Reaction products of cyclohexanone with buta-1,3-diene and hydrogen peroxide and iron sulphate and methanol, hydrolysed, ammonium salts

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:

5 January to 1 May, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

Study was performed according to a standard guideline.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch: 021015
Purity: 50.00% UB-20 ammonium salts; 50.00% Ethylene glycol
Physical State/Appearance: Clear brown liquid
Expiry Date: 13 November 2021
Storage Conditions: Approximately 4°C in the dark

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from pooled replicates at 72 hours for quantitative analysis. An additional sample of each inoculated test concentration was incubated alongside the test from which an aliquot was removed for analysis after 24 and 48 hours exposure. An additional sample of each test concentration to which no algal cells were added was incubated alongside the test to provide samples for uninoculated analysis at 72 hours.

The 0, 24 and 48-Hour test samples were stored frozen, the 72-Hour test samples were analysed on the day. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

The test concentrations used in the definitive test were determined by preliminary range-finding tests.

A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 32, 10, 3.2 and 1.0 mg/L. An aliquot (500 mL) of each stock solution was separately inoculated with algal suspension (2.7 mL) to give an initial nominal cell density of 5.00E+3 cells/mL.

A solvent control group was prepared containing 50 mg of ethylene glycol in order to determine whether any inhibitory effects on growth were due to the presence of the solvent. A nominal amount of ethylene glycol (25 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 50 mg/L ethylene glycol stock solution. The solvent stock solution was inoculated with algal suspension (2.7 mL) to give an initial nominal cell density of 5.00E+3 cells/mL.

The stock solutions and each of the prepared concentrations were 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 P. subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

Approximately 3 to 4 days before the start of the test, inoculum cultures of algae was set up at an initial cell density of ca. 10E+3 cells/mL. Flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (ca. 150 rpm) and constant illumination at 24 ±1°C until the algal cell density was approximately 10E+5 to 10E+6 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

No further remarks

Post exposure observation period:

Not applicable

Hardness:

Not reported

Test temperature:

24 ±1ºC

pH:

Control at test start: 7.7; Control at test end: 8.5;
Test item treatments at test start: 7.2 to 7.4; Test item treatments at test end: 7.5 to 8.6;
Solvent control at test start: 7.5; Control at test end: 8.6

Dissolved oxygen:

Not applicable

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal concentrations: 1.0, 3.2, 10, 32 and 100 mg/L;
Geometric mean measured concentrations: 0.42, 2.2, 9.3, 28 and 88 mg/L

Details on test conditions:

Glass conical flasks (250 mL volume) were used as test vessels. Six flasks (containing 100 mL test preparation) were used for the control and three flasks per treatment group and the solvent control (each containing 100 mL test preparation) were used. The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characerised by a cell density of 9.15E+5 cells/mL. Inoculation of 500 mL of test medium with 2.7 mL of this algal suspension gave an initial nominal cell density of 5.00E+3 cells/mL and had no significant dilution effect on the final test concentration. The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron incubator) at 24 ±1°C under continuous illumination (intensity ca. 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at ca. 150 rpm for 72 hours.

Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominal inoculated cell concentration (5.00E+3 cells/mL) was taken as the starting cell density. To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

The pH of the control, solvent control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (tested twice in a 12 month period to demonstrate satisfactory conditions of the test)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

43 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 30 - 60 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% confidence limits: 12 - 25 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

Test concentration verification:
Analysis of test preparations at 0 and 72-hours showed measured test concentrations to range from 19% to 84% of nominal indicating that the decline in measured test concentrations was due to both instability under the conditions of the test and adsorption of the test item to the algal cells present. Results were therefore based on geometric mean measured test concentrations in order to give a “worst-case” analysis of the data. The measured test concentrations obtained from the 0-hour preparations and the 72-hour preparations to which no algae were added were used for the purposes of this calculation as whilst losses through adsorption were evident it can still be considered that the algae were exposed to the test item concentrations determined. It was considered unnecessary to analyse the additional samples taken at 24 and 48 hours given that the decline in measured concentrations could be attributed to both instability and adsorption. Furthermore, inhibitory effects on growth were only observed in the nominal 32 and 100 mg/L test groups where geometric mean measured test concentrations of 88% of nominal were obtained.

Growth data:
Control replicate R1 was considered, by visual inspection to be an outlier and as such this replicate was removed from all statistical calculations. Solvent control replicate R1 was considered, by visual inspection to be an outlier and as such this replicate was removed from all calculations. The data show that growth rate (r) and yield (y) of Pseudokirchneriella subcapitata were affected by the presence of the test item over the 72-hour exposure period. The solvent control group containing the same nominal quantity of ethylene glycol as the nominal 100 mg/L test group saw a 4% reduction in growth rate over the duration of the test when compared to the control group. As such it can be considered that the presence of ethylene glycol in the sample of test item had no effect on the results obtained and that any inhibitory effects observed were as a direct result of exposure to the UB-20 ammonium salts.

