Orthoboric acid, compound with 2,2',2''-nitrilotriethanol

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:

14 December 2017 to 14 January 2018

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:

2011

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- For the range-finding and definitive tests, samples (approximately 150 mL) of all test solutions (exposure and control) were collected in 500 mL new plastic containers (no preservative).
- Samples were collected immediately prior to test commencement (0 hour before the addition of algae) and at test termination (72 hour, algae present, from pooled replicates) for analysis.
- All samples were stored at 4 ± 2°C in the dark prior to pick up and delivery to the laboratory for analysis.

Vehicle:

no

Details on test solutions:

- Individual test solutions of the test material for both range-finding and definitive tests were prepared from stock solutions (1000 mg/L nominal concentration) prepared in algal nutrient media without the use of any solubilising agent. All stock and test solutions were corrected for purity of the test material (i.e., 88% test material, 12% water). All stock solutions were prepared in 500 mL glass volumetric flasks, and stirred until the test material was visibly dissolved using a stir plate and stir bar. The stock solutions were then used to prepare the test concentrations.
- The 1000 mg/L stock solution used for the range-finding test was prepared by adding 0.5690 g of the test material to 500 mL of algal nutrient media in a 500 mL glass volumetric flask and stirring for 0.5 h. The individual test solutions were prepared by adding an appropriate amount of the stock solution into a glass 500 mL graduated cylinder making this up to volume in algal nutrient media. The 1000 mg/L stock solution used for the definitive test was prepared by adding 0.5683 g of the test material to 500 mL of algal nutrient media in a 500 mL glass volumetric flask and stirring for 0.5 h. The individual test solutions were prepared by adding an appropriate
amount of the stock solution into 400 mL (using 2 x 200 mL glass volumetric flask) volume of algal nutrient media. All test solutions were stirred for an additional 30 minutes using a stir bar and stir plate prior to being dispensed into the individual test vessels (i.e., 250 mL Erlenmeyer flasks covered with Jaece® non-toxic foam plugs).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: CPCC 37
- Source: Pseudokirchneriella subcapitata used in testing were obtained from an in-house culture. The starter culture was purchased from the University of Waterloo Culture Collection (CPCC 37)
- Method of cultivation: Cultures were aseptically transferred twice weekly (typically from 3 – 7 day old donors) and maintained in temperature and light controlled environments isolated from all testing. The axenic nature of the stock culture was verified by plating on Trypticase Soy Agar (TSA) and Plate Count Agar (PCA). Algal growth curves conducted semi-annually ensured that algae were in an exponential growth phase, suitable for testing.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 ± 1 °C

pH:

7.5 ± 0.1

Nominal and measured concentrations:

Nominal: 1.563, 3.125, 6.25, 12.5, 25, 50, 100, and 200 mg/L
Measured (0 hours): 1.28, 2.40, 5.36, 12.11, 23.72, 47.00, 93.77 and 117.18 mg/L
Measured (72 hours): 1.39, 3.10, 5.74, 13.10, 25.72, 50.71, 98.97 and 123.05 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Clear glass 250-mL Erlenmeyer flasks covered with Jaece® non-toxic foam plugs
- Material, size, headspace, fill volume: 50 mL
- Agitation: Continuous shaking of test vessels on a rotary shaker set at 100 rpm
- Initial cells density: 5 x 10^3 to 1 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water obtained from the University of Guelph was used in preparation of the nutrient medium for culture and testing of P. subcapitata. This water was free of particles, ions, organic molecules and microorganisms greater than 0.45 μm in diameter. Preparation of the medium was conducted according to Environment Canada. The nutrient medium was filter sterilised prior to use in cultures and in testing.
- Elemental composition of the test media: N 4.20 mg/L, Mg 2.65 mg/L, Ca 1.20 mg/L, S 1.91 mg/L, P 0.19 mg/L, K 0.47 mg/L, Na 11.00 mg/L, C 2.14 mg/L, B 32.44 µg/L, Mn 115.38 µg/L, Zn 1.57 µg/L, Co 0.35 µg/L, Cu 0.004 µg/L, Mo 2.88 µg/L and Fe 33.1 µg/L.
- Culture medium different from test medium: no
- Intervals of water quality measurement: At test commencement, pH was measured in the control and in each test solution. At the end of the test (72 hours), the pH was determined from the pooled replicate samples from the control and each test concentration.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: Continuous (24 hours)
- Light intensity and quality: Cool-white fluorescent, measured at the surface of the liquid in the flasks: 4400-5310 Lux for testing

