Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate

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:

08 October 2018 to 17 January 2019

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:

Organization for Economic Cooperation and Development. 2011. OECD Guidelines for Testing of Chemicals, Guideline 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test. Adopted 23 March 2006. Revised 28 July 2011.

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Official Journal of the European Communities. 1992. No. L 383 A. Method C.3. Algal Inhibition Test.

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

A primary stock was prepared by dissolving 0.0040 g of the test substance in 4000 mL of freshwater AAP medium to achieve a nominal concentration of 1.0 mg/L. The primary stock was inverted at least 20 times, sonicated for approximately twenty-two minutes, and stirred for approximately thirty-nineminutes. The primary stock appeared clear and colorless with a few oily globules on the bottom upon inversion. Four additional test solutions were prepared at nominal concentrations of 0.063, 0.13, 0.25, and 0.50 mg/L by diluting aliquots of the 1.0 mg/L primary stock with freshwater AAP medium. All four test solutions appeared clear and colorless at the time of preparation. The negative control solution consisted of freshwater AAP medium without test substance added.

Test organisms (species):

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

Details on test organisms:

Test Organism
The freshwater alga, Raphidocelis subcapitata, was selected as the test species for this study. The species is representative of an important group of freshwater algae, and was selected for use in the test based upon a past history of use, and ease of culturing in the laboratory. Original algal cultures were obtained from the University of Texas at Austin, and have been maintained in culture medium at EAG Laboratories, Easton, Maryland since June 2017. Algal cells used in this test were obtained from EAG Laboratories – Easton cultures that had been actively growing in culture medium under similar environmental conditions as used in this test for at least two weeks prior to test initiation. Algal cells for this study were taken from a culture that had been transferred to fresh medium three days prior to test initiation.

Culture Medium
The algal cells were cultured and tested in freshwater AAP medium. Stock nutrient solutions were prepared by adding reagent-grade chemicals to purified EAG Laboratories-Easton well water (NANOpure® water). The test medium then was prepared by adding appropriate volumes of the stock nutrient solutions to NANOpure® water. The pH of the medium was adjusted to 7.5 ± 0.1 with 10% hydrochloric acid. The medium was sterilized by filtration (0.22 μm) and stored refrigerated prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period specified in the study report

Hardness:

Not specified

Test temperature:

Temperatures remained within the 24 ± 2°C range established for the test.

pH:

The pH of the test solutions at test initiation ranged from 7.2 to 7.3. At test termination, pH in the pooled replicates of each respective treatment and control group ranged from 9.9 to 10.2.

Dissolved oxygen:

Not specified

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

TEST CONCENTRATIONS:
Nominal: Negative Control, 0.063 mg/L, 0.13 mg/L, 0.25 mg/L, 0.50 mg/L, 1.0 mg/L

Details on test conditions:

EXPERIMENTAL DESIGN
The freshwater alga, Raphidocelis subcapitata, was exposed to five test concentrations and a negative control (culture medium) for 72 hours. Nominal test concentrations used in the definitive test were 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L and were based upon the results of a preliminary, range-finding test and the results of benchtop solubility tests. The two highest test concentrations were at or slightly above the limit of solubility of Trixene AS in water. Due to the instability of both the test substance and the hydrolysis products, it was not feasible to measure test concentrations during the test.
Three replicate test chambers were maintained in each treatment group and six replicate test chambers were maintained in the negative control group throughout the exposure period. At test initiation, an inoculum of the algal cells was added to each test chamber to achieve a nominal concentration of approximately 10,000 cells/mL. Samples were collected from each replicate test chamber at approximately 24-hour intervals during the test to determine cell densities. Cell densities were used to determine growth rates and yields which were subsequently used to calculate percent inhibition values relative to the negative control over the 72-hour exposure period. EC50 and ErC50 values (i.e., the theoretical concentrations that would produce an 50% reduction in cell density and growth rate, respectively) and their 95% confidence intervals were determined, when possible, at 24, 48, and 72 hours of exposure. The 72-hour EyC50 value and corresponding 95% confidence interval, (i.e., the theoretical concentrations that would produce a 50% reduction in cell yield) were determined. No-observed-effect-concentrations (NOEC) were determined at 72 hours through statistical evaluation of the cell density, growth rate, and yield data, as well as examination of the concentration-response pattern.

Test Apparatus
Test chambers were sterile, 250-mL glass Erlenmeyer flasks plugged with sterile, foam stoppers, and contained 100 mL of test or control medium. The test chambers were labeled with the project number, test concentration, and replicate, and were indiscriminately positioned daily in an environmental chamber designed to maintain the desired test temperature throughout the test. The test flasks were continuously shaken on a mechanical shaker at 100 rpm throughout the duration of the test.