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 0.42, 2.2, 9.3 and 28 mg/L. Enlarged cells and cell debris was observed in the 88 mg/L test cultures and misshapen cells and cells debris was observed in the solvent control cultures.

Water quality:
Temperature was maintained at 24 ±1°C throughout the test. The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 8.5 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 the control cultures were observed to be very green or green dispersions. The 0.42, 2.2 and 9.3 mg/L test cultures were observed to be green dispersions. The 28 mg/L test cultures were observed to be clear colourless solutions whilst the 88 mg/L test cultures were observed to be clear colourless solutions with an oily appearance on the surface. The solvent control cultures were observed to be very green or green dispersions.

Test validity:
The test was considered to be valid given that the cell concentration of the control cultures increased by a factor of 178 after 72-hours, the mean coefficient of variation for section by section specific growth rate for the control cultures was 14%, and the coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 2%.

Results with reference substance (positive control):

The most recent positive control (potassium dichromate) test conducted prior to the current test (between 21 to 24 December, 2020) resulted in 72-h ErC50, NOErC and LOErC values of 1.3 mg/L (95% confidence limits: 1.2 - 1.5 mg/L), 0.125 mg/L and 0.25 mg/L, respectively, and 72-h EyC50, NOEyC and LOEyC values of 0.44 mg/L (95% confidence limits: 0.37 - 0.52 mg/L), 0.125 mg/L and 0.25 mg/L, respectively. Results were within the normal range for this reference item.

Reported statistics and error estimates:

All ECx values and associated confidence limits and the slope of the response curve and its standard error for growth rate were calculated by Weibull analysis using Linear Maximum-Likelihood regression, whilst those values for yield were calculated by Probit analysis using Linear Maximum-Likelihood regression. Any data points identified as outliers in the first phase of data evaluation were disregarded when fitting the concentration-response curve (Control replicate R1). Prior to determination of the Lowest Observed Effect Concentration (LOEC) and No Observed Effect Concentration (NOEC), data for each endpoint was assessed using Shapiro-Wilk’s test on Normal Distribution, Levene’s test on Variance Homogeneity and Trend analysis by contrasts (as necessary) to enable the correct comparison test to be performed. The Williams Multiple Sequential t-test Procedure was used for both growth rate and yield. All statistical analyses were performed using the ToxRat computer software package (ToxRat® Solutions GMBH).

Growth rate:
Statistical analysis of the growth rate data showed no statistically significant differences (P≥0.05), between the control and the 0.42, 2.2 and 9.3 mg/L test concentrations; however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 9.3 mg/L. Correspondingly the LOEC based on growth rate was 28 mg/L.

Yield:
Statistical analysis of the yield data showed no statistically significant differences (P≥0.05), between the control and the 0.42, 2.2 and 9.3 mg/L test concentrations; however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 9.3 mg/L. Correspondingly the LOEC based on yield was 28 mg/L.

Inhibition of Growth Rate and Yield in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.085	-	2.22E+06	-
	R2	0.071		8.44E+05
	R3	0.071		8.48E+05
	R4	0.071		8.54E+05
	R5	0.074		1.02E+06
	R6	0.071		8.48E+05
	Mean	0.072**		8.83E+05**
	SD	0.001**		7.68E+04**
0.42	R1	0.073	[1]	9.54E+05
	R2	0.075	[4]	1.12E+06
	R3	0.075	[4]	1.12E+06
	Mean	0.074	[3]	1.07E+06	[21]
	SD	0.001		9.63E+04
2.2	R1	0.075	[4]	1.11E+06
	R2	0.075	[4]	1.13E+06
	R3	0.073	[1]	9.73E+05
	Mean	0.074	[3]	1.07E+06	[21]
	SD	0.001		8.52E+04
9.3	R1	0.072	0	8.89E+05
	R2	0.071	1	7.98E+05
	R3	0.073	[1]	9.55E+05
	Mean	0.072	0	8.81E+05	0
	SD	0.001		7.89E+04
28	R1	0.033	54	4.86E+04
	R2	0.032	56	4.65E+04
	R3	0.034	53	5.41E+04
	Mean	0.033	54	4.97E+04	94
	SD	0.001		3.95E+03
88	R1	0.024	67	2.25E+04
	R2	0.023	68	2.04E+04
	R3	0.025	65	2.50E+04
	Mean	0.024	67	2.26E+04	97
	SD	0.001		2.32E+03
Solvent control (50)	R1	0.086	[19]	2.51E+06
	R2	0.068	6	6.69E+05
	R3	0.070	3	7.42E+05
	Mean	0.069**	4**	7.06E+05**	20**
	SD	0.001**		5.16E+04**

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated;

R = Replicate;

SD = Standard Deviation;

[ ] = Increase in growth compared to controls;

- = Not applicable;

