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 aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

Organisation for Economic Cooperation and Development. 2004. OECD Guidelines for Testing of Chemicals, Guideline 202: Daphnia sp., Acute Immobilization Test. Adopted 13 April 2004.

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Version / remarks:

U.S. Environmental Protection Agency. 2016. Series 850 – Ecological Effects Test Guidelines, OCSPP 850.1010: Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids.

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:

Three primary stock solutions were prepared by mixing calculated amounts of test substance in 12 L of dilution water at nominal concentrations of 0.23, 0.34 and 0.50 mg formulation/L, the highest three concentrations tested. The stock solution concentrations were not adjusted for the active ingredient of the test substance during preparation, and all concentrations are based on the test substance as received. The weighed amounts of test substance were rinsed into 1-L glass volumetric flasks using a portion of the premeasured dilution water. The flasks were sonicated for approximately 20 minutes and then rinsed into to the respective glass aspirator bottles using a portion of the premeasured dilution water with a final volume of 12 L. The solutions were then stirred on magnetic stir plates for 60 minutes. The stock solutions appeared clear and colorless with a slight oil slick on the water surface increasing in amount with increasing concentration. The resulting primary stock solutions were decanted by removing approximately 1 L of solution from the bottle before collecting test solutions. Aliquots of the 0.23 mg formulation/L primary stock solution were proportionally diluted with dilution water to a final volume of 1 L to prepare two additional test solutions at nominal concentrations of 0.11 and 0.16 mg formulation/L. The solutions were stirred on a magnetic stir plate for approximately 15 minutes. The solutions appeared clear and colorless with no evidence of precipitate or oil slick. Approximately 220 mL of solution was placed in each of four replicate test chambers per treatment group after removal of test solution for analytical and water quality. The negative control solution was dilution water only.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test Organism
The cladoceran, Daphnia magna, was selected as the test species for this study. This species is representative of an important group of aquatic invertebrates and was selected for use in the test based upon past history of use in the laboratory. Daphnids used in the test were <24-hour old neonates obtained from cultures maintained by EAG Laboratories in Easton, Maryland. Identification of the species was verified by the supplier of the original stock culture.
Adult daphnids were cultured 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 19.6 to 20.4ºC, the pH of the water ranged from 8.0 to 8.4, and the dissolved oxygen concentrations were ≥ 6.8 mg/L (≥ 75% of saturation). The five adult daphnids used to supply neonates for the test were held for 21 days prior to collection of the juveniles for testing and had each produced four previous broods. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7-day period prior to the test. The adults showed no signs of disease or stress, no ephippia were produced during the holding period, and there was no mortality in the culture stock in the two-day period prior to test initiation.
Loading in each test chamber during the test was approximately 44 mL of test solution per daphnid.
Feeding
Daphnids in the cultures were fed daily a mixture of yeast, cereal grass media and trout chow (YCT), supplemented with a vitamin stock solution and a suspension of the freshwater green alga, Raphidocelis subcapitata. The adults were fed during the 24-hour period prior to test initiation, but neonates were not fed during the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No post exposure observation period specified

Hardness:

Hardness (mg/L as CaCO3) 144

Test temperature:

Water temperatures were within the 20 ± 1°C range established for the test.

pH:

Measurements of pH ranged from 8.1 to 8.5.

Dissolved oxygen:

Dissolved oxygen concentrations remained ≥ 8.4 mg/L (≥ 93% of saturation) throughout the test.

Salinity:

Not applicable

Conductivity:

Specific Conductance (μS/cm) 336

Nominal and measured concentrations:

Nominal concentrations selected for use in this study were 0.11, 0.16, 0.23, 0.34 and 0.50 mg formulation/L.

Details on test conditions:

Test Apparatus
Test chambers were 250-mL glass beakers, loosely covered with plastic petri dishes and filled with approximately 220 mL of test water. The depth of the test water in a representative chamber was 7.0 cm. All test chambers were labeled with the project number, test concentration and replicate designation. The test chambers were impartially positioned by treatment group in a temperature-controlled environmental chamber to maintain the target water temperature throughout the test period.