Environmental Conditions
Test chambers were held in an environmental chamber at a temperature of 24 ± 2ºC. The temperature of a container of water adjacent to the test chambers in the environmental chamber was measured continuously using an Amega Scientific Corporation centralized monitoring system. The algae were held under continuous cool-white fluorescent lighting throughout the test. The target light intensity was 6,000 lux ± 10%. Light intensity was measured at test solution level at five locations surrounding the test flasks at test initiation using a SPER Scientific 840006C light meter. The pH of the medium in each treatment and control group was measured at test initiation and exposure termination using a Thermo Orion A214 pH meter. At test initiation, pH was measured in the individual batches of test solution prepared for each treatment and control group. At exposure termination, pH was measured in pooled samples of test solution collected from each of the replicates of each treatment and control group.

Inoculation of Test Chambers
Prior to test initiation, the concentration of algal cells in the stock culture (culture identification No. 17-01) was determined using a hemacytometer and microscope, and was 5.15 x 105 cells/mL. In order to achieve the desired initial cell density of approximately 10,000 cells/mL, 1.94 mL of stock culture was added to each replicate test chamber at test initiation with an Eppendorf pipette.

Algal Growth Measurements and Observations
Test medium samples (approximately 5 mL) were collected from six test chambers included in the control group and three test chambers included in each treatment and control group for the determination of algal cell densities. Samples were collected at approximately 24-hour intervals during the 72-hour exposure and were held for a maximum of eight days under dark, refrigerated conditions sufficient to inhibit growth until cell counts could be performed. Cell counts were performed using an electronic particle counter (Coulter Electronics, Inc.). Prior to conducting cell counts, the linearity of the instrument response was determined at settings previously established for Raphidocelis subcapitata. A primary counting standard containing R. subcapitata cells was prepared, the density was verified using a hemacytometer and a microscope, and the standard was subsequently diluted to provide a series of seven counting standards for the determination of instrument linearity. Theoretical densities were assigned to each secondary counting standard based upon the verified density of the primary counting standard and the dilution ratio. The cell densities of the counting standards were measured using the electronic particle counter and were compared to the theoretical densities by performing a least squares regression analysis. Cell counts for samples collected during the test were conducted once instrument linearity was demonstrated (i.e., the R-squared value obtained through the regression analysis was at least 0.99994). A single aliquot of each sample collected during the test was diluted with an electrolyte solution (Isoton®). Three 0.5-mL volumes of the diluted sample were counted, and the resulting counts were averaged. The cell density of the sample was determined by adjusting the mean cell count (cells/mL) obtained using the particle counter, based upon the Y intercept and slope calculated through the regression analysis, and the dilution factor. The following equation was used:
Cell Density of Sample (cells/mL) = (Mean Cell Count (cells/mL) – Y Intercept)/Slope x Dilution Factor
At the end of the exposure period, samples of test solution were collected from each of the replicates per treatment and control group, pooled within their respective treatments, and subsamples were removed and examined microscopically for atypical cell morphology (e.g., changes in cell shape, size or color). Cells in the replicate test chambers also were assessed for aggregation or flocculation of cells, and adherence of the cells to the test chamber.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Cell Density, Yield & Growth Rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Cell Density, Yield & Growth Rate

Details on results:

Observations and Measurements
Temperatures remained within the 24 ± 2°C range established for the test. The pH of the test solutions at test initiation ranged from 7.2 to 7.3. At test termination, pH in the pooled replicates of each respective treatment and control group ranged from 9.9 to 10.2. The observed increase in pH is typical for tests conducted with R. subcapitata and is attributed to the photosynthetic activity of the algae. The light intensity ranged from 5,660 to 6,350 lux, which was within the desired range of 6,000 lux ± 10%.
At test initiation, the 0.063, 0.13, 0.25, and 0.50 mg/L test solutions appeared clear and colorless and otherwise unremarkable, while the 1.0 mg/L test solution appeared clear and colorless with a few oily globules on the bottom. At test termination, no particulates or surface-slicks were observed in any of the test solutions.
The toxicity of Trixene AS to R. subcapitata was determined by evaluating changes in cell density over a 72-hour exposure period. Cell densities were used to determine the growth rates for each 24-hour interval of exposure and yields at 72 hours of exposure.
After 72 hours of exposure, inhibition of cell density in the 0.063, 0.13, 0.25, 0.5, and 1.0 mg/L treatment groups was 2, 7, 5, 9, and 8%, respectively, relative to the negative control. Inhibition of yield in the 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L treatment groups was 2, 7, 5, 9, and 8%, respectively, relative to the negative control. Inhibition of growth rate in the 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L treatment groups was 0, 1, 1, 1, and 1% respectively, relative to the negative control.
After 72 hours of exposure, aggregation, flocculation, or adherence to the test chambers was not observed in the controls or in any treatment groups. There were no noticeable changes in cell morphology in any of the treatment groups when compared to the control replicates during themicroscopic examinations of the cells.