** Values calculated excluding replicate R1 which was determined, by visual inspection, to be an outlier

Analytical results for test samples

Time point (Hours)	Nominal concentration of test item in test sample (mg/L)	Determined concentration of test item in test sample (mg/L)	Percentage of nominal concentrations (%)
0†	Control	<LOQ	-
	1.0	0.961	96
	3.2	3.26	102
	10	12.1	121
	32	29.8	93
	100	96.1	96
72†	Control	<LOQ	-
	1.0	0.0224	2
	3.2	0.165	5
	10	0.812	8
	32	27.9	87
	100	108	108
72† (no algae)	1.0	0.187* / 0.186**	19* / 19**
	3.2	1.54* / 1.54**	48* / 48**
	10	6.91* / 7.19**	69* / 72**
	32	26.8	84
	100	80.6	81

†: All test samples analysed on the same day;

* = Original analysis sample outside the calibration range;

** = Original sample further diluted and re-analysed;

*** = Analysed alongside the re-analysis;

LOQ = Limit of Quantification (0.010 mg/L);

- = Not applicable

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item, UB20-LDB (50%), on the growth of Pseudokirchneriella subcapitata was investigated over a 72-h exposure period, with results based on geometric mean measured test concentrations. The 72-h ErC50, NOErC and LOErC values were calculated to be 43 mg/L (95% confidence limits: 30 - 60 mg/L), 9.3 mg/L and 28 mg/L, respectively. The 72-h EyC50, NOEyC and LOEyC values were calculated to be 17 mg/L (95% confidence limits: 12 - 25 mg/L), 9.3 mg/L and 28 mg/L, respectively.

Executive summary:

The toxicity of UB20-LDB (50%) to the freshwater algae, Pseudokirchneriella subcapitata, was assessed according to OECD Guideline 201 and EU Method C.3. Exponentially growing cultures of the algal were exposed to nominal test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (corresponding to geometric mean measured concentrations of 0.42, 2.2, 9.3, 28 and 88 mg/L) a control and a solvent control (ethylene glycol) (to determine whether any adverse effects of exposure were due to the presence of the solvent) for 72-hours. Samples of the algal populations were taken after 24, 48 and 72-hours for determination of cell densities using a Coulter® Multisizer Particle Counter.

 

Analysis of test preparations at 0 and 72-hours showed measured test concentrations to range from 19% to 84% of nominal indicating that the decline in measured test concentrations was due to both instability under the conditions of the test and adsorption of the test item to the algal cells present. Results were therefore based on geometric mean measured test concentrations. The test was considered to be valid given that the cell concentration of the control cultures increased by a factor of 178 after 72-hours, the mean coefficient of variation for section by section specific growth rate for the control cultures was 14%, and the coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 2%. The solvent control group containing the same nominal quantity of ethylene glycol as the nominal 100 mg/L test group saw a 4% reduction in growth rate over the duration of the test when compared to the control group. As such it can be considered that the presence of ethylene glycol in the sample of test item had no effect on the results obtained and that any inhibitory effects observed were as a direct result of exposure to the UB-20 ammonium salts.

 

The 72-hour ErC50, NOErC and LOErC values were calculated to be 43 mg/L (95% confidence limits: 30 - 60 mg/L), 9.3 mg/L and 28 mg/L, respectively. The 72-hour EyC50, NOEyC and LOEyC values were calculated to be 17 mg/L (95% confidence limits: 12 - 25 mg/L), 9.3 mg/L and 28 mg/L, respectively.

Description of key information

The toxicity ofUB20-LDB (50%) to the freshwater algae, Pseudokirchneriella subcapitata, was assessed according to OECD Guideline 201 and EU Method C.3. Exponentially growing cultures of the algal were exposed to nominal test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (corresponding to geometric mean measured concentrations of 0.42, 2.2, 9.3, 28 and 88 mg/L) a control and a solvent control (ethylene glycol) (to determine whether any adverse effects of exposure were due to the presence of the solvent) for 72-hours. Samples of the algal populations were taken after 24, 48 and 72-hours for determination of cell densities using a Coulter® Multisizer Particle Counter.

 

Analysis of test preparations at 0 and 72-hours showed measured test concentrations to range from 19% to 84% of nominal indicating that the decline in measured test concentrations was due to both instability under the conditions of the test and adsorption of the test item to the algal cells present. Results were therefore based on geometric mean measured test concentrations. The test was considered to be valid given that the cell concentration of the control cultures increased by a factor of 178 after 72-hours, the mean coefficient of variation for section by section specific growth rate for the control cultures was 14%, and the coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 2%. The solvent control group containing the same nominal quantity of ethylene glycol as the nominal 100 mg/L test group saw a 4% reduction in growth rate over the duration of the test when compared to the control group. As such it can be considered that the presence of ethylene glycol in the sample of test item had no effect on the results obtained and that any inhibitory effects observed were as a direct result of exposure to the UB-20 ammonium salts.

  

The 72-hour ErC50, NOErC and LOErC values were calculated to be 43 mg/L (95% confidence limits: 30 - 60 mg/L), 9.3 mg/L and 28 mg/L, respectively. The 72-hour EyC50, NOEyC and LOEyC values were calculated to be 17 mg/L (95% confidence limits: 12 - 25 mg/L), 9.3 mg/L and 28 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

43 mg/L

EC10 or NOEC for freshwater algae:

9.3 mg/L