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 20 ± 1°C. Temperature was measured in each test chamber at the beginning of the test, at approximately 24 hours during the test, and at the end of the test using a digital thermometer. Water temperature also was monitored continuously in a container of water placed adjacent to the test chambers 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 hours 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 at the beginning of the test were measured. 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, neonate daphnids < 24 hours old were collected from the cultures and were impartially distributed one or two at a time to transfer containers until each contained its complement of five daphnids. To initiate the test, the transfer containers were impartially assigned to test chambers, and the daphnids were released into the test chambers. All daphnid transfers were conducted below the water surface using wide-bore pipettes. All organisms were observed periodically during the test to determine the number of immobile organisms in each treatment group. Those daphnids that were not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilized (even if they could still move their antennae). The numbers of individuals exhibiting signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 4, 24 and 48 hours after test initiation.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.5 other: mg formulation/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.5 other: mg formulation/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Nominal concentrations selected for use in this study were 0.11, 0.16, 0.23, 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 no evidence of precipitation observed. The two highest nominal concentrations tested were at or slightly above the limit of solublity of Trixene AS in water.
Water temperatures were within the 20 ± 1°C range established for the test. Measurements of pH ranged from 8.1 to 8.5. Dissolved oxygen concentrations remained ≥ 8.4 mg/L (≥ 93% 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 689 lux at the surface of the water of one representative test chamber.
All daphnids in the negative control group appeared normal throughout the test. All daphnids in the 0.11, 0.16, 0.23, 0.34 and 0.50 mg formulation/L treatment groups also appeared normal throughout the test, with no immobile daphnids or overt signs of toxicity observed. Three floating daphnids were observed in the 0.23 mg formulation/L treatment group at approximately 4 hours after study initiation, but they appeared normal after gentle submersion below the water’s surface. Percent immobility in the 0.11, 0.16, 0.23, 0.34 and 0.50 mg formulation/L treatment groups at test termination was 0, 0, 0, 0 and 0%, respectively. No signs of toxicity were observed among the surviving daphnids in any treatment group at test termination. Therefore in this study, the no-immobility concentration was 0.50 mg formulation/L, the 100% immobility concentration was > 0.50 mg formulation/L and the NOEC was 0.50 mg formulation/L.

Results with reference substance (positive control):

Not specified

Reported statistics and error estimates:

Not applicable, <50% immobility precluded statistical calculation of an EC50 values.

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

Nominal Concentration^1 (mg formulation/L)	Number Immobile in 24-Hour Period/Cumulative Number Immobile/Number Originally Exposed (Observations^3)		Cumulative Percent Immobility
	24 Hours	48 Hours
Negative Control Solvent Control^2 0.0040 0.020 0.10 0.50	0/0/10 (10 AN) 0/0/10 (10 AN) 0/0/10 (10 AN) 0/0/10 (1 C; 9 AN) 0/0/10 (10 AN) 0/0/10 (2 C; 8 AN)	0/0/10 (10 AN) 0/0/10 (10 AN) 0/0/10 (10 AN) 0/0/10 (1 C; 9 AN) 0/0/10 (10 AN) 0/0/10 (1 C; 9 AN)	0 0 0 0 0 0
^1Test solution appearance; all clear and colourless in the test chambers throughout the study. ^2Solvent concentration: 0.1 mL acetone/L ^3Observations: AN = appears normal; C = lethargic

 

