1,2-dihydro-5-nitro-3H-1,2,4-triazol-3-one

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:

07 January 2020 - 02 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EC) No 761/2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Chemring Nobel, Lot DDP17K0188-0002, Charge no. 180004
- Expiration date of the lot/batch: 05 February 2028
- Purity test date: Inspection date 22 February 2018


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group (unadjusted and pH adjusted) from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for immediate quantitative analysis. A set of duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

The 0-Hour test samples were prepared on the day of sampling then analysed after storage at approximately 4°C. The 72-Hour test samples were analysed on the day of sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Nominal amounts of test item (50 and 16 mg) were separately dissolved in culture medium with the aid of ultrasonication for approximately 10 minutes and the volume adjusted to 500 mL to give 100 and 32 mg/L stock solutions respectively. A series of dilutions were made from these stock solutions to give further stock solutions of 10, 3.2 and 1.0 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (2.6 mL) to give an initial nominal cell density of 5.00 x 10^3 cells/mL. No adjustment to pH was made.

A further nominal amount of test item (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 10 minutes and the volume adjusted to 1 liter to give a 100 mg/L stock solution. The pH of this stock solutions was measured (pH 3.8) and adjusted to 7.6. The pH adjusted stock solution was inoculated with algal suspension (5.8 mL) to give an initial nominal cell density of 5.00 x 10^3 cells/mL.

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 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:

TEST ORGANISM
The test was carried out using Raphidocelis subcapitata strain CCAP 278/4. Liquid cultures of Raphidocelis 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 approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1°C until the algal cell density was approximately 10^5 to 10^6 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24±1°C

pH:

A concentration dependent decline in pH was observed in the unadjusted test preparations at the start of the test in the range of pH 8.0 at 1.0 mg/L through to pH 3.7 at 100 mg/L
The pH of the pH adjusted test preparation varied between 7.5 and 8.0.

Nominal and measured concentrations:

Nominal concentrations (mg/L): 0 (Control), 1.0, 3.2, 10, 32, 100, 100 (pH adjusted)
Measured concentrations, 0 hours (mg/L): Not detected, 1.03, 3.19, 9.58, 32.3, 97.6, 99.9
Measured concentrations, 72 hours (mg/L): Not detected, 0.983, 3.26, 10.1, 33.6, 99.6, 101

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass conical flasks, 250 mL capacity, 100 mL fill volume
- Type: Pluged with polyurethane foam bung
- Initial cells density: 5 x 10^3 cells/mL
- Control end cells density:
- No. of vessels per concentration: 3 (non-pH adjusted concentrations), 6 (100 mg/L pH-adjusted level)
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: Not specified
- Adjustment of pH: The majority of the test concentrations were not pH adjusted, however replicate preparations of the highest concentration level (100 mg/L) were pH adjusted to pH 7.5±0.1 with 0.1N NaOH prior to inoculation with algae.
- Photoperiod: Continuous illumination
- Light intensity and quality:Approximately 7000 lux with warm white lighting (380 to 730 nm)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: 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 (5.00 x 103 cells/mL) was taken as the starting cell density.
- Other: 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L. Note that an initial range-finding test showed a flat response curve and potential contamination of the 0.1 mg/L level, so a second range-finding test was conducted using the same concentration levels.
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

A positive control test using potassium dichromate as the reference item was performed twice in a 12 month period to demonstrate satisfactory conditions of the test.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

19 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: pH Unadjusted treatment group

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: pH Unadjusted treatment group

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: pH Unadjusted treatment group

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: pH Adjusted treatment group

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: pH Adjusted treatment group

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: pH Unadjusted treatment group

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: pH Unadjusted treatment group

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: pH Unadjusted treatment group

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: pH Adjusted treatment group

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: pH Adjusted treatment group

Details on results:

- Exponential growth in the control: Cell concentration of the control cultures increased by a factor of 188 after 72 hours; this increase was in line with the OECD Guideline which states that the enhancement must be at least by a factor of 16 after 72 hours.

- Observation of abnormalities (for algal test):
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or unadjusted test cultures at 1.0, 3.2 and 10 mg/L. Cell debris was observed in the unadjusted 32 mg/L test cultures whilst no intact cells were observed in the unadjusted 100 mg/L test cultures. No abnormities were detected in the pH adjusted 100 mg/L test cultures.

