2-Pentanone, 4-methyl-, reaction products with 2-(2-aminoethoxy)ethanol

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:

The study was conducted between 01 November 2015 and 14 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

See below

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

yes

Remarks:

See below

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):Not applicable

Analytical monitoring:

yes

Details on sampling:

Verification of Test ConcentrationsWater samples were taken from the control and the 86% v/v saturated solution test vessel at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for quantitative analysis. The samples were stored frozen prior to analysis.Duplicate samples, and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Definitive TestA nominal amount of test item (2200 mg) was dispersed in 22 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorious Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A dilution was made from this saturated solution to give the required test concentration of 86% v/v saturated solution. Prior to addition of the test organisms, with the agreement of the Sponsor, the pH of the test solution was adjusted to that of the control group with the addition of 1.0 M hydrochloric acid.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Test SpeciesThe test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 07 October 2015. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 27 October 2015 to 03 November 2015. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.The water temperature was controlled at 12 °C to 13 °C with a dissolved oxygen content of greater than or equal to 9.8 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 20 hours prior to the start of the definitive test. This was a deviation to the study plan which required feeding to be discontinued approximately 24 hours prior to the start of the test, however, this was considered not to have adversely affected the outcome of the study. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.3 cm (sd = 0.2) and a mean weight of 1.19 g (sd = 0.14) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.42 g bodyweight/liter.The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

No data

Test temperature:

The water temperature was recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.Temperature was maintained at approximately 14 °C throughout the test.

pH:

The pH was recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The pH was measured using a Hach Flexi handheld meter.There were no treatment related differences for pH (range 7.6 - 8.1).

Dissolved oxygen:

The dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The dissolved oxygen concentration were measured using a Hach Flexi handheld meter.There were no treatment related differences for oxygen concentration (range 9.6 0 10.2 mg O2/L).

Salinity:

No data

Nominal and measured concentrations:

The test was conducted at a single concentration of 86% v/v saturated solution.

Details on test conditions:

Test WaterThe test water used for the definitive test was the same as that used to maintain the stock fish.Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.ProcedurePreliminary Media Preparation TrialPreliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.Definitive TestIn accordance with the recommendations of REACh, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EC50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold concentration and a “Limit test” is conducted at this threshold concentration. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LC50 is greater than the threshold concentration. The EC50 value obtained from the Acute Toxicity to Daphnia magna study (Study Number 41501078) was the lowest of these two EC50 values and hence the test was conducted at a single concentration of 86% v/v saturated solution.Experimental PreparationA nominal amount of test item (2200 mg) was dispersed in 22 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorious Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A dilution was made from this saturated solution to give the required test concentration of 86% v/v saturated solution. Prior to addition of the test organisms, with the agreement of the Sponsor, the pH of the test solution was adjusted to that of the control group with the addition of 1.0 M hydrochloric acid.The concentration and stability of the test item in the test preparations were verified by chemical analysis of the fresh media at 0 and 72 hours and of the old media at 24 and 96 hours.Exposure ConditionsIn the definitive test, 25-30 liter glass exposure vessels containing 20 liters of test media were used for each control and test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at approximately 14 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.The control group was maintained under identical conditions but not exposed to the test item. A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build up of nitrogenous waste products.EvaluationsTest Organism ObservationsAny mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Definitive TestMortality DataThere were no mortalities in 7 fish exposed to a test concentration of 86% v/v saturated solution for a period of 96 hours. Inspection of the mortality data gave the following results:The 3 h LC50 was > 53 mg/LThe 6 h LC50 was > 53 mg/LThe 24 h LC50 was > 53 mg/LThe 48 h LC50 was > 53 mg/LThe 72 h LC50 was > 53 mg/LThe 96 h LC50 was > 53 mg/LThe No Observed Effect Concentration (NOEC) was 53 mg/L.Sub-Lethal EffectsSub-lethal effects of exposure were observed at test concentration of 86% v/v saturated solution. These responses were increased pigmentation and emptying of the stomach contents.

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

Statistical AnalysisAn estimate of the LC50 values was given by inspection of the mortality data.Geometric Mean Measured Test ConcentrationsThe geometric mean measured test concentrations of the samples were calculated as follows:GM = √C0C1Where:GM = geometric mean measured test concentration (mg/L)C0 = measured concentration in the fresh media (mg/L)C1 = measured concentration in the old or expired media (mg/L)The geometric mean measured concentration was determined for each exposure period (0-Hour fresh to 24-Hour old and 72-Hour fresh to 96-Hour old) and the arithmetic average calculated of these values.

Sublethal observations / clinical signs:

Verification of Test Concentrations

Analysis of the freshly prepared test media at 0 and 72 hours showed measured test concentrations to be 79.9 and 87.4 mg/L. A decline in measured test concentration was observed in the old or expired test media at 24 and 96 hours to 39.9 and 27.0 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data.

