O-(ethoxycarbonyl)-N-(1-methyl-2-oxo-2-phenylethylidene)hydroxylamine

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:

25 July 2017 to 16 September 2017

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:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentrations
- Samples were taken from the control and each test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the old pooled replicates at 24 and 48 hours for quantitative analysis and analyzed on the day of sampling.
- Duplicate samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Preliminary Media Preparation Trial
- Preliminary 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.
- A nominal amount of test material (1100 mg) was dispersed, in duplicate, in 11 litres of deionised reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After the stirring was stopped, visual observations made on the preparations indicated that no undissolved test material remained. As such a sample of each preparation was taken for analysis with no pre-treatment. Accordingly the following results were obtained: 24 hours: 19.4 mg/L, 48 hours: 3.48 mg/L.
- It was determined that the test material was unstable in water; hence why near nominal concentrations were not obtained in either sample despite visual observations indicating that the test material had dissolved. A further trial was therefore conducted in order to determine the stirring period required to dissolve the test material and the corresponding concentrations obtained.
Further Trial:
- A nominal amount of test material (1100 mg) was dispersed in 11 litres of deionised reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm. Visual observations were made on an hourly basis. After 4 hours stirring it was considered appropriate to stop despite observations showing that the test material had not completely dissolved as a stirring period in excess of 4 hours was impractical. As undissolved test material remained, a sample was taken for chemical analysis following filtration through a 0.45 μm Gelman Acrocap filter (approximately 100 mL discarded in order to pre-condition the filter). Accordingly the following result was obtained: 4 hours: 59.6 mg/L.

Range-Finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 60 mg/L could be obtained using a saturated solution method of preparation.
The test concentration to be used in the initial test was determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 % v/v saturated solution.
A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 4 hours. After 4 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 mL discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10 % v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Initial Daphnia Test
- Based on these results, in order to attain the maximum dissolved concentration of the parent test material, a saturated solution of the test material was prepared at an initial loading rate of 100 mg/L, stirred for a period of 4 hours prior to the removal of any undissolved test material by filtration through a 0.45 µm Gelman Acrocap filter (first ~100 mL discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution with a nominal concentration of approximately 60 mg/L.

Definitive Daphnia Test
A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 4 hours. After 4 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 mL discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 10, 18, 32 and 56 % v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0, 24 and 48 hours.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: In house
- Age: First instar; gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- Feeding during test: No

ACCLIMATION
- Acclimation conditions: Adult daphnids were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a cycle of 16 hours of light and 8 hours of darkness with 20 minute dawn and dusk transition periods. Culture conditions ensured that reproduction was by parthenogenesis.
- Type and amount of food: Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.
- Feeding frequency: Daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

22 ± 1 °C

pH:

6.6 - 8.2 (0 hours fresh media)

Dissolved oxygen:

8.6 - 9.1 mg O2/L

Nominal and measured concentrations:

Nominal: 10, 18, 32, 56 and 100 % v/v saturated solutions
Geometric mean measured: 0.037, 0.051, 0.066, 0.089 and 0.16 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass beakers
- Type: The test vessels were covered to reduce evaporation.
- Material, size, headspace, fill volume: 100 mL of test preparation.
- Aeration: No
- Renewal of test solution: Semi static test conditions were employed in the test in an effort to maintain dissolved test material concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24 hour old test media into the fresh test media.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water, Elendt M7, contained the following substances in mg/L: H3BO3 0.715, MnCl2.4H2O 0.090, LiCl 0.077, RbCl 0.018, SrCl2.6H2O 0.038, NaBr 0.004, Na2MoO4.2H2O 0.016, CuCl2.2H2O 0.004, ZnCl2 0.013, CoCl2.6H2O 0.010, KI 0.0033, Na2SeO3 0.0022, NH4VO3 0.00058, Na2EDTA.2H2O 0.625, FeSO4.7H2O 0.249, CaCl2.2H2O 293.8, NaHCO3 64.8, MgSO4.7H2O 123.3, Na2SiO3.9H2O 10, KCl 5.8, NaNO3 0.274, K2HPO4 0.184, KH2PO4 0.143, Thiamine hydrochloride 0.075, Cyanocobalamine (vitamin B12) 0.0010 and D(+) biotin (vitamin H) 0.00075.
The pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21 ºC.
- Water quality assessments: The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal and on termination of the test after 48 hours represent those of the used or 24 hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The light intensity during the light period was measured using an ATP Instrumentation Lux meter. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of darkness with 20 minute dawn and dusk transition periods
- Light intensity: 200 to 1200 Lux

