Reaction products of 4,4'-methylenebis(cyclohexylamine) and 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter containing undissolved residue was
kept for possible analysis.

Frequency at t=0 h, t=24 h and t=72 h
Volume 50 mL
Storage Samples were stored in a freezer (≤-15°C) until analysis.

Details on test solutions:

Preparation of test solutions started with loading rates individually prepared at 0.46, 1.0, 2.2,
4.6 and 10 mg/L. The M2 medium used was heated to 70°C before the test item was added.
A 3- day period of magnetic stirring was applied to accelerate dissolution of the test item in
medium. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were filtered
through a 0.45 μm membrane filter (RC55, Whatman) and used as test concentrations.
After preparation, volumes of 50 mL were added to each replicate of the respective test
concentration. Subsequently, 1 mL of an algal suspension was added to each replicate
providing a cell density of 104 cells/mL. All test solutions were clear and colorless at the end
of the preparation procedure.

Test organisms (species):

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

Details on test organisms:

Species Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source In-house laboratory culture.
Reason for selection This system is an unicellular algal species sensitive to toxic
items in the aquatic ecosystem and has been selected as an
internationally accepted species.
Stock culture Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. Light intensity 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Stock culture Algae stock cultures were started by inoculating growth
medium with algal cells from a pure culture on agar. The
suspensions were continuously aerated and exposed to light in a
climate room at a temperature of 21-24°C.
Light intensity 60 to 120 μE/m2/s when measured in the photosynthetically
effective wavelength range of 400 to 700 nm.
Stock culture medium M1; according to the NPR 6505 (“Nederlandse Praktijk
Richtlijn no. 6505”) formulated using Milli-RO water (tapwater
purified by reverse osmosis; Millipore Corp., Bedford,
Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L
Pre-culture 3 days before the start of the test, cells from the algal stock
culture were inoculated in culture medium at a cell density of 1
x 104 cells/mL. The pre-culture was maintained under the same
conditions as used in the test. The cell density was measured
immediately before use.
Pre-culture medium M2; according to the OECD 201 Guideline, formulated using
Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 μg/L
Na2EDTA.2H2O 100 μg/L
H3BO3 185 μg/L
MnCl2.4H2O 415 μg/L
ZnCl2 3 μg/L
CoCl2.6H2O 1.5 μg/L
CuCl2.2H2O 0.01 μg/L
Na2MoO4.2H2O 7 μg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

pH:

8.1

Nominal and measured concentrations:

PACM BADGE adduct WAFs prepared at loading rates of 0.46, 1.0, 2.2, 4.6 and 10 mg/L Controls Test medium without test item or other additives. Replicates 3 replicates of each test concentration.
6 replicates of the control. 1 or 5 replicates of each test concentration without algae. 3 or 5 replicates of each test concentration for sampling purposes at 24 hours of exposure.

The expected EL50 for growth rate and yield inhibition was between loading rates of 1.0 and
10 mg/L. Based on these results samples taken from the WAF prepared at loading rates of 1.0
and 10 mg/L were analyzed. The initially measured concentrations were 0.41 and 0.45 mg/L,
respectively. It should be noted that these concentration were obtained by extrapolation of the
calibration curve. The concentration measured in WAF of 10 mg/L decreased to 63% of
initial at the end of the exposure period. In the lower WAF an unexpected increase of
concentration was observed as the concentrations become slightly turbid.

Details on test conditions:

Test duration 72 hours
Test type Static
Test vessels 100 mL, all-glass, containing 50 mL of test solution
Medium M2
Cell density An initial cell density of 1 x 104 cells/mL.
Illumination Continuously using TLD-lamps with a light intensity
93 μE.m- 2.s-1.
Incubation Capped vessels were distributed at random in the incubator and
daily repositioned. During incubation the algal cells were kept
in suspension by continuous shaking.

Reference substance (positive control):

yes

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

4.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.49 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Growth rate inhibition increased with applied dose from 16% at the lowest loading rate to 94% at the highest. Statistically significant inhibition of growth rate was found in all test item treated groups, hence the NOEL for the growth rate inhibition was below 0.46 mg/L. The EL10 can be used as an alternative to NOEL. In this study EL10 was calculated to be 0.90 mg/L. Inhibition of yield increased with increasing concentration of PACM BADGE adduct from 55% at the loading rate of 0.46 mg/L upwards resulting in 100% inhibition at loading rate of 10 mg/L. Statistically significant inhibition of yield was found at all loading rates tested. Neither the NOEL nor the EL0 for yield inhibition could not be determined based on this study.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a loading rate of 4.6 mg/l when compared to the control.

Samples taken from all groups were analyzed. Despite relatively high water solubility provided by the sponsor, the measured concentration were below 1.0 mg/L at the start of the test, with an exception for the WAF prepared at 2.2 mg/L where 2.2 mg/L was measured. The measured concentrations did not show dose-related increase at the start of the test. Moreover, an increase of test concentrations was observed in time. As clear dose-related effects were observed on algal growth and the measured concentrations did not reflect this trend, it was decided to express the effect parameters in terms of loading rates initially prepared.

Results with reference substance (positive control):

Potassium dichromate inhibited growth rate of this fresh water algae species at nominal
concentrations of 0.56 mg/L and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 1.2 mg/L with a 95% confidence
interval ranging from 1.1 to 1.2 mg/L. The historical ranges for growth rate inhibition lie
between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds
with this range.
The EC50 for yield inhibition (72h-EYC50) was 0.43 mg/L with a 95% confidence interval
ranging from 0.42 to 0.44 mg/L. The historical ranges for yield inhibition lie between 0.43
and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested corresponds with this range.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, PACM BADGE adduct reduced growth rate and inhibited the yield of this fresh water algae species significantly at a loading rate of 0.46 mg/L and higher.
The EL50 for growth rate inhibition (72h-ERC50) was 4.4 mg/L with a 95% confidence interval ranging from 3.9 to 5.1 mg/L.
The EL50 for yield inhibition (72h-EYC50) was 0.49 mg/L with a 95% confidence interval ranging from 0.36 to 0.61 mg/L.
The 72h-NOEL for growth rate inhibition and yield inhibition was lower than 0.46 mg/L.

Executive summary:

Under the conditions of the present study with Pseudokirchneriella subcapitata, PACM BADGE adduct reduced growth rate and inhibited the yield of this fresh water algae species

significantly at a loading rate of 0.46 mg/L and higher.

The EL50 for growth rate inhibition (72h-ERC50) was 4.4 mg/L with a 95% confidence interval ranging from 3.9 to 5.1 mg/L.

The EL50 for yield inhibition (72h-EYC50) was 0.49 mg/L with a 95% confidence interval ranging from 0.36 to 0.61 mg/L.

The 72h-NOEL for growth rate inhibition and yield inhibition was lower than 0.46 mg/L.

Description of key information

Under the conditions of the present study with Pseudokirchneriella subcapitata, PACM BADGE adduct reduced growth rate and inhibited the yield of this fresh water algae species significantly at a loading rate of 0.46 mg/L and higher.

The EL50 for growth rate inhibition (72h-ERC50) was 4.4 mg/L with a 95% confidence interval ranging from 3.9 to 5.1 mg/L.

The EL50 for yield inhibition (72h-EYC50) was 0.49 mg/L with a 95% confidence interval ranging from 0.36 to 0.61 mg/L.

The 72h-NOEL for growth rate inhibition and yield inhibition was lower than 0.46 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

3.9 mg/L

EC10 or NOEC for freshwater algae:

0.46 mg/L