EFFECT PARAMETERS MEASURED:
- For all replicates, cell counts were determined at approximately 24, 48, and 72 hours. Cell counts were conducted using a haemocytometer and a phase-contrast microscope (at 100 - 200 times magnification). Any changes in cell development or appearance, such as cell clumping, cell morphology, cell colour, cell shape, cell size, etc. (or lack thereof) were reported on the bench sheets. Any additional observations relating to the test solutions, such as sedimentation of the test solution, precipitation of cells, solution appearance / colouration or other abnormalities, were also recorded on the bench sheets.

TEST CONCENTRATIONS
- The test consisted of eight nominal concentrations of the test material including 1.563, 3.125, 6.25, 12.5, 25, 50, 100, and 200 mg/L plus a negative control. For this test, four replicate 250 mL Erlenmeyer flasks, each containing 49 mL of the test solution (prepared in nutrient medium) and 1 mL of algal inoculum (5 x 10^3 to 1
x 10^4 algal cells/mL), were established for each concentration. Each control replicate flask contained 49 mL of the same batch of algal nutrient medium and 1 mL of the same algal inoculum, without the addition of the test material.
- Range finding study: The test consisted of five nominal concentrations of the test material including 0.1, 1, 10, 100, 300 mg/L plus a negative control (i.e., nutrient media with no added test material prepared the day of test initiation). For this test, two replicate 250 mL Erlenmeyer flasks, each containing 49 mL of the test solution (prepared in nutrient medium) and 1 mL of algal inoculum (5 x 10^3 to 1 x 10^4 algal cells/mL), were established for each concentration. Each control replicate flask contained 49 mL of the same batch of algal nutrient medium and 1 mL of the same algal inoculum, without the addition of the test material.

Reference substance (positive control):

yes

Remarks:

Sodium chloride

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and cell yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and cell yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and cell yield

Details on results:

RANGE-FINDING TEST
- Confirmation of the Exposure Concentrations: Nominal and measured concentrations of the test material determined on samples collected at the beginning and end of the test are provided in Table 1. The test material was shown to be stable in algal nutrient media for at room temperature for five days. However, samples collected at the beginning of the test did not contain algae, whereas those collected at test termination did. Nominal concentrations of 300 mg/L achieved an initial measured concentration of 254 mg/L (i.e., approximately 85% of the nominal concentration).
- Results of Range-Finding Test: Results of the range-finding test are summarised in Table 2. The test met all of the test validity criteria. Test results expressed as the percent inhibition of growth in terms of the effects on average specific growth rate and cell yield after 72 hours of exposure. Due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results are expressed here in terms of nominal concentrations only, corrected for purity. In terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be > 300 mg/L. For cell yield, the EC10, EC20 and EC50 were estimated to be between 10 and > 300 mg/L. The NOEC and LOEC from the range-finding test were estimated to be 100 and 300 mg/L, respectively for both average specific growth rate and cell yield.

DEFINITIVE TEST RESULTS
- Confirmation of the Exposure Concentrations: Nominal and measured concentrations of the test material determined on samples collected at the beginning and end of the test are provided in Table 3. The spike recovery was 96.1 to 99.7% which was acceptable (between 80 and 100%). In the definitive test, the measured concentrations at the test start were similar at test end showing the test material was stable for the test duration.
- Results of the Definitive Test: Results of the definitive test are summarised in Table 4. Due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results are expressed here in terms of nominal concentrations only, corrected for purity. The test met all of the test validity criteria. The NOEC and LOEC were estimated to be 200 and > 200 mg/L, respectively for both average specific growth rate and cell yield however, the response at 100 mg/L was significantly less than the control response. In terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to all be > 200 mg/L. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 14.3, 91.7 and >200 mg/L, respectively.