- Any stimulation of growth found in any treatment: No

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start of the test all control cultures were observed to be clear colorless solutions. The unadjusted test cultures were observed to range from being clear colorless solutions at 1.0 mg/L through to pale yellow/green solutions at 100 mg/L. The pH adjusted 100 mg/L test cultures were observed to be slightly pale yellow/green solutions.
After the 72-Hour test period all control, 1.0, 3.2 and 10 mg/L test cultures were observed to be green dispersions. The 32 mg/L test cultures were observed to be pale yellow/green solutions whilst the unadjusted 100 mg/L test cultures were observed to be yellow/green solutions. The pH adjusted 100 mg/L test cultures were observed to be bright yellow/green solutions.


- Effect concentrations exceeding solubility of substance in test medium: Not applicable

Results with reference substance (positive control):

The positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. The positive control was conducted between 16 July 2019 and 19 July 2019. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Raphidocelis subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 to 72 hour) : 1.3 mg/L; 95% confidence limits 1.2 to 1.4 mg/L
EyC50 (0 to 72 hour) : 0.55 mg/L; 95% confidence limits 0.49 to 0.63 mg/L

No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item*.

* In-house data from the last 5 years shows a mean ErC50 value of 1.4 mg/L (standard deviation = 0.22) and a mean EyC50 value of 0.67 mg/L (standard deviation = 0.12).

Reported statistics and error estimates:

Inhibition of Growth Rate:
Unadjusted Treatment Groups
ErC10 (0 to 72 hour) : 12 mg/L
ErC20 (0 to 72 hour) : 15 mg/L
ErC50 (0 to 72 hour) : 19 mg/L*
where ErCx is 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 the 1.0, 3.2 and 10 mg/L test concentrations; however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 10 mg/L. Correspondingly the LOEC based on growth rate was 32 mg/L.

pH adjusted Treatment Group
ErC10 (0 to 72 hour) : >100 mg/L
ErC20 (0 to 72 hour) : >100 mg/L
ErC50 (0 to 72 hour) : >100 mg/L
where ErCx is the test concentration that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100 mg/L test concentration using a Students t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). A statistically significant difference (P<0.05) was observed between the control and 100 mg/L test concentration. However, inspection of the data shows that only 4% inhibition of growth rate occurred (mean value for the test group) which is not considered to be significant. As such the NOEC based on growth rate was considered to be 100 mg/L.

Refer to Any other information on results incl. tables for Statistics for Inhibition of Yield.

Reported statistics and error estimates for Inhibition of Yield:
Unadjusted Treatment Groups
EyC10 (0 to 72 hour) : 9.0 mg/L
EyC20 (0 to 72 hour) : 11 mg/L
EyC50 (0 to 72 hour) : 14 mg/L; 95% confidence limits 13 to 15 mg/L
where EyCx is the test concentration that reduced yield by x%.
It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Statistical analysis of the yield data was carried out as for the pH unadjusted treatment groups. There were no statistically significant differences (P≥0.05), between the control and the 1.0, 3.2 and 10 mg/L test concentrations; however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 10 mg/L. Correspondingly the LOEC based on yield was 32 mg/L.

pH adjusted Treatment Group
EyC10 (0 to 72 hour) : <100 mg/L
EyC20 (0 to 72 hour) : >100 mg/L
EyC50 (0 to 72 hour) : >100 mg/L
where EyCx is the test concentration that reduced yield by x%.
It was not possible to calculate an EyL10 value as 18% inhibition of yield was observed.
Statistical analysis of the yield data was carried out as for the pH unadjusted treatment groups. A statistically significant difference (P<0.05) was observed between the control and 100 mg/L test concentration and therefore the NOEC based was yield was determined to be less than 100 mg/L.

 

Cell Densities and Percentage Inhibition of Growth from the Initial Range-finding Test

Nominal Concentration (mg/L)		Cell Densities* (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	5.72E+03	9.65E+05	-	-
	R2	6.10E+03	9.84E+05
	Mean	5.91E+03	9.75E+05
0.10	R1	6.72E+03	9.86E+05	10	26
	R2	7.33E+03	4.62E+05
	Mean	7.03E+03	7.24E+05
1.0	R1	7.39E+03	6.31E+05	10	32
	R2	5.87E+03	6.97E+05
	Mean	6.63E+03	6.64E+05
10	R1	7.13E+03	8.23E+05	7	18
	R2	6.69E+03	7.79E+05
	Mean	6.91E+03	8.01E+05
100	R1	6.83E+03	3.07E+04	83	99
	R2	8.24E+03	5.25E+03
	Mean	7.54E+03	1.80E+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