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

Nominal Concentration (mg formulation/L)	Replicate	0 Hours			24 Hours			48 Hours
		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 C D	20.6 20.6 20.4 20.5	9.1 9.1 9.1 9.1	8.1 8.1 8.1 8.1	20.0 20.0 19.9 20.0	8.7 8.7 8.7 8.7	8.3 8.3 8.3 8.3	20.1 20.1 20.0 20.0	8.7 8.8 8.7 8.7	8.3 8.3 8.4 8.4
0.11	A B C D	20.5 20.7 20.5 20.6	9.0 9.0 9.0 9.0	8.2 8.2 8.2 8.2	19.9 20.0 19.8 19.9	8.7 8.7 8.8 8.8	8.4 8.4 8.4 8.4	20.1 20.1 20.1 20.0	8.5 8.4 8.4 8.4	8.4 8.5 8.4 8.4
0.16	A B C D	20.4 20.5 20.4 20.4	9.0 9.0 9.0 9.0	8.2 8.2 8.2 8.2	19.8 19.9 19.9 19.9	8.8 8.7 8.7 8.7	8.4 8.4 8.4 8.4	20.0 20.0 20.0 20.0	8.7 8.6 8.6 8.5	8.4 8.5 8.5 8.5
0.23	A B C D	19.7 19.8 19.8 19.8	9.1 9.1 9.1 9.1	8.2 8.2 8.2 8.2	20.0 20.0 20.0 20.0	8.7 8.7 8.7 8.8	8.4 8.4 8.4 8.4	20.1 20.1 20.1 20.0	8.5 8.6 8.6 8.6	8.5 8.5 8.5 8.5
0.34	A B C D	19.7 19.8 19.8 19.8	9.1 9.1 9.1 9.1	8.2 8.2 8.2 8.2	19.9 20.0 20.0 20.0	8.7 8.7 8.7 8.8	8.4 8.4 8.4 8.4	20.0 20.0 20.0 20.0	9.0 8.8 8.7 8.7	8.5 8.5 8.5 8.5
0.50	A B C D	19.8 19.8 19.7 19.8	9.1 9.1 9.1 9.1	8.2 8.2 8.2 8.2	20.0 20.0 20.0 20.0	8.8 8.7 8.7 8.9	8.4 8.4 8.4 8.4	20.1 20.1 20.1 20.3	8.7 8.6 8.7 8.7	8.5 8.5 8.5 8.5
^1Manual temperature measurements. Temperature monitored continuously during the test ranged from 19.75 to 19.94 °C, measured to the nearest 0.01 °C. ^2A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation in 20 °C in freshwater.

 

Cumulative Immobility and Observations

Nominal Concentration (mg formulation/L)	Rep.	No. Exposed	~4 Hours		24 Hours		48 Hours		Cumulative Percent Immobile
			Number Immobile^1	Observations^2	Number Immobile^1	Observations^2	Number Immobile^1	Observations^2
Negative Control	A B C D	5 5 5 5	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0
0.11	A B C D	5 5 5 5	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0
0.16	A B C D	5 5 5 5	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0
0.23	A B C D	5 5 5 5	0 0 0 0	5 AN 5 AN 1 Q,AN; 4 AN 2 Q,AN; 3 AN	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0
0.34	A B C D	5 5 5 5	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0
0.50	A B C D	5 5 5 5	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0 0 0 0	5 AN 5 AN 5 AN 5 AN	0
^1Cumulative number of immobile daphnids ^2Observations of surviving organisms: AN = appears normal; Q,AN = trapped at water surface by appear normal after gentle submersion

 

EC50 Values Based on Nominal Test Concentrations

Time	EC50 (mg formulation/L)	95% Confidence Interval (mg formulation/L)	Statistical Method
24 Hours 48 Hours	> 0.50 > 0.50	--^1 --^1	NA^2 NA^2
^195% confidence limits could not be calculated with the immobility data obtained. ^2NA = not applicable, <50% immobility precluded statistical calculation of an EC50 values.

 

Validity criteria fulfilled:

yes

Conclusions:

The cladoceran, Daphnia magna, was exposed for 48 hours under static conditions to five nominal concentrations of Trixene AS ranging from 0.11 to 0.50 mg formulation/L. Based on the nominal concentrations, the 48-hour EC50 value was > 0.50 mg formulation/L, the highest concentration tested. The no-immobility concentration was 0.50 mg formulation/L, and the no observed effect concentration (NOEC) was 0.50 mg formulation/L, which is at or slightly above the limit of solubility.

