Formaldehyde, oligomeric reaction products with 3-aminomethyl-3,5,5-trimethylcyclohexylamine and 4-tert-butylphenol

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 February 2018 - 13 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-aminomethyl-3,5,5-trimethylcyclohexylamine

EC Number:

220-666-8

EC Name:

3-aminomethyl-3,5,5-trimethylcyclohexylamine

Cas Number:

2855-13-2

Molecular formula:

C10H22N2

IUPAC Name:

3-(aminomethyl)-3,5,5-trimethylcyclohexanamine

Constituent 2

Reference substance name:

4-tert-butylphenol

EC Number:

202-679-0

EC Name:

4-tert-butylphenol

Cas Number:

98-54-4

Molecular formula:

C10H14O

IUPAC Name:

4-tert-butylphenol

Constituent 3

Reference substance name:

2-(((5-amino-1,3,3-trimethylcyclohexyl)methylamino)methyl)-4-tert-butylphenol

Molecular formula:

C21H36N2O

IUPAC Name:

2-(((5-amino-1,3,3-trimethylcyclohexyl)methylamino)methyl)-4-tert-butylphenol

Test material form:

liquid: viscous

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L loading rate WAF
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- A nominal amount of test item (20 mg) was added to the surface of 20 liters of culture medium to give the 1.0 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. A series of dilutions was made from the 1.0 mg/L loading rate to give further stock solutions of 0.32, 0.10, 0.032 and 0.010 mg/L loading rate WAF.
An aliquot (450 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (1.1 mL) to give the required test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L loading rate WAF.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium

ACCLIMATION
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ±1 ºC

pH:

7.6 - 7.7 at test start, 7.7 - 7.9 at test end

Nominal and measured concentrations:

0.010, 0.032, 0.10, 0.32 and 1.0 mg/L loading rate WAF

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks containing 100 mL of test preparation
- Aeration: constantly shaken at approximately 150 rpm for 72 hours.
- Type (delete if not applicable): closed
- Initial cells density: 5.00E03 cells/mL
- Control end cells density: 7.25E+05 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Culture medium different from test medium: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement: no
- Other:
Samples were taken at 19, 42 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00E03 cells/mL) was taken as the starting cell density. To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.



TEST CONCENTRATIONS
- Spacing factor for test concentrations: sqrt(10)
- Range finding study
- Test concentrations: 0.010, 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study:
The results showed no effect on growth at 0.010 mg/L loading rate WAF. However, growth was observed to be reduced at 0.10, 1.0, 10 and 100 mg/L loading rate WAF.
Based on this information loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

1.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.062 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

biomass

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits 0.20 to 0.31 mg/L loading rate WAF

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control, 0.010 and 0.032 mg/L test cultures. Cell clumping and enlarged and misshapen cells were observed in the 0.10 mg/L test cultures whilst cell debris and enlarged and misshapen cells were observed in the 0.32 and 1.0 mg/L test cultures.
- Effect concentrations exceeding solubility of substance in test medium:
At both the start and end of the mixing period and following a 1-Hour standing period, the 1.0 mg/L loading rate WAF was observed to have formed a clear colorless media column with oily globules of test item floating at the media surface. Microscopic examination of the WAF showed there to be no micro-dispersions of test item present and as such the aqueous phase was removed by mid-depth siphon.
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and 0.010, 0.032 and 0.10 mg/L loading rate WAF test cultures were observed to be green dispersions. The 0.32 mg/L loading rate WAF test cultures were observed to be pale green dispersions whilst 1.0 mg/L loading rate WAF test cultures were observed to be very pale or extremely pale green dispersions.

Results with reference substance (positive control):

- Results with reference substance valid? yes
ErC50 (0 to 72 hour) : 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour) : 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).
Where appropriate 95% confidence limits for the EL50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

Any other information on results incl. tables

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 145 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Nominal cell density of control at 0 hours : 5.00E03 cells per mL Mean cell density of control at 72 hours : 7.25E05 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 7% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 3% and hence satisfied the validation

