Reaction product of trimethylhexamethylenediisocyanate and (3-mercaptopropyl)trimethoxysilane

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:

01 May 2018 to 28 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Principles of method if other than guideline:

Test was conducted according to a standard OECD guideline.

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Thio Isocyanate Adduct
Batch number: BZA13264 and BZA15779
Expiry date: 23 June 2018 and 23 August 2018
Test item storage: At room temperature under nitrogen
Appearance: Colourless liquid

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from all test concentrations and the control at the start, after 24 hours and at the end of the test period (72 hours). Sample volumes of 2.0 mL where taken from the approximate centre of the test vessels and were subsequently analysed on the day of sampling (i.e. samples were not stored for later analysis). At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at a WAF prepared at a loading rate of 10 mg/L but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Vehicle:

no

Details on test solutions:

Preparation of test solutions for the combined limit/range-finding test and 1st full test were from loading rates that were individually prepared at concentrations ranging between 1.0 to 100 mg/L. A two day period of magnetic stirring was applied to accelerate dissolution of the test item in medium. The obtained mixtures were allowed to settle overnight. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. All test solutions were clear and colourless at the end of the preparation procedures.

Based on the results of a pre-test, test solution preparations used in the final test started with loading rates individually prepared at 1.0, 3.2, 10, 32 and 100 mg/L. A three hour period of magnetic stirring was applied to accelerate dissolution of the test item in medium. The obtained mixtures were allowed to settle for a period of 15 minutes. Thereafter, the aqueous WAFs were collected by siphoning and used as test concentrations. All test solutions were clear and colourless at the end of the preparation procedures.

After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL. Any residual volumes were discarded.

Test organisms (species):

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

Details on test organisms:

Strain: NIVA CHL 1
Source: In-house laboratory culture
Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA).
Pre-culture: 3 – 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

Standard duration

Post exposure observation period:

None, not a standard test requirement

Hardness:

Not reported

Test temperature:

23 to 24°C

pH:

0 h: 8.0 (control), 8.0, 8.0, 8.0, 8.0, 7.9 in test item treatments in ascending concentration order.
72 h: 8.1 (control), 8.0, 8.0, 6.9, 6.9, 7.1 in test item treatments in ascending concentration order.

Dissolved oxygen:

NA

Salinity:

NA

Conductivity:

NA

Nominal and measured concentrations:

WAFs prepared at loading rates of: 1.0, 3.2, 10, 32 and 100 mg/L

Details on test conditions:

Test vessels: 100 mL glass test vessels with aluminium caps, perforated for ventilation, containing 50 mL of test solution.
Test medium: Prepared according to the OECD 201 Guideline and formulated using Milli-RO water.
Cell density: Initial cell density of 1 x 10^4 cells/mL.
Illumination: Tests were under continuous illumination using TLD-lamps with a light intensity within the range of 95 to 97 µE.m-2.s-1.
Incubation: Vessels were distributed at random in the incubator and repositioned daily. During incubation the algal cells were kept in suspension by continuous shaking.

A combined limit/range-finding test was initially conducted, which exposed six replicates of exponentially growing algae to a control and a WAF prepared at a loading rate of 100 mg/L. The test procedure and conditions were similar to those applied in the final test with the exception that:
• Three replicates per concentration were exposed to WAFs individually prepared at loading rates of 1.0 and 10 mg/L in the combined range-finding test,
• Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
• pH was only measured in the control and the highest test concentration.

After 24 hours of exposure in the combined limit/range-finding test, undissolved particles were observed in the highest test concentration that interfered with spectrophotometric measurement. Therefore, algal density was determined by use of a microscope and a counting chamber throughout the test and subsequent tests.

During the full tests, measurements of pH were taken at the beginning and at the end of the test, for all test concentrations and the control and temperature of medium was measured continuously in a temperature control vessel. At the end of the final test microscopic observations of the appearance of the algal cells were performed on the highest test concentration and the control to observe for any abnormal appearance of the algae.

Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.