Executive summary:

The objective of this study was to determine the acute effects of Trixene AS on the cladoceran, Daphnia magna, during a 48-hour exposure period under static test conditions.

 

The study was conducted according to the procedures outlined in the protocol, “Trixene AS: A 48-Hour Static Acute Toxicity Test with the Cladoceran (Daphnia magna)”. The protocol was based on procedures in the OECD Guidelines for Testing of Chemicals, Guideline 202: Daphnia sp., Acute Immobilization Test; the U.S. EPA Series 850 – Ecological Effects Test Guidelines, OCSPP 850.1010: Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids; and ASTM Standard E 729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians.

 

Daphnids were exposed to a geometric series of five test concentrations and a negative control (dilution water) for 48 hours under static conditions. Four replicate test chambers were maintained in each treatment and control group, with five daphnids in each test chamber, for a total of 20 daphnids 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 (Appendix 1). Nominal test concentrations selected were 0.11, 0.16, 0.23, 0.34 and 0.50 mg Trixene AS/L. 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 (hydrolysis study report from Project Number 796C-103 located in Appendix 2), it was not feasible to measure test concentrations during the test.

 

Neonate daphnids <24-hours old were impartially assigned to exposure chambers at test initiation. Observations of immobility and other signs of toxicity were made approximately 4, 24 and 48 hours after test initiation. Cumulative percent immobility observed in the treatment groups was used to determine EC50 values at 24 and 48-hour intervals. The no-immobility concentration and the 100% immobility concentration were determined by visual interpretation of the immobility data. The no-observed-effect concentration (NOEC) was empirically estimated from the immobility data.

 

The cladoceran, Daphnia magna, was exposed for 48 hours under static conditions to five nominal concentrations of Trixene AS ranging from 0.11 to 0.50 mg formulation/L. Based on the nominal concentrations, the 48-hour EC50 value was > 0.50 mg formulation/L, the highest concentration tested. The no-immobility concentration was 0.50 mg formulation/L, and the no observed effect concentration (NOEC) was 0.50 mg formulation/L, which is at or slightly above the limit of solubility.

Description of key information

Based on the nominal concentrations, the 48-hour EC50 value was > 0.50 mg formulation/L and the no observed effect concentration (NOEC) was 0.50 mg formulation/L, which is at or 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 cladoceran, Daphnia magna, during a 48-hour exposure period under static test conditions.

 

Daphnids were exposed to a geometric series of five test concentrations and a negative control (dilution water) for 48 hours under static conditions. Four replicate test chambers were maintained in each treatment and control group, with five daphnids in each test chamber, for a total of 20 daphnids 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 (Appendix 1). Nominal test concentrations selected were 0.11, 0.16, 0.23, 0.34 and 0.50 mg Trixene AS/L. 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 (hydrolysis study report from Project Number 796C-103 located in Appendix 2), it was not feasible to measure test concentrations during the test.

 

Neonate daphnids <24-hours old were impartially assigned to exposure chambers at test initiation. Observations of immobility and other signs of toxicity were made approximately 4, 24 and 48 hours after test initiation. Cumulative percent immobility observed in the treatment groups was used to determine EC50 values at 24 and 48-hour intervals. The no-immobility concentration and the 100% immobility concentration were determined by visual interpretation of the immobility data. The no-observed-effect concentration (NOEC) was empirically estimated from the immobility data.

 

The cladoceran, Daphnia magna, was exposed for 48 hours under static conditions to five nominal concentrations of Trixene AS ranging from 0.11 to 0.50 mg formulation/L. Based on the nominal concentrations, the 48-hour EC50 value was > 0.50 mg formulation/L, the highest concentration tested. The no-immobility concentration was 0.50 mg formulation/L, and the no observed effect concentration (NOEC) was 0.50 mg formulation/L, which is at or slightly above the limit of solubility. It is considered that there are no effects at the limit of solubility in water.