Hexamethylene diisocyanate, trimers, reaction products with 2-hydroxyethyl acrylate

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:

29 Aug 2016 - 13 Apr 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006, corrected 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Germany (10 Dec 2015)

Analytical monitoring:

yes

Remarks:

HPLC-MS/MS

Details on sampling:

- Concentrations: All test item loading rates and control at 0 h (fresh media) and 72 h (aged media).
- Sampling method: For each sampling a retain sample was taken. Additionally, samples were taken from the stock solution at test start.
- Sample storage conditions before analysis: Deep-frozen (≤ - 18 °C) for a maximum storage period of 16 d.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Five stock solutions were prepared by directly weighing appropriate amounts of the test item into test medium and stirring in the dark for 24 h at room temperature according to OECD 23. Then, the undissolved test item was allowed to sediment and/or float for 0.5 h until the phases separated. After the settling, the necessary volumes for the test were withdrawn via a Teflon tube from the medium level of the stock solution. Approximately 50 mL of the prepared solutions were transferred to each test vessel.
- Differential loading: yes
- Controls: yes, untreated test medium
- Evidence of undissolved material: The stock solutions were clear and transparent but not the entire test item went into solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Single cell green alga
- Strain: SAG 61.81
- Source: MBM Sciencebridge GmbH, Göttingen, Germany (commercial supplier)
- Age of inoculum (at test initiation): 3 - 4 d
- Method of cultivation: Algae are grown as liquid, semi-continuous sterile cultures using 100 mL Erlenmeyer flasks. Old medium is periodically replaced by fresh mineral solution to maintain exponential growth. Stock cultures are regularly ordered from the commercial supplier.
- Culture conditions: Continuous illumination (60 - 120 µE*m^-2*s^-1), Temperature: 21 - 24 °C, CO2 supply: Shaking on rotation shaker (ca. 105 rpm)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.4 - 22.8 °C ( 0 h)
22.6 - 22.8 °C (72 h)

pH:

7.62 - 7.73 (0 h)
7.43 - 7.54 (72 h)

Nominal and measured concentrations:

Control, 0.954, 3.05, 9.77, 31.3, and 100 mg/L (nominal)
Control, 0.0306, 0.0253, 0.0654, 0.212, and 0.256 mg/L (geometric mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks with aluminium caps filled with 50 mL test solution.
- Initial mean cell density: 0.55*10E+04 cells/mL
- Control end cell density: 37.25*10E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Culture medium: mineral solution; Test medium: AAP-medium according to OECD 201
- Intervals of water quality measurement: pH was measured at 0 h and 72 h. Temperature was measured continuously. Light intensity was determined at test start.

OTHER TEST CONDITIONS
- Adjustment of pH: The test medium (AAP) was adjusted to 7.5 ± 0.1.
- Photoperiod: continuous illumination
- Light intensity and quality: 88.8 µE*m^-2*s^-1

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Daily, with a fluorimeter (infinite 200Pro) at an emission wavelength of 670 nm
- Morphological appearance: Observed at the end of the test by micrcoscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: yes, non-GLP
- Test concentrations: 0.1, 1.0, 10.0, and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes. No inhibitory effects on growth rate and yield were observed after 72 h.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EL10

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.:

> 0.256 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

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.:

> 0.256 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

9.77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.065 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and biomass

Remarks on result:

other:

Remarks:

based on the geometric mean of each measured concentration

Duration:

72 h

Dose descriptor:

EL10

Basis for effect:

biomass

Remarks on result:

not determinable

Remarks:

Due to an inhibition below 50% and a missing concentration response relation the data set was inappropriate for probit analysis, which hence was not performed.

Duration:

72 h

Dose descriptor:

EL50

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.:

> 0.256 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: Yes, cell numbers between 0 and 72 h were found to increase by a factor of 67.73 for the control, which corresponds to a growth rate of 1.40808 d^-1.
- Observation of abnormalities: No
- Any observations that might cause a difference between measured and nominal values: The prepared stock solutions were clear and transparent but not the entire test item went into solution.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the validity criteria were fulfilled.
- ErC50 (72 h) = 1.64 mg/L (nominal)
- Other: The last routine toxic reference test with potassium dichromate dates from 15 - 18 Nov 2016.