Cell Densities and Percentage Inhibition of Growth from the Second Range-finding Test

Nominal Concentration (mg/L)		Cell Densities* (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	4.96E+03	1.30E+06	-	-
	R2	5.37E+03	1.52E+06
	Mean	5.16E+03	1.41E+06
0.10	R1	5.34E+03	1.05E+06	5	20
	R2	5.78E+03	1.21E+06
	Mean	5.56E+03	1.13E+06
1.0	R1	5.60E+03	1.18E+06	4	11
	R2	5.60E+03	1.33E+06
	Mean	5.60E+03	1.25E+06
10	R1	5.19E+03	1.23E+06	4	8
	R2	6.75E+03	1.35E+06
	Mean	5.97E+03	1.29E+06
100	R1	8.36E+03	5.00E+04	76	99
	R2	5.75E+03	5.40E+03
	Mean	7.05E+03	2.77E+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

Cell Densities and pH Values in the Definitive Test

Nominal Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0 Hours	24 Hours	48 Hours	72 Hours	72 Hours
Control	R1	8.0	2.56E+04	1.53E+05	7.43E+05	9.0
	R2		3.47E+04	1.76E+05	9.94E+05
	R3		3.37E+04	1.91E+05	1.21E+06
	R4		3.37E+04	1.80E+05	1.01E+06
	R5		2.90E+04	1.47E+05	8.25E+05
	R6		2.92E+04	1.64E+05	8.72E+05
	Mean		3.10E+04	1.69E+05	9.42E+05
1.0	R1	8.0	2.85E+04	1.66E+05	8.61E+05	9.1
	R2		3.02E+04	1.61E+05	8.87E+05
	R3		3.56E+04	1.88E+05	1.07E+06
	Mean		3.14E+04	1.72E+05	9.39E+05
3.2	R1	7.8	3.29E+04	1.39E+05	7.69E+05	9.0
	R2		3.37E+04	1.63E+05	8.85E+05
	R3		3.92E+04	1.89E+05	9.27E+05
	Mean		3.53E+04	1.63E+05	8.60E+05
10	R1	7.4	2.75E+04	1.49E+05	7.65E+05	9.0
	R2		3.14E+04	1.41E+05	8.38E+05
	R3		3.19E+04	1.69E+05	7.59E+05
	Mean		3.03E+04	1.53E+05	7.87E+05
32	R1	5.0	6.31E+03	7.71E+03	2.51E+04	4.4
	R2		5.60E+03	2.99E+03	5.13E+03
	R3		4.22E+03	2.17E+03	3.43E+03
	Mean		5.38E+03	4.29E+03	1.12E+04
100	R1	3.7	4.25E+03	2.70E+03	5.60E+03	3.7
	R2		5.31E+03	3.20E+03	6.34E+03
	R3		4.75E+03	2.58E+03	5.60E+03
	Mean		4.77E+03	2.83E+03	5.85E+03
100 pH Adjusted	R1	7.5	3.35E+04	1.44E+05	6.49E+05	8.0
	R2		3.56E+04	1.49E+05	7.46E+05
	R3		2.98E+04	1.62E+05	8.25E+05
	R4		2.98E+04	1.59E+05	8.16E+05
	R5		3.92E+04	1.71E+05	8.53E+05
	R6		2.87E+04	1.21E+05	7.45E+05
	Mean		3.28E+04	1.51E+05	7.72E+05

*  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	0.068	0.074	0.066
	R2	0.081	0.068	0.072
	R3	0.080	0.072	0.077
	R4	0.080	0.070	0.072
	R5	0.073	0.068	0.072
	R6	0.074	0.072	0.070
	Mean	0.076	0.071	0.072

