L-Arginine, N2-(2,3-dihydroxypropyl)-, monohydrochloride

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:

18 February 2020 - XX August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with GLP and to the relevant OECD Test Guideline

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

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentration and stability of the test item in the test preparations (100 mg/L only in the full main study) were verified by chemical analysis at 0 and 72 h.
- Sampling method: Samples were taken from the control and test groups from the bulk test preparation at 0 h and from the pooled replicates at 72 h for immediate quantitative analysis. A set of duplicate samples were taken at 0 and 72 h and stored frozen for further analysis if necessary.
- Sample storage conditions before analysis: The 0 h test samples were analysed on the day of sampling and stored refrigerated at approx. 4 ºC prior to analysis. The 72 h test samples were analysed on the day of sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on the range-finder results (see 'Details on test conditions'), a limit test was conducted. A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1000 mL to give a 100 mg/L stock solution. An aliquot (1000 mL) of the stock solution was separately inoculated with algal suspension (3.1 mL) to give an initial nominal cell density of 5.00 x 10^3 cells/mL.
- Eluate: N/A
- Differential loading: N/A
- Controls: Yes: 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. A concurrent negative control group was maintained under identical conditions but not exposed to the test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The stock solution prepared concentration was inverted several times to ensure adequate mixing and homogeneity. Preliminary solubility work showed that inversion was sufficient to allow the test item to fully dissolve.
- Other relevant information: N/A

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Cultures of R. subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): Approx. 3 - 4 days old
- Method of cultivation: Approx.3 to 4 days before the test start, inoculum cultures of algae was set up at an initial cell density of approx. 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 ºC until the algal cell density was approximately 10^5 to 10^6 cells/mL.

ACCLIMATION
- Acclimation period: N/A
- Culturing media and conditions (same as test or not): The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
- Any deformed or abnormal cells observed: None reported

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

Guideline specified

Post exposure observation period:

None - not required.

Test temperature:

24 ºC

pH:

Control: 8.0 - 8.2
100 mg/L: 7.3 - 7.8

Dissolved oxygen:

Not reported

Salinity:

Not reported/not relevant

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal: 100 mg/L; procedural recoveries 1 and 2: 105 %
Measured:
0 h: 99.6 mg/L; procedural recovery 1: 111 % and 2: 101 %;
72 h: 109 mg/L
The concentrations for the procedural recovery are the actual fortified concentrations. The corresponding percentages of nominal concentration values relate to the percentage of fortified concentration recovered. Analysis of the test preparations at 0 and 72 h showed measured test concentrations to be near nominal, therefore the results are based on the nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flask
- Type: Closed - plugged with polyurethane foam bungs to reduce evaporation
- Material, size, headspace, fill volume: Glass, 250 mL, containing 100 mL test preparation
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): None, static test.
- Initial cells density: 5.00 x 10^3 cells/mL (nominal)
- Control end cells density: The control group was maintained under identical conditions but not exposed to the test item. Mean cell density at 0 h: 5.59 x 10^3; at 72 h: 3.37 x 10^5
- No. of organisms per vessel: See "initial and end cell densities"
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/A

GROWTH MEDIUM
- Standard medium used: yes (AAP-medium, U.E. EPA). The culture medium was prepared using reverse osmosis purified deionized water (Elga Optima 15+ or Elga Purelab Option R-15 BP)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Not specified
- Culture medium different from test medium: No, the culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
- Intervals of water quality measurement: pH was measured for the control and each test preparation at initiation of the test and after 72 h exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. Appearance of the test media was also recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: Not reported
- Adjustment of pH: pH was adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Photoperiod: Continuous illumination provided by warm white lighting (380 to 730 nm)
- Light intensity and quality: Approx. 7000 lux
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron incubator) at 24 °C and constantly shaken at approximately 150 rpm for 72 h.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples of the algal populations were removed daily (at 24, 47 and 72 h) and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter (v3.53). Three determinations were made for each sample. Growth rate and yield data were inspected and ECx values determined.
- Chlorophyll measurement: N/A
- Other: Shape and size of algal cells was inspected microscopically and any abnormalities recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: N/A
- Justification for using less concentrations than requested by guideline: N/A

- Range finding (R-F) study:
The range-finding test was conducted by exposing R. subcapitata cells to nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 h. The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium. A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1000 mL to give a 100 mg/L stock solution, from which a series of dilutions was made to give further stock solutions of 0.10, 1.0 and 10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (2.3 mL) to give an initial nominal cell density of 5.00 x 103 cells/mL. Prior to use, the test item was heated at 80 ºC to aid weighing. The stock solutions and each test concentration were inverted several times to ensure adequate mixing and homogeneity. Preliminary solubility work showed that inversion was sufficient to ensure that test item was fully dissolved. At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron incubator) at 24 oC under continuous illumination (intensity approx. 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 h.
After 72 h the cell density of each flask was determined using a Coulter® Multisizer Particle Counter. A sample of each test concentration was taken for chemical analysis at 0 and 72 h in order to determine the stability of the test item under test conditions.
- R-F test concentrations: 0.10, 1.0, 10 and 100 mg/L (nominal)
- R-F results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and
100 mg/L. Based on the result of the R-F test, a "limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no effect on algal growth was observed.

