Reaction products of cyclohexylamine, alkan-1-amine and 1,1'-methanediylbis(4-isocyanatobenzene)

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:

From 2008-11-24 to 2009-01-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

analysis to confirm test substance concentration not carried out

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

analysis to confirm test substance concentration not carried out

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch No.: 2008-9-16
Purity: >95.0%

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The method of preparation used during the definitive test was based on the results of a range finding test and guidance given in OECD Monograph 23: ‘Guidance document on aquatic toxicity testing of difficult substances and mixtures’. The test substance (200 mg) was mixed with Dimethylsulphoxide (DMSO; 0.1 mL) and rinsed a volumetric flask with OECD medium. These aqueous mixtures were stirred overnight before being left to stand for four hours. An aliquot (1000 mL) was removed midvessel from the mixture and used in the test as a water accommodated fraction (WAF).

- Controls: As an intermediate vehicle was used to facilitate the preparation of the test medium, an additional control group containing the solvent (DMSO) and dilution water (0.1 mL/L) was included in the study. The solvent control vessel was filled with dilution medium containing the same concentration of auxiliary substance as present in the test concentration.
- Chemical name of vehicle: Dimethyl sulphoxide
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.1 mL
- Evidence of undissolved material (e.g. precipitate, surface film, etc): At the start of the test, the test medium was a cloudy (white) homogenous dispersion.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Dunstaffnage Marine
Laboratory, Dunbeg, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): 6 days (3 days primary culture + 3 days secondary culture)
- Method of cultivation: The liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures (100 mL) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth, characterised by a cell density of 0.85 E+6 cells/mL.


ACCLIMATION
- Acclimation period: not reported
- Culturing media and conditions (same as test or not): Sterile algal nutrient medium as recommended in Official Journal No. L383A Part C.3 and
OECD Procedure 201
- Any deformed or abnormal cells observed: No microscopic abnormalities of the cells were detected.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

21.6 - 22.6 °C

pH:

0 h: 7.50 - 7.52
72 h: 9.69-9.78

Nominal and measured concentrations:

Nominal: 100 mg/L (WAF)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): the required number of empty test vessels (250 mL conical flasks), were loosely stoppered with foam bungs, covered with aluminium foil that was secured by autoclave tape and sterilised by autoclaving (121°C for at least 15 minutes). After the addition of the inoculated test medium (100 mL), each flask was then loosely plugged with a foam bung.
- Material, size, headspace, fill volume: fill volume 100mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 0.85 E+6
- Control end cells density: 156.50 E+4 cells/mL
- No. of organisms per vessel: not stated
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: filtered, dechlorinated tap water which had been softened and treated by reverse osmosis, before microfiltration and purification
- Conductivity: resistivity of 18 Megohm/cm


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: none
- Photoperiod: continuous illumination
- Light intensity and quality: 6190 (mean) lux provided by 6 x 30 W “cool white” 1 metre fluorescent tubes


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell densities were measured using a haemacytometer (Improved Neubauer) at 24, 48 and 72 hours .


TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable
- Justification for using less concentrations than requested by guideline: limit test
- Range finding study
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: After 72 hours, algal growth was not inhibited at 100 mg/L

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No microscopic abnormalities of the cells were detected.
- Effect concentrations exceeding solubility of substance in test medium: water accomodated fractions were used

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The data were compiled in an Excel spreadsheet and analysed using SAS 8.2 (SAS Institute 1999).
For AUC and growth rate, Williams'test (1971, 1972) was also used to compare each treated group with solvent control unless there was evidence of a non-monotonic dose-response relationship, in which case Dunnett's test (1955, 1964) was used.

Limit test - algal growth

Individual cell densities for each culture were determined. The calculated area under the growth curve and average specific growth rate values are are expressed in terms of percentage inhibition by comparing the test group value with that of the solvent control curve. The test results have been expressed in terms of nominal concentrations of test item used to prepare initial aqueous mixture from which the WAF was prepared.

The mean coefficient of variation (CoV) for daily growth rates in solvent control cultures ranged between 4.4 and 10.2 during the definitive test and the CoV for the average specific growth rates of solvent control culture was 1.8 during the 72 hour exposure period.