Reported statistics and error estimates:

The statistical evaluation was performed with SAS. A test for normality of the data was performed by calculating the Shapiro-Wilk statistic. The homogeneity of variance of the data was evaluated by calculating the Levene Test. The NOELR and LOELR as well as LOEC were determined by using a multiple comparison method (Dunnetts-t-test, left sided). The calculation of the EL10, 20, 50 as well as the EC10, 20, 50 was not indicated due to a missing concentration-response-relation and since the inhibition was below 50% at all test item loading rates for both yield and growth rate.

ANALYTICAL RESULTS

The measured initial concentration of the test item ranged from 0.293 - 4.35% of nominal. In the aged samples the measured content was 0.244 - 2.37% of nominal (Table 1). Therefore, toxicological endpoints were based both on nominal loading rates and the actual concentrations (geometric mean measured) of the test item.

Table 1. Determined test item concentration.

Nominal test item loading rate [mg/L]	Sampling time [h]	Test item found			Actual test item concentration [mg/L]
		[mg/L]	% of nominal	Geometric mean [%)
Control	0	n.d.	-	-	-
	72	n.d.	-
0.954	0	0.0415	4.35	3.21	0.0306
	72	0.0226	2.37
3.05	0	0.0310	1.02	0.828	0.0253
	72	0.0205	0.672
9.77	0	0.0637	0.652	0.669	0.0654
	72	0.0671	0.687
31.3	0	0.255	0.815	0.667	0.212
	72	0.176	0.562
100	0	0.293	0.293	0.256	0.256
	72	0.224	0.224

 

BIOLOGICAL RESULTS

For the inhibition of growth rate and yield no concentration response relation was observed. The inhibition of growth rate peaked at 7.3% at a nominal loading rate of 31.3 mg/L (Table 3) and the inhibition of yield peaked at 26.0% at a nominal loading rate of 31.3 mg/L (Table 4).

Appropriate statistical analysis revealed significant inhibitory effects on growth rate and yield at test item loading rates of 31.3 mg/L (nominal), corresponding to 0.212 mg/L (actual concentration). The overall LOELR was therefore 31.3 mg/L (nominal) or 0.212 mg/L (actual). The NOELR was 9.77 mg/L (nominal), corresponding to a NOEC of 0.0654 mg/L (actual). The absence of a dose-response relationship and the observed maximum inhibition below 50% did not allow for a statistically reliable determination of EL50 values for inhibition of growth rate and yield.

Due to the absence of effects at a nominal loading rate of 100 mg/L, the observed statistically significant inhibition of growth rate and yield at a nominal loading rate of 31.3 mg/L is more likely due to biological variance than related to test item effects. This is supported by the analytical results, which confirmed a higher amount of test item at a nominal loading rate of 100 mg/L than at a nominal loading rate of 31.3 mg/L. Additionally, the results of the non-GLP range-finding test showed no inhibitory effects on growth rate and yield at nominal loading rates of 31.3 and 100 mg/L.

In conclusion, the statistically observed significant effect at the nominal loading rate of 31.3 mg/L is not considered to be related to the substance itself but is likely to be caused by natural biological variance.

  

Table 2. Cell numbers.

Nominal loading rate [mg/L]	Cell numbers [10^4/mL]				Yield
	0 h	24 h	48 h	72 h	0 – 72 h
Control	0.50	1.89	8.82	32.19	31.69
	0.50	1.88	8.64	38.59	38.09
	0.62	1.16	8.13	38.85	38.23
	0.60	1.03	6.65	38.63	38.03
	0.62	1.85	8.48	37.46	36.84
	0.45	1.58	7.48	37.79	37.34
Mean	0.55^1)	1.57	8.03	37.25	36.70
0.954	0.55	1.88	9.39	40.48	39.93
	0.55	1.72	8.96	32.49	31.94
	0.55	2.34	10.24	45.54	44.99
Mean	0.55	1.98	9.53	39.50	38.95
3.05	0.55	1.93	8.43	38.67	38.12
	0.55	2.23	9.20	41.23	40.68
	0.55	1.95	8.59	34.74	34.19
Mean	0.55	2.04	8.74	38.21	37.66
9.77	0.55	1.69	8.21	30.11	29.56
	0.55	2.09	9.47	35.23	34.68
	0.55	2.02	9.64	38.49	37.94
Mean	0.55	1.93	9.11	34.61	34.06
31.3	0.55	1.49	7.48	27.63	27.08
	0.55	1.91	7.97	30.87	30.32
	0.55	1.81	8.14	24.57	24.02
Mean	0.55	1.74	7.86	27.69	27.14
100	0.55	1.17	7.74	30.86	30.31
	0.55	2.04	8.29	35.27	34.72
	0.55	1.89	8.34	32.12	31.57
Mean	0.55	1.70	8.12	32.75	32.20

