5-methylheptan-3-one oxime

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:

24 March 2014 - 02 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch: PE00083052
Purity: 96.30% (sum of the two isomers)

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

- Sampling method:
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

- Sample storage conditions before analysis:
The samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

An amount of test item (nominally 500 mg) was dissolved in culture medium and the volume adjusted to 5 liters to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 1.0, 3.2, 10 and 32 mg/L. An aliquot (1 liter) of each of the stock solutions was separately inoculated with algal suspension (18.6 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
The stock solution and each of the prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of standard culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Nominal and measured concentrations:

Range finding:
nominal concentration: 0.1 / 1.0 / 10 / 100 mg/L

Definitive study:
nominal concentration: 1.0 / 3.2 / 10 / 32 / 100 mg/L
0h measured concentration: 0.869 / 2.83 / 9.10 / 29.6 / 94.4 mg/L
72h measured concentration: 0.593 / 2.49 / 6.61 / 19.9 / 79.1 mg/L
72h uninoculated measured concentration: 0.855 / 2.89 / 9.13 / 29.5 / 93.6 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

62 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 55-70 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Effects based on yield were also reported (see attached report and executive summary). However, the preferred observational endpoint in the algal inhibition study is growth rate because it is not dependent on test design (ECHA guidance chapter R.7b v2.0, OECD 201 Guideline). The guideline includes the additional response yield, to satisfy current regulatory requirements in some countries. The EU CLP regulation (No 1272/2008 and its adaptation 286/2011) also states that classification should be based on the ErC50. Thus only the effects based on growth rate are presented in the above "effects concentration" table.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.87 to 94 mg/L (87% to 94% of nominal). A decline in measured test concentration was observed at 72 hours to between 0.59 and 79 mg/L (59% to 79% of nominal). Analysis of uninoculated aged test preparations at 72 hours gave measured concentrations of 0.86 to 94 mg/L (86% to 94% of nominal), indicating that the test item adsorbed to the algal biomass in the test suspensions. As the 0-Hour and uninoculated 72-Hour measured test concentration are all within 20% of the nominal concentrations results are based on nominal test concentrations only.

Results with reference substance (positive control):

The health of the algae culture and the overall quality of the test organism is monitored regularly by using the reference toxicant, potassium dichromate. The most recent reference test performed in a separate study (Study Number 41303826, conducted between 21 Oct 2013 and 24 Oct 2013) resulted in an ErC50 (0-72h) of 1.1 mg/l, EyC50 (0-72h) of 0.51 mg/L and NOEC of 0.25 mg/L. The results were within the normal range for this reference item.

RESULTS

Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure ofPseudokirchneriella subcapitatato the test item during the range-finding test are given in the table below:

Nominal Concentration (mg/L)		Cell Densities (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	5.23E+03	2.69E+05	-	-
	R2	5.63E+03	3.02E+05
	Mean	5.43E+03	2.86E+05
0.10	R1	5.38E+03	3.43E+05	[7]	[39]
	R2	5.60E+03	4.47E+05
	Mean	5.49E+03	3.95E+05
1.0	R1	5.47E+03	3.37E+05	[7]	[37]
	R2	5.73E+03	4.43E+05
	Mean	5.60E+03	3.90E+05
10	R1	5.90E+03	1.52E+05	25	60
	R2	6.11E+03	8.28E+04
	Mean	6.01E+03	1.17E+05
100	R1	5.79E+03	6.16E+03	105	100
	R2	6.16E+03	3.44E+03
	Mean	5.98E+03	4.80E+03

The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/L, however, growth was observed to be reduced at 10 and 100 mg/L.

Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Chemical analysis of the 1.0, 10 and 100 mg/L test preparations at 0 hours showed measured concentration of 5.1, 26 and 85 mg/L and at 72 hours showed measured concentration of 4.3, 21 and 79 mg/L indicating that the test item was either unstable under test conditions or was adsorbing to the algal cells present. The high measured concentrations at 1.0 and 10 mg/L are possibly as a result of incomplete mixing of the 100 mg/L stock solution.