Results

Table 1           Cell densities

 

 

Exposure concentrations (mg test item/L) Nominal#	Replicate number	Cell densities (1E+04 cells/ml)
		24 hours	48 hours	72 hours
Control	R1 R2 R3 R4 R5 R6   Mean	8.5 8.38 7.25 6.25 7.00 6.38   7.29	32.25 34.50 34.88 34.75 36.88 31.25   34.58	165 25 155.75 174.00 142.00 155.50 146.50   156.50
Solvent control	R1 R2 R3 R4 R5 R6   Mean	7.00 8.38 7.38 8.25 7.13 8.63   7.79	39.5 30.75 33.13 34.88 37.63 42.00   36.31	168.75 131.25 150.00 139.00 139.25 137.00   144.21
100	R1 R2 R3 R4 R5 R6   Mean	8.13 7.38 6.38 7.13 7.13 7.38   7.25	30.75 34.75 31.25 35.75 29.50 30.88   32.15	154.75 123.75 117.25 152.75 132.75 139.00   136.71

 

R1-R6: replicate number.

# :Nominal concentration of the aqueous mixture from which the WAF was prepared

Note : the initial cell density was estimated to be 1 E+4 /mL.

Table 2 Cell densities

Parameter	Concentration #	Sample size	Mean	% Inhibition	p
Area under curve to 72	Control	6	28.2	-3.4	0.423
hours	Solvent control	6	27.3	0.0	-
	100 mg/L	6	25.3	7.4	0.096
Growth rate to 72 hours	Control	6	0.070	-1.7	0.144
	Solvent control	6	0.069	0.0	-
	100 mg/L	6	0.068	1.1	0.322
Rate 0 to 24 hours	Control	6	0.082	3.4	0.255
	Solvent control	6	0.085	0.0	-
	100 mg/L	6	0.082	3.5	0.248
Rate 24 to 48 hours	Control	6	0.065	-1.7	0.740
	Solvent control	6	0.064	0.0	-
	100 mg/L	6	0.062	3.1	0.540
Rate 48 to 72 hours	Control	6	0.063	-9.2	0.063
	Solvent control	6	0.058	0.0	-
	100 mg/L	6	0.060	-4.6	0.331

Environmental parameters

The measurements of water quality (temperature and pH) in control and test flasks were recorded; they remained within acceptable limits throughout the study although the pH of the control and test cultures increased by more than 1.5 pH units. Since the validity criteria for this type of study were met by the control cultures, this increase in pH is not thought to have affected either the validity or integrity of the study. The temperature of the incubator ranged between: 22.1 to 23.8°C. At the start of the test, the test medium was a cloudy (white) homogenous dispersion.

Validity criteria fulfilled:

not applicable

Remarks:

limit test

Conclusions:

After 72 hours of exposure to test item, neither the EbC50 nor ErC50 could be determined and so can only be stated as being >100 mg/L*.
The "no observed effect concentration" (NOEC) for both area under the growth curve and growth rate was 100 mg/L*.
* : Nominal concentrations of the aqueous mixtures from which the WAFs were prepared.

Executive summary:

The effect of test item on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed under non-axenic conditions. The study was conducted in accordance with EC C3 and OECD 201. Six algal cultures, with an initial cell density of 1 E+04/mL, were exposed to water accommodated fractions of test item prepared from an aqueous mixture with an initial nominal concentration of 100 mg/L. The test media were prepared in OECD medium to aid dissolution/dispersion, a solvent (Dimethylsulphoxide) and overnight stirring were employed. Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the area under the growth curve and growth rate.

After 72 hours of exposure to test item, neither the EbC50 nor ErC50 could be determined and so can only be stated as being >100 mg/L*.

The "no observed effect concentration" (NOEC) for both area under the growth curve and growth rate was 100 mg/L*.

* : Nominal concentrations of the aqueous mixtures from which the WAFs were prepared.

Description of key information

Conducted in accordance with OECD Guideline 201 (Alga, Growth Inhibition Test) and EU Method C.3 (Algal Inhibition test).

72h ErC50 >100 mg/L, NOEC = 100 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L