A final full test was performed based on the results of the preceding combined limit/range-finding test and the 1st full test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs individually prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Loading rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Yield

Remarks on result:

other: Loading rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Loading rate

Remarks:

Based on biological relevance

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Yield

Remarks on result:

other: Loading rate

Remarks:

Based on biological relevance

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Loading rate

Remarks:

Based on statistical significance

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Yield

Remarks on result:

other: Loading rate

Remarks:

Based on statistical significance

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.2 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: First constituent

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.2 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Yield

Remarks on result:

other: First constituent

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.2 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: First constituent

Remarks:

Based on biological relevance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.2 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Yield

Remarks on result:

other: First constituent

Remarks:

Based on biological relevance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.054 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: First constituent

Remarks:

Based on statistical relevance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.054 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Yield

Remarks on result:

other: First constituent

Remarks:

Based on statistical relevance

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 59 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Second constituent

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 59 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Yield

Remarks on result:

other: Second constituent

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

59 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Second constituent

Remarks:

Based on biological relevance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

59 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Yield

Remarks on result:

other: Second constituent

Remarks:

Based on biological relevance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

11 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Second constituent

Remarks:

Based on statistical relevance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

11 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Yield

Remarks on result:

other: Second constituent

Remarks:

Based on statistical relevance

Details on results:

In the combined limit/range finding test, no inhibition of algal growth rate or yield was found at the WAF prepared at a loading rate of 1.0 mg/L. Inhibition of algal growth rate and yield was found at WAFs from 10 mg/L upwards at the end of the test. An inhibition of 34 and 81% was observed at the highest test concentration, respectively.

In the 1st full test, no inhibition of algal growth rate or yield was found at the WAFs prepared at loading rates of 1.0 and 3.2 mg/L. Inhibition of algal growth rate and yield was found at loading rates of 10, 32 and 100 mg/L at the end of the test. A growth rate inhibition of 5.8%, 39% and 32% and yield inhibition of 24%, 84% and 77% was observed, respectively.

In the final test, statistically significant inhibition of growth rate and yield was found at the highest test concentration. However, growth rate inhibition was considered to be biologically not relevant, since the measured inhibition was below 10%. The NOEC based on biological relevance was thus set 100 mg/L based on the highest prepared WAF loading rate, at 0.20 µg/L for the first constituent and 59 µg/L for the second constituent.
Although inhibition for both growth rate and yield was higher during the combined limit/range-finding test and 1st full test, actual exposure concentrations were higher during the final test due to the change in solution preparation. Therefore, effect parameters were calculated based on the results of the final test.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the highest test concentration when compared to the control.

Results with reference substance (positive control):

The positive control used potassium dichromate as the reference item at nominal concentrations of 0.18, 0.32, 0.56, 1.0, 1.8, 3.2 mg/L and to a control. Exposure conditions were similar to those in the definitive test. Exposure of Pseudokirchneriella subcapitata to the reference item resulted in inhibited growth rate and yield at nominal concentrations 0.18 mg/L and higher and 72 hour ErC50 and EyC50 values of 0.90 mg/L (95% confidence limits: 0.88 to 0.93 mg/L) and 0.30 mg/L (95% confidence limits: 0.29 to 0.31 mg/L), respectively. Resulting 72 hour NOEC and LOEC values were 0.25 mg/L and 0.5 mg/L respectively, based on both growth rate and yield. The results from the positive control were within the normal range for this reference item.

Reported statistics and error estimates:

The determination of the NOEC and the ECx followed the approaches recommended in the OECD guideline 201. An effect was considered significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). Calculation of ECx-values was based on probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test item. The ERCx-values could not be determined because the observed effects were below 10%. The EYC50-value could not be determined because the observed effect was below 50%. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Growth rate and percentage inhibition for the total test period of the final test

Thio Isocyanate Adduct Loading Rate (mg/L)	Mean	Std. Dev.	n	% Inhibition
Control	1.677	0.0288	6
1.0	1.699	0.0350	3	-1.3
3.2	1.622	0.0687	3	3.3
10	1.640	0.0410	3	2.2
32	1.629	0.0364	3	2.9
100	1.515	0.0362	3	9.7^#

^#- effect was statistically significant however biologically not relevant (<10%).

Yield and percentage inhibition for the total test period of the final test

 

Thio Isocyanate Adduct Loading Rate (mg/L)	Mean	Std. Dev.	n	% Inhibition
Control	152.5	12.90	6
1.0	163.2	16.69	3	-7.0
3.2	130.5	27.12	3	14
10	136.8	16.31	3	10
32	132.0	14.08	3	13
100	93.4	9.99	3	39*

* - effect was statistically significant.

