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

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:

September 17, 2018 to December 19, 2018

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:

Organisation for Economic Cooperation and Development. 1992. OECD Guidelines for Testing of Chemicals, Guideline 203: Fish, Acute Toxicity Test. Adopted 17 July 1992.

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Version / remarks:

U.S. Environmental Protection Agency. 2016. Series 850-Ecological Effects Test Guidelines, OCSPP 850.1075: Freshwater and Saltwater Fish Acute Toxicity 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:

Individual test solutions were prepared directly in each test chamber. Test solution concentrations were not adjusted for the active ingredient of the test substance during preparation and are based on the test substance as received. Solutions were prepared at nominal concentrations of 0.34 and 0.50 mg formulation/L by rinsing a calculated amount of test substance using a portion of the premeasured dilution water into 1-L glass volumetric flasks. The flasks were sonicated for approximately 20 minutes. The contents of the flasks were then rinsed into the appropriate test chambers using a portion of the premeasured dilution water with a final volume of 15 L. The solutions were stirred with electric top-down mixers for approximately 60 minutes. The negative control solution was dilution water only.

Test organisms (species):

Pimephales promelas

Details on test organisms:

The fathead minnow (Pimephales promelas) was selected as the test species for this study. This species is representative of an important group of aquatic vertebrates and was selected for use in the test based upon past history of use in the laboratory. Fathead minnows used in the test were received as juveniles from Osage Catfisheries, Inc. of Osage Beach, Missouri. Identification of the species was verified by the supplier.
All fish used in the test were juveniles from the same source and year class, and the length of the longest fish measured was no more than twice the length of the shortest. The average total length of 10 negative control fish measured at the end of the test was 2.6 cm, with a range of 2.1 to 3.0 cm. The average wet weight (blotted dry) of 10 negative control fish measured at the end of the test was 0.120 grams, with a range of 0.060 to 0.187 grams. Loading was defined as the total wet weight of fish per liter of test solution, and was 0.040 g fish/L.
The fish were held for at least 14 days prior to the test in water from the same source and at approximately the same temperature as used during the test. During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 21.8 to 22.8°C, the pH of the water ranged from 8.4 to 8.6, and the dissolved oxygen concentrations were ≥ 8.4 mg/L (≥ 96% of saturation). During this 2-week period, the fish in the lot used for the test showed no signs of disease or stress and mortality was < 5%.
Feeding
Daily during the holding period, except during periods of fasting prior to testing, the fish were fed brine shrimp nauplii (Artemia sp.) supplied by Brine Shrimp Direct, Ogden, Utah. The fish were not fed for at least two days prior to the test or during the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Hardness (mg/L as CaCO3): 128

Test temperature:

Water temperatures were within the 22 ± 1ºC range established for the test.

pH:

Measurements of pH ranged from 8.4 to 8.8 during the test.

Dissolved oxygen:

Dissolved oxygen concentrations remained ≥ 7.6 mg/L (≥ 87% of saturation) throughout the test.

Salinity:

Not applicable

Conductivity:

Specific Conductance (μS/cm): 298

Nominal and measured concentrations:

Nominal concentrations selected for use in this study were 0.34 and 0.50 mg formulation/L.

Details on test conditions:

Environmental Conditions
The test systems were illuminated using fluorescent tubes that emit wavelengths similar to natural sunlight. The lights were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in light intensity. Light intensity was measured at the water surface of one representative test chamber at the beginning of the test using a SPER Scientific Model 840006 light meter.
The test was conducted at a target water temperature of 22 ± 1°C. Temperature was measured in each test chamber at the beginning of the test, at approximately 24-hour intervals during the test, and at the end of the test using a digital thermometer. Temperature also was monitored continuously in one negative control test chamber using a validated environmental monitoring system (AmegaView Central Monitoring System). The system measurements were calibrated prior to exposure initiation with a digital thermometer.
Dissolved oxygen and pH were measured in each test chamber at the beginning of the test, at approximately 24-hour intervals during the test, and at the end of the test. Dissolved oxygen was measured using a Thermo Scientific Orion Star A213 Benchtop RDO/DO meter, and measurements of pH were made using a Thermo Scientific Orion DUAL STAR pH/ISE meter.
Hardness, alkalinity and specific conductance in the dilution water were measured at the beginning of the test. Hardness and alkalinity measurements were made by titration based on methods in Standard Methods for the Examination of Water and Wastewater. Specific conductance was measured using a Thermo Scientific Orion Star A122 portable conductivity meter.