Definitive Test

Verification of Test Concentrations

Analysis of the test preparations at 0 hour showed measured test concentrations to range from 0.87 to 94 mg/L (87% to 94% of nominal). A decline in measured test concentration was observed at 72 hours to between 0.59 and 79 mg/L (59% to 79% of nominal). Analysis of uninoculated aged test preparations at 72 hours gave measured concentrations of 0.86 to 94 mg/L (86% to 94% of nominal), indicating that the test item adsorbed to the algal biomass in the test suspensions. As the 0-Hour and uninoculated 72-Hour measured test concentration are all within 20% of the nominal concentrations results are based on nominal test concentrations only.

Growth Data

Cell density values determined at each sampling time and pH values at 0 and 72 hours, Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in the table below:

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)		pH	Cell Densities* (cells per mL				pH
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*	0 h	0 h	24 h	45 h	72 h	72 h
Control	R1	0.065		5.21E+05		7.2	6.59E+03	2.91E+04	1.07E+05	5.27E+05	7.9
	R2	0.068		6.47E+05			5.58E+03	2.96E+04	1.24E+05	6.53E+05
	R3	0.068		6.68E+05			5.38E+03	2.81E+04	1.16E+05	6.73E+05
	R4	0.067	-	6.05E+05	-		5.20E+03	2.73E+04	1.09E+05	6.11E+05
	R5	0.069		7.09E+05			5.95E+03	2.82E+04	1.20E+05	7.15E+05
	R6	0.068		6.75E+05			7.06E+03	3.00E+04	1.23E+05	6.82E+05
	Mean	0.068		6.38E+05			5.96E+03	2.87E+04	1.17E+05	6.43E+05
	SD	0.001		6.67E+04			-	-	-	-
1.0	R1	0.069	[1]	7.24E+05		7.3	7.62E+03	3.43E+04	1.42E+05	7.32E+05	7.9
	R2	0.07	[3]	7.87E+05			5.79E+03	2.87E+04	1.21E+05	7.93E+05
	R3	0.069	[1]	7.09E+05			6.53E+03	3.36E+04	1.46E+05	7.15E+05
	Mean	0.069	[2]	7.40E+05	[16]		6.65E+03	3.22E+04	1.36E+05	7.47E+05
	SD	0.001		4.16E+04			-	-	-	-
3.2	R1	0.071	[4]	8.28E+05		7.3	5.94E+03	3.49E+04	1.43E+05	8.34E+05	7.9
	R2	0.072	[6]	9.09E+05			7.23E+03	3.28E+04	1.50E+05	9.16E+05
	R3	0.072	[6]	8.58E+05			6.78E+03	3.76E+04	1.48E+05	8.65E+05
	Mean	0.072	[5]	8.65E+05	[36]		6.65E+03	3.51E+04	1.47E+05	8.71E+05
	SD	0.001		4.11E+04			-	-	-	-
10	R1	0.069	[1]	7.15E+05		7.3	5.92E+03	3.32E+04	1.48E+05	7.21E+05	7.9
	R2	0.069	[1]	7.17E+05			7.36E+03	2.80E+04	1.27E+05	7.25E+05
	R3	0.071	[4]	8.52E+05			6.11E+03	3.29E+04	1.35E+05	8.58E+05
	Mean	0.07	[2]	7.61E+05	[19]		6.46E+03	3.14E+04	1.37E+05	7.68E+05
	SD	0.001		7.86E+04			-	-	-	-
32	R1	0.058	15	3.26E+05		7.3	5.58E+03	2.53E+04	8.01E+04	3.32E+05	7.9
	R2	0.057	16	2.87E+05			5.97E+03	2.14E+04	6.54E+04	2.93E+05
	R3	0.057	16	3.05E+05			5.65E+03	2.78E+04	8.61E+04	3.11E+05
	Mean	0.057	16	3.06E+05	52		5.73E+03	2.48E+04	7.72E+04	3.12E+05
	SD	0.001		1.95E+04			-	-	-	-
100	R1	0.023	66	1.96E+04		7.3	5.82E+03	1.30E+04	1.35E+04	2.55E+04	7.8
	R2	0.014	79	7.14E+03			6.75E+03	1.10E+04	9.60E+03	1.39E+04
	R3	0.009	87	3.15E+03			6.30E+03	9.12E+03	9.81E+03	9.46E+03
	Mean	0.015	77	9.98E+03	98		6.29E+03	1.10E+04	1.10E+04	1.63E+04
	SD	0.007		8.61E+03			-	-	-	-

From the data given in the table above, it is clear that the growth rate (r) and yield (y) ofPseudokirchneriella subcapitata(CCAP 278/4) were affected by the presence of the test item at a nominal test concentration of 32 mg/L over the 72-Hour exposure period.