Results with reference substance (positive control):

Reference substance not tested

Reported statistics and error estimates:

Mean cell density, mean growth rate, and mean yield, were significantly reduced (Jonckheere –Terpstra Step-Down Trend test; p ≤ 0.05) in the 0.13, 0.25, 0.50, and 1.0 mg/L treatment groups at 72 hours, when compared to the negative control. Although, reductions in mean cell density, mean growth rate, and mean yield were statistically significant in the 0.13, 0.25, 0.50, and 1.0 mg/L treatment groups, these reductions were not considered to be biologically significant. Percent inhibition of all three endpoints was <10% in all Trixene AS treatment groups after 72 hours of exposure. The 72-hour NOEC was determined to be 1.0 mg/L and was based on evaluation of the dose-response.

Non-GLP Range-Finding Test Data

Nominal Concentration (mg/L)	72-Hour Cell Density (cells/mL)	Percent Inhibition vs. Control
Negative Control 10 100 1000	3,626,667 3,925,000 3,620,000 3,610,000	-- -6 0 0

 

Temperature Measurements

Time (Days)	Temperature (°C)^1
	Minimum	Maximum	Mean
0 1 2 3	23.65 23.59 23.59 23.65	23.90 23.72 23.78 23.78	23.69 23.69 23.69 23.70
0-3	23.59	23.90	23.69
^1Temperature was continuously monitored throughout the study in a container of water located adjacent to the test chambers.

 

pH Measurements

Nominal Concentration (mg/L)	pH Measurements
	Day 0^1	Day 3^2
Negative Control 0.063 0.13 0.25 0.50 1.0	7.2 7.3 7.2 7.2 7.2 7.3	10.1 10.2 10.2 9.9 10.1 1.0
^1Day 0 samples were collected from the individual batches of test solutions prepared for each treatment and control group at test initiation. ^2Day 3 samples were composites of test solution collected from each replicate per treatment and control group at test termination.

 

Light Intensity Measurement on Day 0

Shaker Table ID#	Light Intensity (lux)^1
	Measurement No. 1	Measurement No. 2	Measurement No. 3	Measurement No. 4	Measurement No. 5	Mean ± S.D.
AQL#14	6350	5880	6210	5720	5660	5964 ± 303.5
^1Measurements taken at five locations surrounding the test flasks at test solution level.

 

Mean Cell Density, Mean Yield, and Percent Inhibition

Nominal Concentration (mg/L)	24 Hours		48 Hours		72 Hours		0-72 Hours
	Mean Cell Density (cells/mL)	Percent Inhibition^1,2	Mean Cell Density (cells/mL)	Percent Inhibition^1,2	Mean Cell Density (cells/mL)	Percent Inhibition^1,2	Mean Yield (cells/mL)	Percent Inhibition^1,2
Negative Control 0.063 0.13 0.25 0.50 1.0	121,689 134,066 122,951 127,902 117,184 124,173	-- -10 -1 -5 4 -2	1,067,951 1,068,919 1,092,087 1,071,450 1,072,939 1,087,965	-- 0 -2 0 0 -2	4,162,605 4,084,619 3,881,253* 3,964,032* 3,804,550* 3,823,782*	-- 2 7 5 9 8	4,152,605 4,074,619 3,871,253* 3,954,032* 3,794,550* 3,813,782*	-- 2 7 5 9 8
^1Calculations were performed using SAS Version 9.4. Manual calculations may differ slightly. ^2Inhibition was calculated relative to the mean negative control response. * Treatment group mean was significantly reduced (Jonckheere-Terpstra Step-Down Trend test, p ≤ 0.05) when compared to the negative control mean. Significance only evaluated at 72 hours of exposure.

 

Mean Growth Rate (per Hour) and Percent Inhibition

Nominal Concentration (mg/L)	0-24 Hours		0-48 Hours		0-72 Hours
	Mean Growth Rate (hour^-1)	Percent Inhibition^1,2	Mean Growth Rate (hour^-1)	Percent Inhibition^1,2	Mean Growth Rate (hour^-1)	Percent Inhibition^1,2
Negative Control 0.063 0.13 0.25 0.50 1.0	0.1041 0.1080 0.1045 0.1062 0.1025 0.1050	-- -4 0 -2 1 -1	0.0973 0.0973 0.0978 0.0973 0.0974 0.0977	-- 0 0 0 0 0	0.0837 0.0835 0.0828* 0.0831* 0.0825* 0.0826*	-- 0 1 1 1 1
^1Calculations were performed using SAS Version 9.4. Manual calculations may differ slightly. ^2Inhibition was calculated relative to the mean negative control response. * Treatment group mean was significantly reduced (Jonckheere-Terpstra Step-Down Trend test p ≤ 0.05) when compared to the negative control mean. Significance only evaluated at 72 hours of exposure.