EFFECT PARAMETERS MEASURED
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilised if they were unable to swim within 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 % v/v saturated solution.
- In the range-finding test five daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item.
A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions and analyzed on the day of sampling.
- Results used to determine the conditions for the definitive study: Yes. Based on the results of the range finding test a "limit test" was conducted at a concentration of 100 % v/v saturated solution to confirm that at highest attainable test concentration no immobilisation or adverse reactions to exposure were observed, however, in this test significant immobilisation was observed in the 100 % v/v saturated solution and therefore a full range definitive test was conducted.
- Based on the results of the initial test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 % v/v saturated solution.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

RANGE-FINDING TEST
- No immobilisation or sub-lethal effects of exposure were observed throughout the test.
- Based on this information, a single test concentration of four replicates, of 100 % v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at highest attainable test concentration, no immobilization or adverse reactions to exposure were observed.
- Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from less than the LOQ of the analytical method employed were obtained which was determined to be 0.018 to 0.89 mg/L. Chemical analysis of the old test preparations at 48 hours showed all measured test concentrations had declined to less than the LOQ indicating that the test material was not stable under test conditions.

INITIAL TEST
- In the initial test no immobilisation was observed in the control, however, 20 % immobilisation was observed in the 100 % v/v saturated solution, therefore failing the limit test pass criteria.
- Based on this information test concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution were selected for the definitive test.

DEFINITIVE TEST
- Verification of Test Concentrations:
Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.098 to 2.2 mg/L. Analysis of the old test preparations at 24 and 48 hours showed measured test concentrations had declined, to between less than the limit of quantification (LOQ) of the analytical method employed and 0.023 mg/L. The LOQ of the analytical method was determined to be 0.018 mg/L.
The measured concentrations determined in both the range-finding and definitive tests were considerably lower than the measured concentration determined during the media preparation trial. Given the relative consistency of the measured concentrations of the 100 % v/v saturated solution preparation across the range-finding (0.89 mg/L) and definitive tests (2.2 and 0.96 mg/L) it was considered likely that the lower measured concentrations were as a result of a media effect.
Given the decline in measured concentration throughout the exposure period, it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.
- Immobilisation Data:
There was no significant immobilisation in 20 daphnids exposed to a test concentration of 0.16 mg/L for a period of 48 hours. This study showed that there were no toxic effects at saturation.
- A sub lethal effect of exposure was observed in the 0.089 mg/L test concentration. This response was seven daphnia trapped at the water surface at 48 hours.
- Validation Criteria: The test was considered to be valid given that no more than 10 % of the control daphnids showed immobilisation or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.
- Water Quality Criteria: Temperature was maintained at 20 to 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH. Throughout the test the light intensity was observed to be in the range 343 to 375 Lux.
- Observations on Test Material Solubility: At the start and throughout the test all control and test solutions were observed to be clear colourless solutions.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilization data was carried out using the Trimmed Spearman-Karber method at 24 hours and the Binomial Distribution method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:
- The 48 h-EC50 for the positive control was 0.75 mg/L with 95 % CL of 0.56 to 1.0 mg/L; the NOEC was 0.56 mg/L and the LOEC was 1.0 mg/L. The results from the positive control with potassium dichromate were within the normal range for this reference material.

Reported statistics and error estimates:

An estimate of the EC50 values was given by inspection of the immobilisation data.

