Cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked

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:

2012-05-04 to 2012-05-18

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The saturated solution, further dilution levels and the control were verified at the beginning and the end of the test via LC-MS/MS analysis.
- Sampling method: At test start samples were taken out of additional replicates provided without algae. At the end of the test samples were taken from pooled replicates.
- Sample storage conditions before analysis: All stabilized samples were stored at 6 ± 2 °C until sample preparation.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution with a nominal loading of 3.9 mg test item/L was prepared with dilution water three days prior to application. The test item dispersion was stirred for three days with a magnetic stirrer at 1100 rpm at room temperature. After a separation of the phases after 1 min of standing, the saturated solution was taken from the homogeneous phase.

- Eluate: Dilutioon water
- Differential loadings: 226 - 399 - 638 - 1268 - 2088 µg/L (geom mean measured)
- Controls: Six replictes without test item were tested under the same test conditions as the test replicates.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDAK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Not the same, standard growth medium
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not measured

Test temperature:

Mean: 22.8 °C, Min: 22.5 °C, Max: 23.0 °C

pH:

Geometric mean measured
test item concentration pH-value
[µg/L] Start; 0 h End; 72 h
2088 7.83 9.32
1268 7.90 9.29
638 7.92 9.41
399 7.93 9.40
226 7.94 9.43
Control 7.92 9.36

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to information on materials and methods

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): sealed with cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 5466 cells/mL
- Control end cells density: Mean 2139925 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: Yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
According to the guideline (OECD)

Composition of Dilution water (OECD)

Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  2 H2O 1 x 10-5
NaHCO3 50
pH-value 8.1  0.2
This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0 – 72 h)


Saturated solution (nominal)
[mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 9 38
10 -1 -4

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2 088 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2 088 µg/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):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.718 (CI 0.699 - 0.737) mg/L
Yield Inhibition: 0.422 (CI 0.386 - 0.466) mg/L

Reported statistics and error estimates:

NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

NOEC, LOEC and EC-Values (0-72 hours)

based on geometric mean measured test item concentration [mg/L] 

	Rate-related inhibition
NOEC	2088
LOEC	> 2088
ErC10	> 2088
ErC20	> 2088
ErC50	> 2088
	Inhibition of yield
NOEC	2088
LOEC	> 2088
EyC10	1335 (544 - > 2088)
EyC20	> 2088
EyC50	> 2088

Cell Densities

Geometric mean measured test item concentration	Replicate	Cell density [cells/mL]
[µg/L]	No.	0 hours	24 hours	48 hours	72 hours
2088	1	5466	18511	175631	1465644
	2	5466	21022	206579	1979843
	3	5466	19785	222119	2062240
	Mean	5466	19773	201443	1835909
1268	1	5466	19945	209261	1818885
	2	5466	18757	194212	1781933
	3	5466	17937	235671	2141768
	Mean	5466	18880	213048	1914195
638	1	5466	22303	234419	2219360
	2	5466	21346	240730	1993700
	3	5466	21975	251741	2131189
	Mean	5466	21875	242297	2114750
399	1	5466	20429	220592	2055348
	2	5466	21148	268168	2221483
	3	5466	20940	270798	2010648
	Mean	5466	20839	253186	2095826
226	1	5466	23115	255026	2024990
	2	5466	25238	297171	2485586
	3	5466	23160	305768	2182222
	Mean	5466	23838	285988	2230933
Control	1	5466	20955	233451	1839261
	2	5466	19871	277317	2174362
	3	5466	23041	335561	2017502
	4	5466	25838	314648	2163150
	5	5466	21990	304099	2363555
	6	5466	23756	288983	2281717
	Mean	5466	22575	292343	2139925

Evaluation after 72 hours

                                Statistically significant differences of growth rates and yield compared to

                                control values are marked (+), not significant differences are marked (-).

Geometric mean measured test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[µg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
2088	1		1.86	6.29		1460178	31.6
	2		1.96	1.25		1974377	7.50
	3		1.98	0.56		2056774	3.64
	Mean	(-)	1.94	2.70	(-)	1830443	14.2
1268	1		1.94	2.67		1813419	15.0
	2		1.93	3.01		1776467	16.8
	3		1.99	-0.07		2136302	-0.09
	Mean	(-)	1.95	1.87	(-)	1908729	10.6
638	1		2.00	-0.67		2213894	-3.72
	2		1.97	1.13		1988234	6.85
	3		1.99	0.01		2125723	0.41
	Mean	(-)	1.99	0.16	(-)	2109284	1.18
399	1		1.98	0.62		2049882	3.96
	2		2.00	-0.68		2216017	-3.82
	3		1.97	0.99		2005182	6.06
	Mean	(-)	1.98	0.31	(-)	2090360	2.07
226	1		1.97	0.87		2019524	5.38
	2		2.04	-2.57		2480120	-16.2
	3		2.00	-0.38		2176756	-1.98
	Mean	(-)	2.00	-0.69	(-)	2225467	-4.26
Control	1		1.94			1833795
	2		2.00			2168896
	3		1.97			2012036
	4		1.99			2157684
	5		2.02			2358089
	6		2.01			2276251
	Mean		1.99			2134459