Procedures and Biological Observations
Prior to test initiation, juvenile fathead minnows were collected from the holding tanks. To initiate the test, the fish were impartially distributed one or two at a time to the test chambers until each contained five fish. All organisms were observed periodically during the test to determine the number of mortalities in each treatment group. Mortality was defined as the lack of visible movement (e.g., lack of fin or opercular movement) and the lack of response to gentle prodding in the fish. The numbers of individuals exhibiting signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 4, 24, 48, 72 and 96 hours after test initiation.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.5 other: mg formulation/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.5 other: mg formulation/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Nominal concentrations selected for use in this study were 0.34 and 0.50 mg formulation/L. Test solutions in the test chambers at these nominal concentrations appeared clear and colorless during the test, with an oil slick on the water surface that increased with concentration. The nominal concentrations tested were slightly above the limit of solubility of Trixene AS in water.
Water temperatures were within the 22 ± 1ºC range established for the test. Measurements of pH ranged from 8.4 to 8.8 during the test. Dissolved oxygen concentrations remained ≥ 7.6 mg/L (≥ 87% of saturation) throughout the test. The measurements of hardness, alkalinity and specific conductance in the dilution water at the beginning of the test were typical of EAG Laboratories-Easton well water. Light intensity at the beginning of the test was 603 lux at the surface of the water of one representative test chamber.
All fathead minnows in the negative control group appeared normal throughout the test. All fish in the 0.34 and 0.50 mg formulation/L treatment groups also appeared normal throughout the test, with no mortalities or overt signs of toxicity observed. Therefore in this study, the no-mortality concentration was 0.50 mg formulation/L, the 100% mortality concentration was > 0.50 mg formulation/L and the NOEC was 0.50 mg formulation/L. LC50 values at 24, 48, 72 and 96 hours were determined from the mortality data.

Results with reference substance (positive control):

Not required

Reported statistics and error estimates:

Not applicable; <50% mortality precluded statistical calculation of an LC50 value.

Sublethal observations / clinical signs:

Mortality and Observations During a Non-GLP Range-Finding Test

Nominal Concentration^1 (mg formulation/L)	Number Dead in 24-Hour Period/Cumulative Number Dead/Number Originally Exposed (Observations)^3				Cumulative Percent Mortality
	24 Hours	48 Hours	72 Hours	96 Hours
Negative Control Solvent Control^2 0.0040 0.020 0.10 0.50	0/0/5 (5 AN) 0/0/5 (5 AN) 0/0/5 (5 AN) 0/0/5 (5 AN) 0/0/5 (5 AN) 0/0/5 (5 AN)	0/0/5 (5 AN) 0/0/5 (5 AN) 0/0/5 (5 AN) 0/0/5 (5 AN) 0/0/5 (5 AN) 0/0/5 (5 AN)	0/0/5 (5 AN) 0/0/5 (5 AN) 0/0/5 (5 AN) 0/0/5 (5 AN) 0/0/5 (5 AN) 0/0/5 (5 AN)	0/0/5 (5 AN) 0/0/5 (5 AN) 0/0/5 (5 AN) 0/0/5 (5 AN) 0/0/5 (5 AN) 0/0/5 (5 AN)	0 0 0 0 0 0
^1Test solution appearance: all clear and colourless in the test chambers throughout the study. A light oil sheen was observed on the surface of the 0.50 mg formulation/L test solution. ^2Solvent Control = 0.1 ml Acetone/L ^3Observations: AN = appears normal

