Reaction mass of disodium 2-[[2-hydroxy-3-[[methyl-(2-oxido-2-oxoethyl)amino]methyl]-5-nonylphenyl]methyl-methylamino and sodium 2-[(2-hydroxy-5-nonylphenyl)methyl-methylamino]acetate

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:

6 Aug 2018 to 17 Apr 2019

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:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

NA (study was performed according to guidelines).

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch: 99101243
Purity: 76.39%
Physical State/Appearance: Beige coloured paste
Expiry date: 23 November 2019
Storage: Room temperature in the dark under nitrogen

Analytical monitoring:

yes

Details on sampling:

Samples were taken at 24, 48 and 72 hours and cell densities determined using a Coulter Multisizer Particle Counter. Three determinations were made for each sample. The nominal inoculated cell concentration (5.00 x 10E3 cells/mL) was taken as the starting cell density. A sample was removed from each test and control culture (replicates pooled) at the end of the test to determine the potential effect of the test item on algal cell appearance.

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

Samples were taken for quantitative analysis from the control and each test group at 0 hours and from the pooled replicates at 72 hours. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.


Two additional samples of the 10 mg/L stock solution prepared in deionized reverse osmosis water were taken prior to the addition of the culture medium stock solutions. Two additional samples were also taken of the 10 mg/L stock solution to which the culture medium component solutions had been added were taken prior to inoculation with algal cells. For these additional samples, one sample was provided for immediate analysis at 0 hours, the other was incubated as a stability control alongside the test to provide a sample for analysis at 72 hours.

Vehicle:

no

Details on test solutions:

The test concentrations used in the definitive test were determined by a preliminary range-finding test, which exposed Pseudokirchneriella subcapitata cells to nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for 72 hours. Based on the range-finding test results, nominal test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were assigned to the definitive test.

It was considered likely that the test item was interacting with the salts in the OECD algal test medium and that this had caused measured concentrations to fall short of nominals in the range finding test. Thus, the test item was solubilised in deionized reverse osmosis water prior to the addition of the culture medium stock solutions and additional stability control groups dosed with the test item at 10 mg/L were incubated in the test design to give insight into possible causes for the decline in concentration.

A nominal amount of test item (20 mg for the definitive test, 200 mg for the range-finding test) was dispensed onto a glass slide and suspended in 2 litres of deionised reverse osmosis water (2 litres of culture medium for the range-finding test) to give a 10 mg/L (100 mg/L for the range-finding test) stock solution. The preparation was stirred via magnetic stirrer for 23 hours to ensure complete dissolution of the test item. For the definitive test, appropriate volumes of culture medium component solutions were added to 1.5 litres of the 10 mg/L stock solution and pH adjusted to 7.5 ± 0.1. A series of dilutions was made from the resulting 10 mg/L primary test medium, to give further test media containing the test item at concentrations of 3.2, 1.0, 0.32 and 0.10 mg/L. An aliquot of each test media and a negative control medium prepared in the same way, but without addition of the test item, was separately inoculated with algal suspension to give the required initial algal cell density.

Two additional variants of the 10 mg/L treatment without algae served as stability controls: one comprised the primary stock solution of the test item in deionized reverse osmosis water, the second was the primary medium comprising the primary stock solution amended by addition of the mineral components of the algal test medium. Both were incubated beside the inoculated test flasks under identical conditions.

The stock solution and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

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

Test organisms (species):

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

Details on test organisms:

Liquid cultures of Pseudokirchneriella subcapitata (strain CCAP 278/4) 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 periodic replenishment of culture medium, under constant agitation by orbital shaker and illumination at 24 ±1°C.

Prior to test initiation, a master algal culture was added to ca. 100 mL volumes of culture media to give an initial cell density of ca. 10E3 cells/mL. The flasks were plugged with polyurethane foam stoppers and constantly agitated by orbital shaker and illuminated at 24 ±1°C until algal cell density was ca. 10E4 to 10E5 cells/mL.