Results with reference substance (positive control):

- Sodium chloride was the reference toxicant used in this study. The 72-hour EC25 was compared to historical data and was deemed acceptable based on results falling within the 95% confidence interval (warning limit) calculated for the last seventeen reference toxicant tests.
- In conjunction with the definitive test, the 72-hour EC25 for the P. subcapitata reference toxicant test was 956 mg/L.

Reported statistics and error estimates:

- Due to the nature of the test material (UVCB), measured concentrations were not necessarily representative of the whole substance. Therefore, all endpoints for cell yield and average specific growth rate, from the range-finding and definitive tests, were generated using nominal concentrations (corrected for purity). For all concentrations and the controls, average specific growth rate (0 to 3 days) and cell yield (72-hour) and were calculated for each replicate and then averaged. Percent inhibition (I) and Coefficient of Variation (CV; %) were also calculated for average specific growth rate and cell yield. For the control flasks, the section-by-section specific growth rates (e.g., 0-1, 1-2, and 2-3 days) and mean % CV were determined. Concentration response curves were plotted for average specific growth rate and for cell yield.
- All data satisfied the assumptions for normality and homogeneity of variance. For the range-finding test, the NOEC and LOEC endpoints for average specific growth rate and cell yield were determined using ANOVA/ Dunnett Multiple Comparison Test (α=0.05). The ECx range endpoints, for cell yield (EyCx) and average specific growth rate (ErCx), were estimated from the inhibition of the average response at each test concentration compared to the control. For the definitive test, the NOEC and LOEC endpoints for average specific growth rate and cell yield were determined using ANOVA/Dunnett Multiple Comparison Test (α=0.05). Since there was no test concentration showing > 10% inhibition of average specific growth rate, all ErCx results were based on intuitive evaluation of the data (i.e., no statistical analysis was conducted). Statistical analyses to estimate EyC10 and EyC20 endpoints were calculated using Linear Interpolation. Since there was no concentration showing greater than 50% inhibition of cell yield, the EyC50 result was based on intuitive evaluation of the data (i.e., no statistical analysis was conducted).

Table 1: Summary of the Nominal and Measured Concentrations of the Test Material in the Range-Finding Test Solutions

Nominal Concentration (mg/L)	Measured Concentration
	0 Hours	72 Hours
300	254.17d	256.78e
100	87.78	90.01
10.0	7.48	9.29
1.0	0.68	1.08
0.10	0.11	0.17
0 (Negative control)	< MDL	< MDL

d Average of duplicate analyses. Relative percent difference (RPD) was 0.4%

e Average of duplicate analyses. RPD was 0.2%

MDL (Method Detection Limit) = 0.06 mg/L.

 

Table 2: Summary of Range-Finding Test Results Showing Percent Inhibition of Growth of P. subcapitata in Terms of Average Specific Growth Rate and Cell Yield After 72 Hours of Exposure to the Test Material

Test Endpoint	Nominal Concentration (mg/L)
	0.1	1.0	10.0	100	300
% Inhibition in Average Specific Growth Rate (0 to 72 hours)	-1.72	0.54	0.31	3.12	8.00
% Inhibition in Cell Yield (72 hour)	-9.04	2.42	1.32	14.33	32.84

  

Table 3: Summary of the Nominal and Measured Concentrations of the Test Material in the Definitive Test Solutions

Nominal Concentration (mg/L)	Measured Concentration (mg/L)
	0 Hours	72 Hours
200	117.18	123.05
100	93.77d	98.97e
50.0	47.00	50.71
25.0	23.72	25.72
12.5	12.11	13.10
6.25	5.36	5.74
3.125	2.40	3.10
1.563	1.28	1.39
0 (Negative Control)	< MDL	< MDL

d Average of duplicate analyses, RPD = 2.5%

e Average of duplicate analyses, RPD = 1.1%

MDL (Method Detection Limit) = 0.40 mg/L

 