Table 1: Cumulative Immobilisation Data and Observations in the Definitive Test after 24 Hours

Geometric Mean Measured Concentration (mg/L)	24 Hours
	Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	1	0	0	0	5	5N	4 N	5 N	5 N
0.037	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.051	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.066	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.089	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.16	0	0	0	0	0	0	5 N	5 N	5 N	5 N

R = Replicate

N = Normal

Table 2:Cumulative Immobilisation Data and Observations in the Definitive Test after 48 Hours

Geometric Mean Measured Concentration (mg/L)	48 Hours
	Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	1	0	0	0	5	5 N	4 N	5 N	5 N
0.037	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.051	1	0	0	0	0	5	4 N	5 N	5 N	5 N
0.066	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.089	0	0	0	0	0	0	3 N 2T	4 N 1 T	3 N 2 T	3 N 2 T
0.16	0	0	0	0	1	5	5 N	5 N	5 N	4 N

R = Replicate

N = Normal

T = Trapped at surface

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the test material based on the geometric mean measured test concentrations gave a 48 hour EC50 value of greater than 0.16 mg/L. The NOEC was 0.16 mg/L. This study showed that there were no toxic effects at saturation.

Executive summary:

The acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions using an acute daphnia immobilisation study.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 60 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range finding test and an initial limit test conducted at a concentration of 100 % v/v saturated solution, 20 daphnids (4 replicates of 5 animals) were exposed to aqueous solutions of the test material at nominal concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution for 48 hours at a temperature of 20 to 22 °C under semi static test conditions. The test material solutions were prepared by stirring an excess (100 mg/L) of test material in test water using a propeller stirrer at approximately 1500 rpm for 4 hours. After the stirring period any undissolved test material was removed by filtration through a 0.2 µm Gelman Acrocap filter, first approximate 100 mL discarded in order to pre condition the filter) to produce a 100 % v/v saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the required test concentrations. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.098 to 2.2 mg/L. Analysis of the old test preparations at 24 and 48 hours showed measured test concentrations had declined, to between less than the limit of quantification (LOQ) of the analytical method employed and 0.023 mg/L. The LOQ of the analytical method was determined to be 0.018 mg/L. Given the decline in measured concentration throughout the exposure period, it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured concentrations were determined to be 0.037, 0.051, 0.066, 0.089 and 0.16 mg/L.

Under the conditions of this study, the test material based on the geometric mean measured test concentrations gave a 48 hour EC50 value of greater than 0.16 mg/L. The NOEC was 0.16 mg/L. This study showed that there were no toxic effects at saturation.

Description of key information

Under the conditions of this study, the test material based on the geometric mean measured test concentrations gave a 48 hour EC50 value of greater than 0.16 mg/L. The NOEC was 0.16 mg/L. This study showed that there were no toxic effects at saturation.

Additional information

The acute toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions using an acute daphnia immobilisation study. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 60 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range finding test and an initial limit test conducted at a concentration of 100 % v/v saturated solution, 20 daphnids (4 replicates of 5 animals) were exposed to aqueous solutions of the test material at nominal concentrations of 10, 18, 32, 56 and 100 % v/v saturated solution for 48 hours at a temperature of 20 to 22 °C under semi static test conditions. The test material solutions were prepared by stirring an excess (100 mg/L) of test material in test water using a propeller stirrer at approximately 1500 rpm for 4 hours. After the stirring period any undissolved test material was removed by filtration through a 0.2 µm Gelman Acrocap filter, first approximate 100 mL discarded in order to pre condition the filter) to produce a 100 % v/v saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the required test concentrations. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.098 to 2.2 mg/L. Analysis of the old test preparations at 24 and 48 hours showed measured test concentrations had declined, to between less than the limit of quantification (LOQ) of the analytical method employed and 0.023 mg/L. The LOQ of the analytical method was determined to be 0.018 mg/L. Given the decline in measured concentration throughout the exposure period, it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured concentrations were determined to be 0.037, 0.051, 0.066, 0.089 and 0.16 mg/L.

Under the conditions of this study, the test material based on the geometric mean measured test concentrations gave a 48 hour EC50 value of greater than 0.16 mg/L. The NOEC was 0.16 mg/L. This study showed that there were no toxic effects at saturation.