Effect parameters

Parameter (µg/L)		NOEC*	NOEC^#	EC10	EC20	EC50
Growth rate,	Value	0.054	0.20	>0.20	>0.20	>0.20
First Constituent	lower 95%-cl
(MRM m/z 375.0 --> m/z 165.0)	upper 95%-cl
Yield,	Value	0.054	0.20	0.029	0.070	>0.20
First Constituent	lower 95%-cl			0.014	0.042
(MRM m/z 375.0 --> m/z 165.0)	upper 95%-cl			0.063	0.12
Growth rate,	Value	11	59	>59	>59	>59
Second Constituent	lower 95%-cl
(MRM m/z 393.0 --> m/z 133.0)	upper 95%-cl
Yield,	Value	11	59	3.3	12	>59
Second Constituent	lower 95%-cl			0.99	5.8
(MRM m/z 393.0 --> m/z 133.0)	upper 95%-cl			11	27
Parameter (mg/L)		NOELR*	NOELR^#	EL10	EL20	EL50
Growth rate	Value	32	100	>100	>100	>100
	lower 95%-cl
	upper 95%-cl
Yield	Value	32	100	6.8	27	>100
	lower 95%-cl			1.7	12
	upper 95%-cl			27	58

cl – confidence limit,* - based on statistical significance,^#- based on biological relevance.

Validity criteria fulfilled:

yes

Remarks:

The study fulfilled all of the validity described by the OECD Guideline.

Conclusions:

Exposure of the freshwater algae Pseudokirchneriella subcapitata to the test item (Thio Isocyanate Adduct) over a 72 hour period resulted in measured (TWA) EL50 values of >100 mg/L for both growth rate inhibition and yield. The 72 hour EC50 based on the first constituent was >0.20 µg/L for both growth rate inhibition and yield. The 72 hour EC50 based on the second constituent was 59 µg/L for both growth rate inhibition and yield. The resulting 72 hour NOELR values for growth rate inhibition and yield were 32 mg/L based on statistical significance and 100 mg/L based on biological relevance. The resulting 72 hour NOEC values based on the first constituent for growth rate inhibition and yield was 0.054 µg/L based on statistical significance and 0.20 µg/L based on biological relevance. The resulting 72 hour NOEC values based on the first constituent for growth rate inhibition and yield was 11 µg/L based on statistical significance and 59 µg/L based on biological relevance.

Executive summary:

The toxicity of Thio Isocyanate Adduct to the freshwater algae, Pseudokirchneriella subcapitata, was assessed via an OECD Guideline 201 (‘Freshwater Alga and Cyanobacteria, Growth Inhibition Test’) study. The study was performed at a GLP accredited laboratory, fulfilled the validity criteria described by the guideline and is well documented and is therefore considered to be reliable.  

Exposures of P. subcapitata to the test item resulted in Time Weighted Average (TWA) measured 72 hour EL50 values of >100 mg/L for both growth rate inhibition and yield. The 72 hour EC50 based on the first constituent was >0.20 µg/L for both growth rate inhibition and yield. The 72 hour EC50 based on the second constituent was 59 µg/L for both growth rate inhibition and yield. The resulting 72 hour NOELR values for growth rate inhibition and yield were 32 mg/L based on statistical significance and 100 mg/L based on biological relevance. The resulting 72 hour NOEC values based on the first constituent for growth rate inhibition and yield was 0.054 µg/L based on statistical significance and 0.20 µg/L based on biological relevance. The resulting 72 hour NOEC values based on the first constituent for growth rate inhibition and yield was 11 µg/L based on statistical significance and 59 µg/L based on biological relevance.

Description of key information

The toxicity of Thio Isocyanate Adduct to the freshwater algae, Pseudokirchneriella subcapitata, was assessed via an OECD Guideline 201 (‘Freshwater Alga and Cyanobacteria, Growth Inhibition Test’) study. The study was performed at a GLP accredited laboratory, fulfilled the validity criteria described by the guideline and is well documented and is therefore considered to be reliable.  

Exposures ofP. subcapitatato the test item resulted in Time Weighted Average (TWA) measured 72 hour EL50 values of >100 mg/L for both growth rate inhibition and yield. The 72 hour EC50 based on the first constituent was >0.20 µg/L for both growth rate inhibition and yield. The 72 hour EC50 based on the second constituent was 59 µg/L for both growth rate inhibition and yield. The resulting 72 hour NOELR values for growth rate inhibition and yield were 32 mg/L based on statistical significance and 100 mg/L based on biological relevance. The resulting 72 hour NOEC values based on the first constituent for growth rate inhibition and yield was 0.054 µg/L based on statistical significance and 0.20 µg/L based on biological relevance. The resulting 72 hour NOEC values based on the first constituent for growth rate inhibition and yield was 11 µg/L based on statistical significance and 59 µg/L based on biological relevance.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L