Accordingly the following results were determined from the data:

Inhibition of Growth Rate

ErC10(0 - 72 h) : 27 mg/L

ErC20(0 - 72 h) : 36 mg/L

ErC50(0 - 72 h) : 62 mg/L; 95% confidence limits 55 - 70 mg/L

where ErCxis the test concentration that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences (P≥0.05), between the control and 1.0, 3.2 and 10 mg/L test concentration however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 10 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 32 mg/L.

Inhibition of Yield

EyC10(0 - 72 h) : 28 mg/L

EyC20(0 - 72 h) : 29 mg/L

EyC50(0 - 72 h) : 32 mg/L; 95% confidence limits 31 - 33 mg/L

Where:

EyCxis the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as described above. There were no statistically significant decreases in yield (P≥0.05), between the control and 1.0, 3.2 and 10 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 10 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 32 mg/L.

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 108 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours : 5.96 x 103cells per mL

Mean cell density of control at 72 hours : 6.43 x 105cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 8% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2 and 10 mg/L, however swollen and deformed cells was observed to be present in the test cultures at 32 mg/L, additionally swollen and deformed cells and cell debris was observed to be present in the test cultures at 100 mg/L.

Water Quality Criteria

The pH values of the control and each test preparation are given in the table above. Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures (see Table 2) was observed to increase from pH 7.2 at 0 hours to pH 7.9 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Observations on Test Item Solubility

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, 1.0, 3.2 and 10 mg/L test cultures were green dispersions, the 32 mg/L test cultures were pale green dispersions whereas the 100 mg/L test cultures were clear colourless solutions.

Green coloration of the test preparations occurs over the test period due to the growth of the algal cells present, the depth of coloration depending upon the increase in cell density.

CONCLUSION

The effect of the test item on the growth ofPseudokirchneriella subcapitatahas been investigated over a 72-Hour period and gave the following results:

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	62	55 - 70	10	32
Yield	32	31 - 33	10	32

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and the 72h EC50 for growth rate was found to be 62mg/L. The corresponding NOEC is 10 mg/L.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Results

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.87 to 94 mg/L (87% to 94% of nominal). A decline in measured test concentration was observed at 72 hours to between 0.59 and 79 mg/L (59% to 79% of nominal). Analysis of uninoculated aged test preparations at 72 hours gave measured concentrations of 0.86 to 94 mg/L (86% to 94% of nominal), indicating that the test item adsorbed to the algal biomass in the test suspensions. As the 0-Hour and uninoculated 72-Hour measured test concentration are all within 20% of the nominal concentrations results are based on nominal test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	62	55 - 70	10	32
Yield	32	31 - 33	10	32

Description of key information

The effects of the test item on the growth of the green alga, Pseudokirchneriella subcapitata, were determined during a 72 hour growth inhibition test. The preferred observational endpoint is growth rate because it is not dependent on test design (ECHA guidance chapter R.7b v1.1). The EU CLP regulation (No 1272/2008 and its adaptation 286/2011) also state that classification should be based on the ErC50. Thus the 72h EC50 and NOEC based on growth rate, which were determined to be 62 and 10 mg/L respectively, have been selected as the key values for the chemical safety assessment.

Key value for chemical safety assessment

EC50 for freshwater algae:

62 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.87 to 94 mg/L (87% to 94% of nominal). A decline in measured test concentration was observed at 72 hours to between 0.59 and 79 mg/L (59% to 79% of nominal). Analysis of uninoculated aged test preparations at 72 hours gave measured concentrations of 0.86 to 94 mg/L (86% to 94% of nominal), indicating that the test item adsorbed to the increasing algal biomass in the test suspensions. Given that disappearance of the test substance from solution by adsorption to the algal biomass does not mean that it is lost from the test system and that the algae are still exposed to the test substance and given that the 0-Hour and uninoculated 72-Hour measured test concentrations are all within 20% of the nominal concentrations, it is considered appropriate to base the results on nominal test concentrations only.