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.069	-	7.38E+05	-
	R2	0.074		9.89E+05
	R3	0.076		1.20E+06
	R4	0.074		1.01E+06
	R5	0.071		8.20E+05
	R6	0.072		8.67E+05
	Mean	0.073		9.37E+05
	SD	0.003		1.64E+05
1.0	R1	0.072	1	8.56E+05
	R2	0.072	1	8.82E+05
	R3	0.074	[1]	1.06E+06
	Mean	0.073	0	9.34E+05	0
	SD	0.001		1.12E+05
3.2	R1	0.070	4	7.64E+05
	R2	0.072	1	8.80E+05
	R3	0.073	0	9.22E+05
	Mean	0.072	2	8.55E+05	9
	SD	0.002		8.17E+04
10	R1	0.070	4	7.60E+05
	R2	0.071	3	8.33E+05
	R3	0.070	4	7.54E+05
	Mean	0.070	4	7.82E+05	16
	SD	0.001		4.42E+04
32	R1	0.022	70	2.01E+04
	R2	0.000	100	1.33E+02
	R3	-0.005	107	-1.57E+03
	Mean	0.006	92	6.21E+03	99
	SD	0.014		1.20E+04
100	R1	0.002	97	6.03E+02
	R2	0.003	96	1.34E+03
	R3	0.002	97	6.03E+02
	Mean	0.002	97	8.47E+02	100
	SD	0.001		4.23E+02
100 pH Adjusted	R1	0.068	7	6.44E+05
	R2	0.070	4	7.41E+05
	R3	0.071	3	8.20E+05
	R4	0.071	3	8.11E+05
	R5	0.071	3	8.48E+05
	R6	0.069	5	7.40E+05
	Mean	0.070	4	7.67E+05	18
	SD	0.001		7.48E+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

- = Not applicable

[ ] = Increase in growth as compared to the control

Validation Criteria

Nominal cell density of control at 0 hours	5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours	9.42 x 10^5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 6% 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 to 72 hour) was 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Raphidocelis subcapitata has been investigated over a 72-Hour period under both unadjusted and pH adjusted conditions.

Exposure of Raphidocelis subcapitata to the test item with no pH adjustment gave the following results:
Growth rate: EC50 = 19 mg/L; NOEC = 10 mg/L
Yield: EC50 = 14 mg/L; NOEC = 10 mg/L

Exposure of Raphidocelis subcapitata to the test item with pH adjustment gave the following results:
Growth rate: EC50 > 100 mg/L; NOEC = 100 mg/L
Yield: EC50 > 100 mg/L; NOEC < 100 mg/L

In comparing the results obtained from both tests it was evident that the acidic nature of the test item had a significant effect on algal growth. The pH values in the unadjusted treatment groups ranged from 8.0 at 1.0 mg/L through to 3.7 at 100 mg/L. The pH of the adjusted treatment group was determined to be 3.8 at the time of preparation and was adjusted to 7.6 prior to inoculation with algae.
The results of the unadjusted test can be considered to represent the effects of the test item in the immediate vicinity of environmental release particularly where there is little or no pH buffering capacity.
The results of the pH adjusted test can be considered to represent the effects of the test item when released in to the aquatic environment and transported away from the direct exposure area, or when released onto a well buffered environment.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

According to the Biotechnology Research Institute of NRC, 2010, the 96h EC50 was found to be 2323.7 mg/L.

The toxicity to aquatic algae was also examined by AnalyCen Ecotox AS. The72 hours EC50 value for the test substance NTO, Nitrotriazolone was found to be 113 mg/L.

According to Haley Mark V.,Kuperman Roman G., Checkai Ronald T. (Aquatic Toxicity of 3-Nitro-l,2,4-triazol-5-one ), 96h NOEC for the test substance to Scenedesmus capricornutum is 1375 mg/L; 96h LOEC for the test substance to Scenedesmus capricornutum is 2680 mg/L.

The calculation by EPIWIN was showing EC50 of 1329.272 mg/L after 96 hours exposure.

The toxicity of NTO to aquatic plants was assessed (Covance, 2020) according to OECD test guideline 201 and EC method C.3. Cultures of Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) were exposed to NTO at nominal concentrations of 1, 3.2, 10, 32 and 100 mg/L for 72 hours; a supplementary 100 mg/L concentration level. adjusted to approximately neutral pH prior to incubation was also tested concurrently.

In the groups without pH adjustment the 72 -hour EC50 was 19 mg/L in terms of growth rate and 14 mg/L in terms of yield. The NOEC was 10 mg/L in terms of both rate and yield.

In the pH-adjusted group, the EC50 in terms of growth rate and yield was > 100 mg/L. The NOEC in terms of growth rate was 100 mg/L. A NOEC could not be calculated in terms of yeild as a statistically significant difference between the 100 mg/L level and the control was observed.

It was observed that the test material had caused a significant decrease in the pH in the unadjusted groups; it is considered that this may be representative of the situation which may arise if the substance were released into a closed aquatic system with no significant buffering capacity and no dilution over time. It is considered that this situation is unlikely to be representative of actual environmental release; actual environmental release into fresh or marine water would typically be associated with significant dilution and / or pH buffering from the aquatic environment. On this basis the results of the pH-adjusted test group will be used for the purposes of hazard assessment.