Cell Densities in the Definitive Test

Nominal Loading Rate (mg/L)		Cell Densities* (cells per mL)
		24 h	48 h	72 h
Control	R1	2.85E+04	1.39E+05	7.93E+05
	R2	2.59E+04	1.19E+05	5.60E+05
	R3	2.72E+04	1.38E+05	8.36E+05
	R4	2.95E+04	1.30E+05	8.11E+05
	R5	2.74E+04	1.34E+05	7.00E+05
	R6	2.97E+04	1.26E+05	6.51E+05
	Mean	2.80E+04	1.31E+05	7.25E+05
0.01	R1	3.26E+04	1.21E+05	6.99E+05
	R2	3.16E+04	1.26E+05	6.66E+05
	R3	3.37E+04	1.42E+05	8.64E+05
	Mean	3.26E+04	1.30E+05	7.43E+05
0.032	R1	2.80E+04	1.20E+05	6.09E+05
	R2	2.52E+04	9.75E+04	4.72E+05
	R3	2.11E+04	1.05E+05	5.44E+05
	Mean	2.48E+04	1.07E+05	5.41E+05
0.1	R1	2.77E+04	1.31E+05	6.30E+05
	R2	2.51E+04	1.27E+05	6.71E+05
	R3	3.24E+04	1.45E+05	7.51E+05
	Mean	2.84E+04	1.34E+05	6.84E+05
0.32	R1	2.17E+04	5.67E+04	3.53E+05
	R2	9.39E+03	3.01E+04	1.89E+05
	R3	1.08E+04	3.51E+04	1.92E+05
	Mean	1.40E+04	4.06E+04	2.45E+05
1	R1	1.31E+04	1.36E+04	2.60E+04
	R2	2.05E+04	5.83E+04	2.45E+05
	R3	1.78E+04	1.70E+04	8.66E+04
	Mean	1.71E+04	2.96E+04	1.19E+05

* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R = Replicate

Inhibition of Growth Rate and Yield in the Definitive Test

 

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.07	-	7.88E+05	-
	R2	0.066		5.55E+05
	R3	0.071		8.31E+05
	R4	0.071		8.06E+05
	R5	0.069		6.95E+05
	R6	0.068		6.46E+05
	Mean	0.069		7.20E+05
	SD	0.002		1.08E+05
0.01	R1	0.069	0	6.94E+05	[2]
	R2	0.068	1	6.61E+05
	R3	0.072	[4]	8.59E+05
	Mean	0.07	[1]	7.38E+05
	SD	0.002		1.06E+05
0.032	R1	0.067	3	6.04E+05	25
	R2	0.063	9	4.67E+05
	R3	0.065	6	5.39E+05
	Mean	0.065	6	5.36E+05
	SD	0.002		6.90E+04
0.1	R1	0.067	3	6.25E+05	6
	R2	0.068	1	6.66E+05
	R3	0.07	[1]	7.46E+05
	Mean	0.068	1	6.79E+05
	SD	0.002		6.18E+04
0.32	R1	0.059	14	3.48E+05	67
	R2	0.05	28	1.84E+05
	R3	0.051	26	1.87E+05
	Mean	0.053	23	2.40E+05
	SD	0.005		9.38E+04
1	R1	0.023	67	2.10E+04	84
	R2	0.054	22	2.40E+05
	R3	0.04	42	8.16E+04
	Mean	0.039	44	1.14E+05
	SD	0.016		1.13E+05

 

 

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R = Replicate

- = Not applicable

SD= Standard deviation

[ ] = Increase in growth compared to controls

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Pseudokirchneriella subcapitata to Mannich base with IPDA-PTBP gave the following results:

ErL10 (0 to 72 hour) : 0.15 mg/L loading rate WAF
ErL20 (0 to 72 hour) : 0.33 mg/L loading rate WAF
ErL50 (0 to 72 hour) : 1.2 mg/L loading rate WAF*
It was not possible to calculate 95% confidence limits for the ErL50 value as the data generated did not fit the models available for the calculation of confidence limits.

EyL10 (0 to 72 hour) : 0.062 mg/L loading rate WAF
EyL20 (0 to 72 hour) : 0.10 mg/L loading rate WAF
EyL50 (0 to 72 hour) : 0.25 mg/L loading rate WAF; 95% confidence limits 0.20 to 0.31 mg/L loading rate WAF

Executive summary:

A study was performed to assess the effect of Mannich base with IPDA-PTBP on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.0016 to 0.15 mg/L. A decline in measured test concentrations was observed at 72 hours in the range of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.00021 mg/L, to 0.042 mg/L indicating that the test item was unstable under the conditions of the test.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

 

ErL10 (0 to 72 hour) : 0.15 mg/L loading rate WAF

ErL20 (0 to 72 hour) : 0.33 mg/L loading rate WAF

ErL50 (0 to 72 hour) : 1.2 mg/L loading rate WAF*

It was not possible to calculate 95% confidence limits for the ErL50 value as the data generated did not fit the models available for the calculation of confidence limits.

 

EyL10 (0 to 72 hour) : 0.062 mg/L loading rate WAF

EyL20 (0 to 72 hour) : 0.10 mg/L loading rate WAF

EyL50 (0 to 72 hour) : 0.25 mg/L loading rate WAF; 95% confidence limits 0.20 to 0.31 mg/L loading rate WAF