference substance (positive control):

For the evaluation of the reliability of the applied test system and experimental conditions, potassium dichromate is tested at least twice a year. The date of the last study with reference item Potassium dichromate was: September 23 - 26, 2014.
The 72h ErC50: 1.48 mg/L, (95 % confidence limits: 1.30 – 1.69 mg/L)
The 72h EyC50: 0.69 mg/L, (95 % confidence limits: 0.61 – 0.78 mg/L

Reported statistics and error estimates:

Mean values and standard deviations were calculated for each treatment at the start, 24 and 48 hours after treatment and at the end of the test using Excel for Windows software.
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 (α = 0.05) using TOXSTAT software. The data were checked for normality by Chi-Square Test, for homogeneity of variance by Hartley’s Test.
As the inhibition of growth of the algal cells was lower than 50 % regarding growth rate and yield, there was no hence to perform Probit analysis.

Validity criteria fulfilled:

yes

Conclusions:

In the 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with the test item, the 72-h EC50 value based on growth rate and yield was determined to be > 0.16 mg/L. The overall NOEC was determined to be 0.03 mg/L. The overall LOEC was determined to be 0.06 mg/L. The results are based on the measured concentrations.

Executive summary:

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours and according to OECD 201. Cultures of Pseudokirchneriella subcapitata were exposed to the test item over several generations under defined conditions. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 104 cells/ mL at the start of the test in all of the test cultures. Glass flasks with total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by microscope in each testing flask during the 72-hour test, in 24-hour intervals. In the 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with the test item, the 72-h EC50 value based on growth rate and yield was determined to be > 0.16 mg/L. The overall NOEC was determined to be 0.03 mg/L. The overall LOEC was determined to be 0.06 mg/L. The results are based on the measured concentrations.

Description of key information

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours and according to OECD 201. The test design

included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 104 cells/ mL at the start of the test in all of the test cultures. Glass flasks with total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by microscope in each testing flask during the 72-hour test, in 24-hour intervals.
In the 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with the test item, the 72-h EC50 value based on growth rate and yield was determined to be > 0.16 mg/L. The overall NOEC was determined to be 0.03 mg/L. The overall LOEC was determined to be 0.06 mg/L. The results are based on the measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.16 mg/L

EC10 or NOEC for freshwater algae:

0.03 mg/L