1) The mean cell density of all control replicates is used as initial cell density for all treatment groups.

 

 Table 3. Percentage inhibition of growth rate.

Loading rate [mg/L]	% inhibition of growth rate
	0 – 24 h	0 – 48 h	0 – 72 h
Control	0.0	0.0	0.0
0.954	-23.7	-6.1	-1.0
3.05	-27.1	-2.9	-0.3
9.77	-21.8	-4.3	2.1
31.3	-11.3	1.0	7.3*
100	-7.0	-0.2	3.3

*statistically significantly different from control

 

 Table 4. Percentage inhibition of yield.

Loading rate [mg/L]	% inhibition of growth rate
	0 – 24 h	0 – 48 h	0 – 72 h
Control	0.0	0.0	0.0
0.954	-40.2	-19.9	-6.4
3.05	-46.1	-9.3	-2.6
9.77	-35.3	-14.3	7.2
31.3	-16.7	2.4	26.0*
100	-12.7	-1.1	12.3

*statistically significantly different from control

 

Validity criteria fulfilled:

yes

Description of key information

EL10 (72 h) > 100 mg/L (nominal, OECD 201, P. subcapitata)

EL50 (72 h) > 100 mg/L (nominal, OECD 201, P. subcapitata)

Additional information

There is one experimental study available, in which the toxicity of Hexamethylene diisocyanate, trimers, reaction products with 2-hydroxyethyl acrylate (CAS 162492-01-5) was assessed according to the OECD guideline 201 and GLP.

In a static test, Pseudokirchneriella subcapitata was exposed to the five nominal loading rates of 0.954, 3.05, 9.77, 31.3, and 100 mg/L test item for 72 h (initial cell density: 0.5 * 10E+04 cells/mL). The actual test item concentrations were analytically verified by HPLC-MS/MS at 0 h (fresh test solution) and 72 h (aged test solution).

The measured initial test item concentration in fresh test solutions at 0 h ranged from 0.293 – 4.35% of nominal. In the aged samples, the measured test item content ranged between 0.224 – 2.37% of nominal. Therefore, the toxicological endpoints were based both on the nominal loading rates and the geometric mean measured concentrations of the test item.

After 72 h, no dose-response curve was obtained for inhibition but statistically significant inhibition of growth rate and yield was determined at the test item loading rate of 31.3 mg/L (nominal), corresponding to an actual concentration of 0.212 mg/L (geometric mean measured). The absence of a dose-response relationship and the observed maximum inhibition well below 50% did not allow for a statistically reliable determination of EL10 and EL50 values for inhibition of growth rate and yield (i.e. the inhibition of growth rate peaked at 7.3%). Since no effects were observed at a nominal loading rate of 100 mg/L, this obtained statistically significant inhibition at 31.3 mg/L is assumed to more likely result from natural biological variance than from intrinsic effects of the test item. This is also supported by the analytical results, which show a higher actual test item concentration at the nominal loading rate of 100 mg/L compared to that at 31.3 mg/L (nominal). Furthermore, the non-GLP range-finding test did not record any inhibitory effects on growth rate or yield after 72 h either.

In conclusion, the statistically significant inhibition observed at the nominal loading rate of 31.3 mg/L is not considered to be related to the substance itself, resulting in a NOELR/NOEC (72 h) of 9.77 mg/L (nominal), corresponding to 0.256 mg/L (geometric mean measured concentrations) and in an ErL10 (72 h)/ErL50 (72 h) of > 100 mg/L (nominal) and > 0.256 mg/L (geometric mean measured concentrations). Thus, no effects are anticipated up to the limit of water solubility of the substance.