 

Temperature, Dissolved Oxygen and pH of Water in the Test Chambers

Nominal Concentration (mg formulation/L)	Replicate	0 Hours			24 Hour			48 Hour			72 Hour			96 Hour
		Temp^1 (°C)	DO^2 (mg/L)	pH	Temp^1 (°C)	DO^2 (mg/L)	pH	Temp^1 (°C)	DO^2 (mg/L)	pH	Temp^1 (°C)	DO^2 (mg/L)	pH	Temp^1 (°C)	DO^2 (mg/L)	pH
Negative Control	A B	21.9 21.8	8.5 8.5	8.7 8.8	21.6 21.6	8.1 8.1	8.6 8.7	21.6 21.7	8.2 8.2	8.6 8.6	21.7 21.7	8.0 7.7	8.5 8.5	21.6 21.6	8.2 8.2	8.5 8.5
0.34	A B	21.9 21.9	8.6 8.5	8.8 8.7	21.6 21.7	8.0 8.1	8.7 8.6	21.6 21.7	8.2 8.2	8.6 8.6	21.7 21.7	7.9 7.6	8.5 8.5	21.6 21.6	8.1 8.1	8.4 8.5
0.50	A B	21.9 21.8	8.6 8.6	8.7 8.7	21.6 21.7	8.0 8.1	8.7 8.6	21.7 21.7	8.1 8.2	8.6 8.6	21.7 21.7	7.7 7.9	8.5 8.5	21.6 21.6	8.1 8.1	8.5 8.5
^1Manual temperature measurements. Temperature monitored continuously during the test ranged from 21.58 to 21.68 °C, measured to the nearest 0.01 °C. ^2A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation at 22 °C in freshwater.

 

Cumulative Mortality and Observations

Nominal Concentration (mg formulation/L)

Rep.

No. Exposed

~4 Hours

24 Hours

48 Hours

72 Hours

96 Hours

Cumulative Percent Mortality

No. Dead^1

Observations^2

No. Dead^1

Observations^2

No. Dead^1

Observations^2

No. Dead^1

Observations^2

No. Dead^1

Observations^2

Negative Control

A B

5 5

0 0

5 AN 5 AN

0 0

5 AN 5 AN

0 0

5 AN 5 AN

0 0

5 AN 5 AN

0 0

5 AN 5 AN

0

0.34

A B

5 5

0 0

5 AN 5 AN

0 0

5 AN 5 AN

0 0

5 AN 5 AN

0 0

5 AN 5 AN

0 0

5 AN 5 AN

0

0.50

A B

5 5

0 0

5 AN 5 AN

0 0

5 AN 5 AN

0 0

5 AN 5 AN

0 0

5 AN 5 AN

0 0

5 AN 5 AN

0

^1Cumulative number of dead fish. ^2Observations of surviving organisms: AN = appear normal.

 

LC50 Values Based on Nominal Test Concentrations

Time	LC 50 (mg formulation/L)	95% Confidence Interval (mg formulation/L)	Statistical Method
24 Hours 48 Hours 72 Hours 96 Hours	> 0.50 > 0.50 > 0.50 > 0.50	--^1 --^1 --^1 --^1	NA^2 NA^2 NA^2 NA^2
^195% confidence limits could not be calculated with the mortality data obtained. ^2NA = not applicable. <50% mortality precluded statistical calculation of an LC 50 value.

 

Validity criteria fulfilled:

yes

Conclusions:

Fathead minnows (Pimephales promelas) were exposed for 96 hours under static conditions to two nominal concentrations of Trixene AS ranging from 0.34 to 0.50 mg formulation/L. Based on the nominal concentrations, the 96-hour LC50 value was > 0.50 mg formulation/L, the highest concentration tested. The no-mortality concentration was 0.50 mg formulation/L, and the no observed effect concentration (NOEC) was 0.50 mg formulation/L, which is slightly above the limit of solubility.