A positive control (potassium dichromate) was performed twice in a 12 month period to demonstrate satisfactory test conditions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

NA

Post exposure observation period:

NA

Hardness:

Not specified

Test temperature:

24 ±1ºC

pH:

0 hours: 7.6 to 7.8
72 hours: 7.6 to 8.6

Dissolved oxygen:

Not specified

Salinity:

NA

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal test concentrations: 0.10, 0.32, 1.0, 3.2, 10 mg/L
Geometric mean measured test concentrations: 0.060, 0.15, 0.41, 2.1, 8.0 mg/L

Details on test conditions:

Definitive tests were conducted in 250 mL glass conical flasks. Six flasks containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group. The control group was maintained under identical conditions but was not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 9.92E5 cells/mL. Inoculation of 500 mL of test medium with 2.5 mL of this algal suspension gave an initial nominal cell density of 5.00E3 cells/mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated at 24 ±1°C under continuous illumination provided by warm white lighting and constantly shaken at ca. 150 rpm for 72 hours.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.57 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.:

2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Limits could not be determined

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.15 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.:

0.41 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% Confidence Limits (mg/L): 0.28 - 0.40

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.41 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Test concentration verification:
In the range-finding test, chemical analysis of the 0.10, 1.0 and 10 mg/L test preparations at 0 hours showed measured test concentrations to range from 70–77% of nominal. A decline in measured test concentrations was observed at 72 hours in the range of 30–72% of nominal (42–93% of the 0-Hour measured test concentrations).

In the definitive test, analysis of the test preparations at 0 hours showed measured test concentrations to range from 96–114% of nominal. Measured test concentrations at 72 hours were in the range of 18–63% of nominal, indicating a decline during the course of the incubation. Additional samples of the 10 mg/L stock solution to which culture medium stock solutions had been added obtained measured concentrations of 97% and 56% of nominal at 0 and 72 hours, respectively. Additional samples of the 10 mg/L stock solution obtained measured concentrations of 99% and 71% of nominal at 0 and 72 hours, respectively.

A comparable reduction in test item concentration was seen at 10 mg/L in the presence of algae (102% reduced to 63% of nominal) and in the 10 mg/L stability control in algal growth medium (97% reduced to 56% of nominal). These results exclude adsorption to the algal cells as a potential cause for the observed decline. The reduction that occurred in the stock solution prepared in deionized water (99% reduced to 71% of nominal – a fall of 28%) accounts for ca. two-thirds of the decline recorded in the growth medium stability control. The remaining one third is presumed to have resulted from an interaction between the test item and the mineral constituents of the medium. Photolysis under the conditions of the test may have played a role in the background reduction observed in RO water that is assumed to have occurred in all treatments. A separate study (Fox (2019), Envigo Study Number WS61JT) indicates that hydrolysis is unlikely to have been a significant factor.

As per regulatory advice, since a decline in measured concentrations was observed, results were based on geometric mean measured concentrations in order to give a “worst case” analysis of the data.


Growth data:
Growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were shown to be affected by the presence of the test item over the 72-hour exposure period.


Validation criteria:
All OECD 201 guideline validity criteria were fulfilled. Algal cell concentration of control cultures increased by a factor of 133 after 72 hours. The mean coefficient of variation for section by section specific growth rate for the control cultures was 10%. The coefficient of variation for average specific growth rate for the control cultures over the test period was 6%.


Culture observations:
No abnormalities were detected in the control and 0.060 mg/L test cultures after 72-hours exposure. Some enlarged cells were observed in test cultures at 0.15 mg/L and severely misshapen cells were observed in test cultures at 0.41 mg/L. No intact cells were observed in the test cultures at 2.1 and 8.0 mg/L.


Water quality criteria:
Control cultures pH increased from pH 7.8 at 0 hours to pH 8.2 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.


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, 0.060 and 0.15 mg/L test cultures were green or slightly pale green dispersions. The 0.41 mg/L test cultures were extremely pale green dispersions whilst the 2.1 and 8.0 mg/L test cultures remained as clear colourless solutions. There was no conspicuous visible evidence of precipitation of the test item in any of the test vessels during the test.

Results with reference substance (positive control):

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the positive control (potassium dichromate) was tested between 12 November 2018 and 3 December 2018, using nominal concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L and exposure conditions and data evaluation similar to those in the definitive test. Tests resulted in 72-hour ECr50 and EyC50 values of 1.5 mg/L (95% confidence limits: 1.3 – 1.7 mg/L) and 0.76 mg/L (95% confidence limits: 0.69 – 0.85 mg/L), respectively. The 72-hour NOEC and LOEC values based on both growth rate and yield were 0.5 and 1.0 mg/L, respectively. These results were within the normal ranges for potassium dichromate.

