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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Apr - 04 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

17 Jul 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 126 Short Guidance On The Threshold Approach for Acute Fish Toxicity, ENV/JM/MONO (2010)17

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment No. 23

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Germany (10 Oct 2013)

Analytical monitoring:

yes

Remarks:

HPLC-MS/MS

Details on sampling:

- Concentrations: Control, 100 mg/L
- Sampling method: Analytical samples were taken in 24 h intervals from fresh and aged test solutions. Retain samples were also taken.
- Sample storage conditions before analysis: The samples (10 mL) were stored at ≤ 18 °C until analysis. The maximum storage period from sampling to analysis was 10 d.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item is a mixture of compounds with partly low water solubility. Therefore, the test item was applied as water soluble fraction (WSF), which was prepared as follows: A stock solution of 200 mg test item in 2000 mL test medium was stirred at room temperature in the dark for 48 h. Then, undissolved test item was allowed to sediment for 1 h and the WSF was withdrawn from the middle of the vessel (according to OECD 23). The withdrawn WSF was used as application solution. This procedure was repeated after 24, 48, 72, and 96 h.
- Controls: yes, untreated test medium
- Evidence of undissolved material: The stock solution was clear and transparent. During withdrawal of the WSF, transfer of undissolved test item was avoided. The WSF was a clear solution and no precipitation was observed throughout the test.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Wild strain (WIK)
- Source: Karlsruhe Institute of Technology, Germany
- Length at study initiation: 19.9 ± 2.9 mm
- Weight at study initiation: 0.0707 ± 0.0293 g
- Method of breeding: Fish were reared in aerated holding water composed of dechlorinated drinking water and deionised water. The water parameters were controlled 3 times a week. The water temperature was 21 - 25 °C. The pH value was 6.0 - 8.5 and total hardness was 140 - 250 mg/L CaCO3. Dissolved oxygen was > 80% saturation for at least 7 d prior testing. The fish were exposed to light for 12 - 16 h daily.
- Feeding of the brood fish: Daily feeding with granular rearing food at approx. 2% of their body-weight. The last feeding was performed 2 d prior test.

FEEDING DURING TEST
No feeding during test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

250 mg/L CaCO3, corresponding to 14 °dH

Test temperature:

22.0 ± 0.6 °C (mean)

pH:

7.46 (mean control) - 7.52 (mean treatments)

Dissolved oxygen:

100 ± 2% saturation (mean)

Conductivity:

718 µS/cm

Nominal and measured concentrations:

Control, 100 mg/L (nominal)
< LOQ, 0.315 mg/L (arithmetic mean measured initial concentrations 0 - 96 h)

Details on test conditions:

TEST SYSTEM
- Test vessel: 2 L glass beaker filled with 1.5 L test solution
- Aeration: Continuous aeration of the test tanks with a membrane pump using a Pasteur pipette.
- Renewal rate of test solution: daily water renewal
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.39 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test medium was reconstituted water consisting of analytical grade salts dissolved in purified water.
- Ca/Mg ratio: 4:1
- Na:K ratio: 10:1
- Culture medium different from test medium: The holding water was composed of dechlorinated drinking water and deionised water. The test medium was reconstituted water.
- Intervals of water quality measurement: The water parameters of the holding water were controlled three times a week. Temperature, pH and oxygen saturation of the test medium were measured in 24 h intervals in fresh and aged solutions. Water hardness of the untreated control was determined at the beginning of the test.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light daily

EFFECT PARAMETERS MEASURED:
- Mortality and visible abnormalities recorded after 0, 4, 24, 48, 72, and 96 h

TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study: The loading rate was selected based on the lowest exisiting and reliable EL50 value for algae or acute invertebrate toxicity data.

Reference substance (positive control):

not required

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

>= 0.315 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other:

Remarks:

Actual concentration based on the arithmetic mean initial measured concentrations of each concentration level

Details on results:

- Behavioural abnormalities: No sub-lethal effects (clinical signs) were observed.
- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The WAF was clear and no precipitation was observed during the test.

Reported statistics and error estimates:

Since no mortality was recorded the effect loading rates were derived based on the raw data. No statistical analysis was considered necessary.

Sublethal observations / clinical signs:

ANALYTICAL RESULTS

The measured test item concentration in fresh solutions was between 0.19 - 0.46% of nominal with an arithmetic mean of 0.315%. The measured test item content in aged test solutions was between 0.33 - 0.52% of nominal (Table 1). Therefore, toxicological endpoints were evaluated using both the nominal test item loading rate and the actual concentration of the test item (mean measured initial concentrations in fresh media).

Table 1. Analytical results. 

Nominal test item loading rate (nominal) [mg/L]	Sampling	Test item		Actual test item concentration [mg/L]^1
		[mg/L]	% of nominal
0	0 h fresh	n.d.	-	-
	24 h aged	n.d.	-
	24 h fresh	n.d.	-
	48 h fresh	n.d.	-
	72 h fresh	n.d.	-
	96 h aged	n.d.	-
100	0 h fresh	0.402	0.400	0.315
	24 h aged	0.332	0.330
	24 h fresh	0.464	0.460
	48 h fresh	0.206	0.210
	72 h fresh	0.187	0.190
	96 h aged	0.515	0.520

LOQ = 0.01 mg/L test item corresponding to 0.01 mg/L analyte

- = not calculable, n.d. = not detectable

¹⁾based on arithmetic mean value

BIOLOGICAL RESULTS

No mortality or sublethal (clinical) effects occurred in the control and the threshold loading rate of 100 mg/L. The established LC50 is > 0.315 mg/L (actual). The corresponding NOELR (96 h) is ≥ 100 mg/L (nominal) and the NOEC is ≥ 0.315 mg/L (actual). The absence of mortality at the threshold loading rate of 100 mg/L (nominal) indicates that fish are not the most sensitive trophic level and that the LL50 is greater than the limit test loading rate.

Validity criteria fulfilled:

yes

Description of key information

LL50 (96 h) > 100 mg/L (nominal, OECD 203, D. rerio)

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) to fish was assessed according to OECD guideline 203 and GLP.

In a semi-static test with daily test medium renewal, D. rerio was exposed to a nominal limit concentration of 100 mg test item/L for 96 h. Since the substance is a UVCB consisting of poorly soluble compounds, the test item was applied as water soluble fraction (WSF). A control was run in parallel. The actual test item concentrations in the test solutions were analytically verified by HPLC-MS/MS detection in 24 h intervals in both fresh and aged media.

The measured test item concentration in fresh medium was between 0.19 – 0.46% of nominal with an arithmetic mean of 0.315%. The measured test item concentration in aged solutions was between 0.33 – 0.52% of nominal. Therefore, the effect concentrations were based on both nominal loading rates and actual mean measured initial concentrations.

After 96 h no mortality or sublethal (clinical) effects occurred in the control and the threshold loading rate of 100 mg/L. The established LL50 (96 h) is > 100 mg/L (nominal) and the LC50 (96 h) > 0.315 mg/L (actual). The corresponding NOELR (96 h) is≥ 100 mg/L (nominal) and the NOEC (96 h) is ≥ 0.315 mg/L (actual). The absence of effects indicates that fish are not the most sensitive trophic level and that the LL50 is greater than the limit loading rate/concentration.