Reference substance (positive control):

yes

Remarks:

potassium dichormate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): None observed
- Unusual cell shape: None observed
- Colour differences: After 72 h, all control and test cultures were pale green dispersions.
- Flocculation: Not reported
- Adherence to test vessels: Not reported
- Aggregation of algal cells: Not reported
- Other: From the results obtained, it is concluded that the growth rate (r) and yield (y) of R. subcapitata (CCAP 278/4) were not inhibited by the presence of the test item at a nominal test concentration of 100 mg/L over the 72 h exposure period.
- Any stimulation of growth found in any treatment: Yes
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50: (0 to 72 h) 1.2 mg/L
- Other:
Exposure of Raphidocelis subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.2 mg/L; 95% confidence limits 1.1 to 1.3 mg/L
EyC50 (0 to 72 hour) : 0.51 mg/L; 95% confidence limits 0.45 to 0.58 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.125 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.25 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item based on in-house data.

Reported statistics and error estimates:

A Students t-test was carried out on the growth rate and yield data after 72 h for the control and the 100 mg/L test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package v8.2 (SAS, 1999 - 2001).

ErCx was determined as the test concentration that reduced growth rate by x %. A significant difference was found between the control and the test group, however, this was due to an increase in growth and therefore the NOEC based on growth rate was 100 mg/L.
EyCx was determined as the test concentration that reduced yield by x %. A significant difference was found between the control and test group however, this was due to an increase in yield and therefore the NOEC based on yield was 100 mg/L.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations of 100 and 105 % of nominal respectively, therefore the results are based on the nominal concentrations.

The 72 h E_rC₁₀, E_rC₂₀, E_rC₅₀ (growth rate) were all concluded to be > 100 mg/L. The 72 h E_yC₁₀, E_yC₂₀, E_yC₅₀ (yield) were all concluded to be > 100 mg/L. The NOEC was concluded to be 100 mg/L.

Table 1. Cell densities and pH values in the definitive test

Nominal Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH	Temperature ºC
		0 h	24 h	47 h	72 h	72 h	0 - 72 h
Control	R1	8.0	1.61E+04	6.40E+04	3.10E+05	8.2	24 ºC
	R2		1.99E+04	8.38E+04	3.47E+05
	R3		1.93E+04	7.89E+04	3.91E+05
	R4		1.93E+04	7.44E+04	3.51E+05
	R5		1.83E+04	6.74E+04	3.12E+05
	R6		1.84E+04	8.82E+04	3.89E+05
	Mean		1.85E+04	7.61E+04	3.50E+05
100	R1	7.3	2.35E+04	1.23E+05	6.90E+05	7.8	24 ºC
	R2		2.59E+04	1.21E+05	6.61E+05
	R3		2.47E+04	8.84E+04	4.47E+05
	R4		2.17E+04	8.76E+04	3.73E+05
	R5		2.34E+04	1.21E+05	5.68E+05
	R6		2.00E+04	1.04E+05	6.13E+05
	Mean		2.32E+04	1.08E+05	5.59E+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

Table 2. Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth rate (cells/mL/h)		Yield (cells/mL)
		0 to 72 h	% Inhibition	0 to 72 h	% Inhibition*
Control	R1	0.057	-	3.05E+05	-
	R2	0.059		3.42E+05
	R3	0.061		3.86E+05
	R4	0.059		3.46E+05
	R5	0.057		3.07E+05
	R6	0.060		3.84E+05
	Mean	0.059		3.45E+05
	SD	0.002		3.51E+04
100	R1	0.068	[15]	6.85E+05
	R2	0.068	[15]	6.56E+05
	R3	0.062	[5]	4.42E+05
	R4	0.060	[2]	3.68E+05
	R5	0.066	[12]	5.63E+05
	R6	0.067	[14]	6.08E+05
	Mean	0.065	[11]	5.54E+05	[61]
	SD	0.003	-	1.25E+05

*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

- = N/A

Validity criteria fulfilled:

yes

Remarks:

The control culture cell concentration increased by a factor of 70 after 72 h. The mean coefficient of variation for section by section specific growth rate for the control cultures was 8 %. The coefficient of variation for average specific growth rat

Conclusions:

Based on the conditions of the test, the test item ErC50 and EyC50 were determined to be > 100 mg/L and the NOEC was determined to be ≥ 100 mg/L.