Reported statistics and error estimates:

Statistical analysis of the growth rate data was conducted using one-way ANOVA incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control. There were no statistically significant decreases in growth rate (P≥0.05), between the control, 0.060 and 0.15 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) from the control, therefore the NOEC based on growth rate was 0.15 mg/L. Correspondingly the LOEC based on growth rate was 0.41 mg/L.

There were no statistically significant decreases in yield (P≥0.05), between the control, 0.060 and 0.15 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) from the control, therefore the NOEC based on yield was 0.15 mg/L. Correspondingly the LOEC based on yield was 0.41 mg/L.

Effect of test item on growth of Pseudokirchneriella subcapitata over a 72 -hour exposure period

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	2.0	*	0.15	0.41
Yield	0.33	0.28 – 0.40	0.15	0.41

 

*95% confidence limits could not be determined for the ErC50 endpoint because of a poor fit of the data to the models available.

 

Cell densities and percentage inhibition of growth from the range-finding test

Nominal Concentration (mg/L)		Cell Densities* (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	6.69E+03	2.36E+06	-	-
	R2	7.86E+03	2.55E+06
	Mean	7.27E+03	2.45E+06
0.10	R1	6.92E+03	2.70E+06	[1]	[9]
	R2	7.51E+03	2.63E+06
	Mean	7.22E+03	2.66E+06
1.0	R1	7.25E+03	7.92E+05	22	72
	R2	7.39E+03	5.97E+05
	Mean	7.32E+03	6.95E+05
10	R1	6.98E+03	3.30E+04	74	99
	R2	6.10E+03	2.70E+04
	Mean	6.54E+03	3.00E+04
100	R1	7.30E+03	1.66E+04	84	100
	R2	7.27E+03	2.02E+04
	Mean	7.29E+03	1.84E+04

* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks

R = Replicate

- = Not applicable

[ ] = Increase in growth compared to controls 

 

 

Cell densities and pH values in the definitive test

Geometric Mean Measured Test Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0 Hours	24Hours	48Hours	72Hours	72Hours
Control	R1	7.8	2.50E+04	1.41E+05	8.06E+05	8.2
	R2		2.38E+04	1.30E+05	4.88E+05
	R3		2.52E+04	1.58E+05	8.20E+05
	R4		2.92E+04	1.57E+05	8.13E+05
	R5		2.40E+04	1.47E+05	5.57E+05
	R6		2.44E+04	1.45E+05	5.03E+05
	Mean		2.53E+04	1.46E+05	6.64E+05
0.060	R1	7.8	2.60E+04	1.69E+05	8.82E+05	8.5
	R2		3.14E+04	1.68E+05	1.00E+06
	R3		3.23E+04	1.75E+05	1.10E+06
	Mean		2.99E+04	1.71E+05	9.95E+05
0.15	R1	7.7	2.89E+04	1.58E+05	7.80E+05	8.6
	R2		2.87E+04	1.50E+05	7.24E+05
	R3		2.80E+04	1.39E+05	6.17E+05
	Mean		2.85E+04	1.49E+05	7.07E+05
0.41	R1	7.7	2.59E+04	1.18E+05	2.81E+05	8.0
	R2		2.90E+04	1.09E+05	2.30E+05
	R3		3.02E+04	1.07E+05	2.57E+05
	Mean		2.84E+04	1.11E+05	2.56E+05
2.1	R1	7.6	1.90E+04	4.35E+04	5.53E+04	7.8
	R2		1.71E+04	4.51E+04	5.60E+04
	R3		1.95E+04	4.34E+04	4.13E+04
	Mean		1.85E+04	4.40E+04	5.09E+04
8.0	R1	7.6	9.68E+03	1.50E+04	1.34E+04	7.6
	R2		9.36E+03	1.59E+04	1.43E+04
	R3		9.15E+03	1.51E+04	1.48E+04
	Mean		9.40E+03	1.53E+04	1.42E+04

* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks

R = Replicate 

 

Daily specific growth rates for the control cultures in the definitive test

Treatment		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 to 1	Day 1 to 2	Day 2 to 3
Control	R1	0.067	0.072	0.073
	R2	0.065	0.071	0.055
	R3	0.067	0.076	0.069
	R4	0.074	0.070	0.068
	R5	0.065	0.076	0.055
	R6	0.066	0.074	0.052
	Mean	0.067	0.073	0.062

R = Replicate 

 