 

NOEC, EC50, E_rC50 and E_yC50 Values, based on Nominal Concentrations

Duration of Exposure	Cell Density (mg/L)		Yield (mg/L)		Growth rate (mg/L)
	EC50	95% Confidence Interval	EyC50	95% Confidence Interval	ErC50	95% Confidence Interval
24 hours 48 Hours 72 Hours	>1.0 >1.0 >1.0	N/A N/A N/A	--^1 --^1 >1.0	--^1 --^1 N/A	>1.0 >1.0 >1.0	N/A N/A N/A
	NOEC (mg/L)		NOEC (mg/L)		NOEC (mg/L)
72 Hours	1.0		1.0		1.0
^1EyC50 values only determined at 72 hours per study protocol.

 

Validity criteria fulfilled:

yes

Conclusions:

The freshwater alga, Raphidocelis subcapitata, was exposed to a series of five nominal concentrations of Trixene AS ranging from 0.063 to 1.0 mg/L. Effects were evaluated based on cell density, yield, and growth rate. The 72-hour EC50, EyC50, and ErC50 values were determined to >1.0, >1.0, and >1.0 mg/L, respectively. The 72-hour NOEC was determined to be 1.0 mg/L, which is above the limit of solubility. Statistically signification reductions in cell density, growth rate, and yield were observed in the 0.13, 0.25, 0.50, and 1.0 mg/L Trixene AS treatment groups, however, inhibition of all three endpoints was <10% when compared to the negative control after 72 hours of exposure. These reductions were statistically significant, but they were not considered to be biologically significant.

Executive summary:

The objective of the study was to determine the toxicity of Trixene AS, to the freshwater alga, Raphidocelis subcapitata, over a 72-hour exposure period.

 

The study was conducted according to the procedures outlined in the protocol, “Trixene AS: A 72-Hour Toxicity Test with the Freshwater Alga (Raphidocelis subcapitata)”. The protocol was based on procedures outlined in OECD Guidelines for Testing of Chemicals, Guideline 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test and the Official Journal of the European Communities No. L 838 A, Method C.3.: Algal Inhibition Test.

 

The freshwater alga, Raphidocelis subcapitata, was exposed to a series of five nominal concentrations of Trixene AS ranging from 0.063 to 1.0 mg/L. Effects were evaluated based on cell density, yield, and growth rate. The 72-hour EC50, EyC50, and ErC50 values were determined to >1.0, >1.0, and >1.0 mg/L, respectively. The 72-hour NOEC was determined to be 1.0 mg/L, which is above the limit of solubility. Statistically signification reductions in cell density, growth rate, and yield were observed in the 0.13, 0.25, 0.50, and 1.0 mg/L Trixene AS treatment groups, however, inhibition of all three endpoints was <10% when compared to the negative control after 72 hours of exposure. These reductions were statistically significant, but they were not considered to be biologically significant.

Description of key information

he 72-hour EC50, EyC50, and ErC50 values were determined to >1.0, >1.0, and >1.0 mg/L, respectively. The 72-hour NOEC was determined to be 1.0 mg/L, which is above the limit of solubility. It is considered that there are no effects at the limit of solubility in water.

Additional information

The objective of the study was to determine the toxicity of Trixene AS, to the freshwater alga, Raphidocelis subcapitata, over a 72-hour exposure period.

 

The freshwater alga, Raphidocelis subcapitata, was exposed to a series of five nominal concentrations of Trixene AS ranging from 0.063 to 1.0 mg/L. Effects were evaluated based on cell density, yield, and growth rate. The 72-hour EC50, EyC50, and ErC50 values were determined to >1.0, >1.0, and >1.0 mg/L, respectively. The 72-hour NOEC was determined to be 1.0 mg/L, which is above the limit of solubility. Statistically signification reductions in cell density, growth rate, and yield were observed in the 0.13, 0.25, 0.50, and 1.0 mg/L Trixene AS treatment groups, however, inhibition of all three endpoints was <10% when compared to the negative control after 72 hours of exposure. These reductions were statistically significant, but they were not considered to be biologically significant. It is considered that there are no effects at the limit of solubility in water.