Executive summary:

A test was performed in accordance with OECD 201 (2006), in order to determine the aquatic toxicity of the test item to algae.

Following a preliminary range-finding test, Raphidocelis subcapitata (strain CCAP 278/4) was exposed to an aqueous solution of the test item at a nominal concentration of 100 mg/L (6 replicate flasks) for 72 h, under constant illumination and shaking at a temperature of 24 °C. Inoculation of 1000 mL of test medium with 3.1 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 cells per mL.

Samples of the algal populations were removed daily (24, 47 and 72 h) and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded. pH was recorded at the start and end of the test.

A concurrent negative control was run in the test, and 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.

Chemical analysis of the test preparations at 0 and 72 h showed measured test concentrations to be 100 and 105 % of nominal respectively and so the results are based on nominal test concentrations only.

The results of the test were considered valid based on fulfillment of the following performance criteria:

 

• The  data demonstrated that the cell concentration of the control cultures increased by a factor of 70 after 72 h. This increase was in line with the OECD Guideline that states the increase must be at least by a factor of 16 after 72 h (nominal cell density of control at 0 h: 5.00 x 10³ cells/mL; mean cell density of control at 72 h: 3.50 x 10⁵ cells/mL).

• The mean coefficient of variation for section by section specific growth rate for the control cultures was 8 % (i.e. <= 35 % stated in OECD Guideline).


• 
  
  
  

  
  

  
  

  The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 h) was 3 % (i.e. <= 7 % stated in OECD Guideline).

  
  

  
  

  
  
  
  

Following microscopic inspection, no abnormalities were detected in any test and control cultures. Growth rate (r) and yield (y) of R. subcapitata were not affected by the presence of the test item at a nominal test concentration of 100 mg/L over the 72 h exposure period. EC₅₀ values were based on the nominal test concentrations of greater than 100 mg/L. The NOEC was concluded to be ≥ 100 mg/L. The E_rC₅₀, E_yC₅₀, E_rC₁₀ and E_yC₁₀ were all concluded to be > 100 mg/L.

Under the conditions of this study, the test item does not cause toxicity to freshwater algae and would not be classified under CLP Regulation (EC) 1272/2008 based on freshwater algae alone.

Description of key information

OECD 201:

E_rC₅₀ > 100 mg/L, E_rC₁₀ > 100 mg/L and NOEC ≥ 100 mg/L

E_yC₅₀ > 100 mg/L, E_yC₁₀ > 100 mg/L and NOEC ≥ 100 mg/L

(Ablitt, 2020)

Additional information

A test was performed in accordance with OECD 201 (2006), in order to determine the aquatic toxicity of the test item to algae.

Following a preliminary range-finding test, Raphidocelis subcapitata (strain CCAP 278/4) was exposed to an aqueous solution of the test item at a nominal concentration of 100 mg/L (6 replicate flasks) for 72 h. A concurent negative control was run and results from a seperate study was available for a positive control.

Samples of the algal populations were removed daily (24, 47 and 72 h) and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Chemical analysis of the test preparations at 0 and 72 h showed measured test concentrations to be 100 and 105 % of nominal, respectively, so the results are based on nominal test concentrations.

The results of the test were considered valid based on fulfillment of the following performance criteria as precribed by the OECD 201 (2006 Annex 5 corrected 2011):

• The  data demonstrated that the cell concentration of the control cultures increased by a factor of 70 after 72 h. This increase was in line with the OECD Guideline that states the increase must be at least by a factor of 16 after 72 h (nominal cell density of control at 0 h: 5.00 x 10³ cells/mL; mean cell density of control at 72 h: 3.50 x 10⁵ cells/mL).

• The mean coefficient of variation for section by section specific growth rate for the control cultures was 8 % (i.e. <= 35 % stated in OECD Guideline).


• 
  
  
  

  
  

  
  

  The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 h) was 3% (i.e. <= 7 % stated in OECD Guideline).

  
  

  
  

  
  
  
  

Following microscopic inspection, no abnormalities were detected in any test and control cultures. Growth rate (r) and yield (y) of R. subcapitata were not affected by the presence of the test item at a nominal test concentration of 100 mg/L over the 72 h exposure period. The NOEC was concluded to be ≥ 100 mg/L. The E_rC₅₀, E_yC₅₀, E_rC₁₀ and E_yC₁₀ were all concluded to be > 100 mg/L.

Under the conditions of this study, no effects on growth or yield were observed and therefore it was concluded that the test item does not cause toxicity to freshwater algae. The test item would therefore not be classified under CLP Regulation (EC) 1272/2008 for aquatic toxicity, based on freshwater algae alone.