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	0.071	-	8.01E+05		-
	R2	0.064		4.83E+05
	R3	0.071		8.15E+05
	R4	0.071		8.08E+05
	R5	0.065		5.52E+05
	R6	0.064		4.98E+05
	Mean	0.068		6.59E+05
	SD	0.004		1.64E+05
0.060	R1	0.072	[6]	8.77E+05
	R2	0.074	[9]	9.97E+05
	R3	0.075	[10]	1.09E+06
	Mean	0.074	[8]	9.90E+05		[50]
	SD	0.002		1.09E+05
0.15	R1	0.070	[3]	7.75E+05
	R2	0.069	[1]	7.19E+05
	R3	0.067	1	6.12E+05
	Mean	0.069	[1]	7.02E+05		[7]
	SD	0.002		8.26E+04
0.41	R1	0.056	18	2.76E+05
	R2	0.053	22	2.25E+05
	R3	0.055	19	2.52E+05
	Mean	0.055	20	2.51E+05		62
	SD	0.002		2.51E+04
2.1	R1	0.033	51	5.03E+04
	R2	0.034	50	5.10E+04
	R3	0.029	57	3.63E+04
	Mean	0.032	53	4.59E+04		93
	SD	0.003		8.26E+03
8.0	R1	0.014	79	8.38E+03
	R2	0.015	78	9.34E+03
	R3	0.015	78	9.84E+03
	Mean	0.015	78	9.19E+03		99
	SD	0.001		7.46E+02

* 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 

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Pseudokirchneriella subcapitata to the test item over a 72-hour period, based on geometric mean measured test concentrations, resulted in ErC50 and EyC50 values of 2.0 and 0.33 mg/L respectively for growth inhibition. The 72-hour NOEC and LOEC values based on both growth rate and yield were 0.15 and 0.41 mg/L, respectively.

Executive summary:

The effect of the test item, Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate, on the growth of the green alga Pseudokirchneriella subcapitata was assessed at a GLP accredited laboratory according to the OECD Guideline 201, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”. All validity criteria were fulfilled. 

 

Following a preliminary range-finding test, P. subcapitata was exposed to nominal test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L for 72-hours, under constant illumination and shaking at a temperature of 24 ±1°C. Algal population samples were removed from each control and treatment group daily and cell concentrations determined using a Coulter® Multisizer Particle Counter. Chemical analysis of test samples showed a decline in measured test concentrations throughout the tests, therefore as per regulatory guidance it was considered justifiable to base study results on geometric mean measured test concentrations to give a “worst-case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.060, 0.15, 0.41, 2.1 and 8.0 mg/L (between 41% and 80% of the nominal test concentrations).

 

Exposure of P. subcapitata to the test item over a 72-hour period resulted in ErC₅₀ and EyC₅₀ values of 2.0 and 0.33 mg/L respectively for growth inhibition, based on geometric mean measured test concentrations. The 72-hour NOEC and LOEC values based on both growth rate and yield were 0.15 and 0.41 mg/L, respectively. Positive control (potassium dichromate) test results were within the normal ranges, thus demonstrating satisfactory conditions of the test.

Description of key information

The inhibitory effect of the test item, Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate, on the growth of the green alga Pseudokirchneriella subcapitata was assessed at a GLP accredited laboratory according to the OECD Guideline 201, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”. All validity criteria were fulfilled. 

 

Following a preliminary range-finding test,P. subcapitatawas exposed to nominal test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L for 72-hours, under constant illumination and shaking at a temperature of 24 ±1°C. Algal population samples were removed from each control and treatment group daily and cell concentrations determined using a Coulter® Multisizer Particle Counter. Chemical analysis of test samples showed a decline in measured test concentrations throughout the tests, therefore as per regulatory guidance it was considered justifiable to base study results on geometric mean measured test concentrations to give a “worst-case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.060, 0.15, 0.41, 2.1 and 8.0 mg/L (between 41% and 80% of the nominal test concentrations).

 

Exposure of P. subcapitata to the test item over a 72-hour period resulted in ErC₅₀ and EyC₅₀ values of 2.0 and 0.33 mg/L respectively for growth inhibition, based on geometric mean measured test concentrations. The 72-hour NOEC and LOEC values based on both growth rate and yield were 0.15 and 0.41 mg/L, respectively. Positive control (potassium dichromate) test results were within the normal ranges, thus demonstrating satisfactory conditions of the test.

Key value for chemical safety assessment

EC50 for freshwater algae:

2 mg/L

EC10 or NOEC for freshwater algae:

0.15 mg/L