Section-by-Section and Average Specific Growth Rates of the Control Group
(0 – 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.34	2.41	2.06	1.94	0.544	28.1	29.1
	2	1.29	2.64	2.06	2.00	0.675	33.8
	3	1.44	2.68	1.79	1.97	0.639	32.4
	4	1.55	2.50	1.93	1.99	0.477	23.9
	5	1.39	2.63	2.05	2.02	0.618	30.5
	6	1.47	2.50	2.07	2.01	0.517	25.7
				Mean	1.99
				SD ±	0.03
				CV [%]	1.51

Validity criteria fulfilled:

yes

Conclusions:

In this study VESTANAT B 1370 (solvent free) was not found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata significantly, when tested with the saturated solution and further dilution levels. The effect values after 72 hours (geometric mean measured test item concentrations) were: The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 hours were > 2088 µg/L for both endpoints. The NOEC-values for inhibition of specific growth rate and yield after 72 hours were 2088 µg/L (highest test concentration) for both endpoints. All effect values are given based on geometric mean measured concentrations of VESTANAT B 1370 (solvent free).

Executive summary:

The toxicity of Cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked  to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 from 2012-05-04 to with the definitive exposure phase from to at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany. The aim of the study was the determination of NOEC, LOEC, EC₁₀ - , EC₂₀- and EC₅₀ - values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5466 cells/mL. A saturated solution* (i.e. 100 %) and further four dilution levels of 9.96 - 17.7 - 31.6 - 56.2 % of the saturated solution were tested, corresponding to geometric mean measured test item concentrations of 226 - 399 – 638 – 1268 – 2088 µg/L. Three replicates for each dilution level and six replicates for the control were tested. Environmental conditions were determined to be within the acceptable limits.

The concentrations of VESTANAT B 1370 (solvent free) were analytically verified by LC-MS/MS analysis at test start and test end of all dilution levels and the control. The measured concentrations of VESTANAT B 1370 (solvent free) at test start were in the range of 180 – 2154 µg/L. The measured concentrations at the end of the test were in the range of 283 – 2024 µg/L. All effect values given are based on the geometric mean measured concentrations of the test item VESTANAT B 1370 (solvent free).

Table 1:    NOEC, LOEC and EC-Values (0-72 hours)

based on geometric mean measured test item concentration [µg/L] 

	Rate-related inhibition
NOEC	2088
LOEC	> 2088
ErC10	> 2088
ErC20	> 2088
ErC50	> 2088
	Inhibition of yield
NOEC	2088
LOEC	> 2088
EyC10	1335 (544 - > 2088)
EyC20	> 2088
EyC50	> 2088

Description of key information

The toxicity of Cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201. The aim of the study was the determination of NOEC, LOEC, EC10 - , EC20- and EC50 - values of growth rate and yield over a period of 72 hours.
The study was conducted under static conditions with a saturated solution* (i.e. 100 %) and further four dilution levels of 9.96 - 17.7 - 31.6 - 56.2 % of the saturated solution, corresponding to geometric mean measured test item concentrations of 226 - 399 – 638 – 1268 – 2088 µg/L.
The concentrations of the test item were analytically verified by LC-MS/MS analysis at test start and test end of all dilution levels and the control. The measured concentrations at test start were in the range of 180 – 2154 µg/L. The measured concentrations at the end of the test were in the range of 283 – 2024 µg/L. All effect values given are based on the geometric mean measured concentrations of the test item.

Key value for chemical safety assessment

EC50 for freshwater algae:

2 088 µg/L

EC10 or NOEC for freshwater algae:

2 088 µg/L

Additional information

In this study the test item was not found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata significantly, when tested with the saturated solution and further dilution levels. The effect values after 72 hours (geometric mean measured test item concentrations) were: The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 hours were > 2088 µg/L for both endpoints. The NOEC-values for inhibition of specific growth rate and yield after 72 hours were 2088 µg/L (highest test concentration) for both endpoints.