Executive summary:

The objective of this study was to determine the acute effects of Trixene AS on the fathead minnow (Pimephales promelas) during a 96-hour exposure period under static test conditions.

 

The study was conducted according to the procedures outlined in the protocol, “Trixene AS: A 96-Hour Static Acute Toxicity Test with the Fathead Minnow (Pimephales promelas)”. The protocol was based on procedures in the OECD Guidelines for Testing of Chemicals, Guideline 203: Fish, Acute Toxicity Test; the U.S. EPA Series 850 - Ecological Effects Test Guidelines, OCSPP 850.1075: Freshwater and Saltwater Fish Acute Toxicity Test; and ASTM Standard E 729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians.

 

Fathead minnows were exposed to a geometric series of two test concentrations and a negative control (dilution water) for 96 hours under static conditions. Two replicate test chambers were maintained in each treatment and control group, with five fish in each test chamber, for a total of 10 fish per concentration. Nominal test concentrations were selected in consultation with the Sponsor based on exploratory range-finding toxicity data and the results of benchtop solubility tests. Nominal test concentrations selected were 0.34 and 0.50 mg formulation/L, which are at or slightly above the limit of solubility 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.

 

Juvenile fathead minnows were impartially assigned to exposure chambers at test initiation. Observations of mortality and other signs of toxicity were made approximately 4, 24, 48, 72 and 96 hours after test initiation. Cumulative percent mortality observed in the treatment groups was used to determine LC50 values at 24, 48, 72 and 96-hour intervals. The no-mortality concentration and the 100% mortality concentration were determined by visual interpretation of the mortality data. The no-observed-effect concentration (NOEC) was empirically estimated from the mortality data.

 

Fathead minnows (Pimephales promelas) were exposed for 96 hours under static conditions to two nominal concentrations of Trixene AS ranging from 0.34 to 0.50 mg formulation/L. Based on the nominal concentrations, the 96-hour LC50 value was > 0.50 mg formulation/L, the highest concentration tested. The no-mortality concentration was 0.50 mg formulation/L, and the no observed effect concentration (NOEC) was 0.50 mg formulation/L, which is slightly above the limit of solubility.

Description of key information

96-hour LC50 value was > 0.50 mg formulation/L, and the no observed effect concentration (NOEC) was 0.50 mg formulation/L, which is slightly 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 this study was to determine the acute effects of Trixene AS on the fathead minnow (Pimephales promelas) during a 96-hour exposure period under static test conditions.

Fathead minnows were exposed to a geometric series of two test concentrations and a negative control (dilution water) for 96 hours under static conditions. Two replicate test chambers were maintained in each treatment and control group, with five fish in each test chamber, for a total of 10 fish per concentration. Nominal test concentrations were selected in consultation with the Sponsor based on exploratory range-finding toxicity data and the results of benchtop solubility tests. Nominal test concentrations selected were 0.34 and 0.50 mg formulation/L, which are at or slightly above the limit of solubility 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.

Juvenile fathead minnows were impartially assigned to exposure chambers at test initiation. Observations of mortality and other signs of toxicity were made approximately 4, 24, 48, 72 and 96 hours after test initiation. Cumulative percent mortality observed in the treatment groups was used to determine LC50 values at 24, 48, 72 and 96-hour intervals. The no-mortality concentration and the 100% mortality concentration were determined by visual interpretation of the mortality data. The no-observed-effect concentration (NOEC) was empirically estimated from the mortality data.

 

Fathead minnows (Pimephales promelas) were exposed for 96 hours under static conditions to two nominal concentrations of Trixene AS ranging from 0.34 to 0.50 mg formulation/L. Based on the nominal concentrations, the 96-hour LC50 value was > 0.50 mg formulation/L, the highest concentration tested. The no-mortality concentration was 0.50 mg formulation/L, and the no observed effect concentration (NOEC) was 0.50 mg formulation/L, which is slightly above the limit of solubility. It is considered that there are no effects at the limit of solubility in water.