Table 4: Summary of Definitive Test Results Showing Percent Inhibition of Growth of P. subcapitata in Terms of Average Specific Growth Rate and Cell Yield After 72 Hours of Exposure to the Test Material

Nominal Concentration (mg/L)	% Inhibition on Average Specific Growth Rate (0 to 72 hours)	% Inhibition on Cell Yield (72 hour)
200	3.25	15.97
100	5.65	26.45
50.0	1.83	9.20
25.0	2.84	13.88
12.5	0.89	5.28
6.25	2.28	10.31
3.125	2.51	13.32
1.563	1.71	8.99
0 (Negative Control)	0.00	0.00

TEST VALIDITY CRITERIA

All of the validity criteria for the range-finding and definitive tests were met:

- Biomass, as determined by cell number, in control test vessels increased by a factor of at least 16 times after 72 hours

- The mean CV (%) for section-by-section specific growth rates (days 0-1, 1-2, and 2-3) in the control cultures did not exceed 35%

- The CV (%) of average specific growth rates during the whole test period (0-3 days) in replicate control cultures did not exceed 7%

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, from the results of the definitive test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to all be > 200 mg/L. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 14.3, 91.7 and > 200 mg/L, respectively. The NOEC and LOEC were estimated to be 200 and > 200 mg/L, respectively, for both average specific growth rate and cell yield.

Executive summary:

The toxicity of the test material to algae was determined in accordance with the standardised guideline OECD 201, under GLP conditions in a study using freshwater green alga, Pseudokirchneriella subcapitata. 

Both the range-finding and definitive tests met the validity criteria.

Analytical concentrations were verified, however, due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results are expressed here in terms of nominal concentrations only, corrected for purity (88% test material; 12% water).

For the range-finding test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be > 300 mg/L. For cell yield, the EC10, EC20 and EC50 were estimated to be between 10 and > 300 mg/L. The NOEC and LOEC from the range-finding test were estimated to be 100 and 300 mg/L, respectively for both average specific growth rate and cell yield.

Under the conditions of this study, from the results of the definitive test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to all be > 200 mg/L. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 14.3, 91.7 and > 200 mg/L, respectively. The NOEC and LOEC were estimated to be 200 and > 200 mg/L, respectively, for both average specific growth rate and cell yield.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Description of key information

Under the conditions of this study, from the results of the definitive test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to all be > 200 mg/L. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 14.3, 91.7 and > 200 mg/L, respectively. The NOEC and LOEC were estimated to be 200 and > 200 mg/L, respectively, for both average specific growth rate and cell yield.

Key value for chemical safety assessment

EC50 for freshwater algae:

200 mg/L

EC10 or NOEC for freshwater algae:

200 mg/L

Additional information

The toxicity of the test material to algae was determined in accordance with the standardised guideline OECD 201, under GLP conditions in a study using freshwater green alga, Pseudokirchneriella subcapitata. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Both the range-finding and definitive tests met the validity criteria.

Analytical concentrations were verified, however, due to the nature of the test material (UVCB; Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Material), measured concentrations were not necessarily representative of the whole substance. Therefore, the results are expressed here in terms of nominal concentrations only, corrected for purity (88% test material; 12% water).

For the range-finding test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be > 300 mg/L. For cell yield, the EC10, EC20 and EC50 were estimated to be between 10 and > 300 mg/L. The NOEC and LOEC from the range-finding test were estimated to be 100 and 300 mg/L, respectively for both average specific growth rate and cell yield.

Under the conditions of this study, from the results of the definitive test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to all be > 200 mg/L. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 14.3, 91.7 and > 200 mg/L, respectively. The NOEC and LOEC were estimated to be 200 and > 200 mg/L, respectively, for both average specific growth rate and cell yield.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.