The geometric mean measured test concentration was determined to be 53 mg/L.

Validation Criteria

The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was ≥60% of ASV in the control and test vessels.

Cumulative Mortality Data in the DefinitiveTest

Nominal Concentration (% v/v Saturated Solution)	Geometric Mean Measured Test Concentration (mg/L)	Cumulative Mortality (Initial Population = 7)						% Mortality
		3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	-	0	0	0	0	0	0	0
86	53	0	0	0	0	0	0	0

Sub-lethal Effects of Exposure in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)	Sub-lethal Effects	Time (Hours)
		1	3	6	24	48	72	96
Control	No abnormalities detected	7/7	7/7	7/7	7/7	7/7	7/7	7/7
86	No abnormalities detected		7/7	7/7	7/7	5/7	4/7	4/7
	Emptying of stomach contents	7/7
	Increased pigmentation					2/7	3/7	3/7

Water Quality Measurements

Nominal Concentration (% v/v Saturated Solution)		Time (Hours)
		0 Hours (Fresh Media)				24 Hours (Old Media)
		pH		mg O2/L		T ºC		pH		mg O2/L		T°C
Control		7.6		9.7		14		8.0		9.9		14
86		7.6		10.2		15		7.9		9.6		14

 

Nominal Concentration (% v/v Saturated Solution)		Time (Hours)
		24 Hours (Fresh Media)				48 Hours (Old Media)
		pH		mg O2/L		T ºC		pH		mg O2/L		T°C
Control		7.7		9.6		14		7.8		9.9		14
86		7.6		9.9		14		7.9		9.8		14

 

Nominal Concentration (% v/v Saturated Solution)		Time (Hours)
		48 Hours (Fresh Media)				72 Hours (Old Media)
		pH		mg O2/L		T ºC		pH		mg O2/L		T°C
Control		7.8		9.6		13		8.1		9.9		14
86		7.8		10.0		14		8.0		9.9		14

 

Nominal Concentration (% v/v Saturated Solution)		Time (Hours)
		72 Hours (Fresh Media)				96 Hours (Old Media)
		pH		mg O2/L		T ºC		pH		mg O2/L		T°C
Control		7.7		9.6		13		8.1		10.0		14
86		7.7		10.0		14		8.0		9.8		14

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and geometric mean measured test concentrations gave a 96-Hour LC50 of greater than 53 mg/L. The No Observed Effect Concentration was 53 mg/L.

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008. 

 

Methods…….

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

 

The test item was known to rapidly degrade in water. Given this, and in accordance with current regulatory guidance, the concentration of the degradation product was determined. A preliminary media preparation trial indicated that a dissolved degradation product concentration of approximately 86 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of the degradation product under test conditions.

 

In accordance with the recommendations of REACh, the test was conducted according to the threshold approach recommended by ECHA.  Using this approach the lowest EC₅₀value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold concentration and a “Limit test” is conducted at this threshold concentration. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LC₅₀is greater than the threshold concentration. The EC₅₀value obtained from the Acute Toxicity to Daphnia magna study (Study Number41501078) was the lowest of these two EC₅₀values and hence the test was conducted at a single concentration of 86% v/v saturated solution.

 

Seven fish were exposed to an aqueous solution of the test item, at a single concentration of 86% v/v saturated solution for a period of 96 hours at a temperature of approximately 14 ºC under semi-static test conditions. The test item solution was prepared by stirring an excess (100mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item from which a dilution was made to give the required test concentration. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

 

Results…….

Analysis of the freshly prepared test media at 0 and 72 hours showed measured test concentrations to be 79.9 and 87.4 mg/L. A decline in measured test concentration was observed in the old or expired test media at 24 and 96 hours to 39.9 and 27.0 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data.

 

Exposure ofrainbow trout (Oncorhynchus mykiss) to the test item gave LC₅₀values based on the geometric mean measured test concentrations of greater than 53 mg/L. The No Observed Effect Concentration was 53 mg/L.

Description of key information

The acute toxicity of the test item was determined with rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the appropriate OECD 203 Guideline for Testing of Chemicals.

In accordance with the recommendations of REACh, the test was conducted according to the threshold approach recommended by ECHA.  Using this approach the lowest EC50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold concentration and a “Limit test” is conducted at this threshold concentration

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. The test item was known to rapidly degrade in water. Given this, and in accordance with current regulatory guidance, the concentration of the degradation product was determined in test medium and used for endpoint derivation. A preliminary media preparation trial indicated that a dissolved degradation product concentration of approximately 86 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of the degradation product under test conditions.

No mortalities were observed during the test which indicates that under the conditions of this test the 96 -hour LC50 is >53 mg/L based on mean measured concentrations. 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

53 mg/L

Additional information

Endpoint is the the highest tested concentration, no effects were observed and therefore the NOEC is 53 mg/L.

The test item is not toxic to Rainbow Trout.