Decanedioic acid, compound with ethanolamine / Decanedioic acid, compound with 2-aminoethanol

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:

03 August 2017 to 13 August 2017

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 250 mL glass jars (pre-cleaned with Teflon lined caps) provided by ISI (no preservative). The vials and jars were filled, leaving some headspace.
- 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 0.56820 g of the test material to 500 mL of algal nutrient media in a 500 mL glass volumetric and stirring for 0.5 h. The individual test solutions were prepared by adding an appropriate amount of the stock solution into a 500 mL volumetric flask making this up to volume in algal nutrient media. The 1000 mg/L stock solution used for the definitive test was prepared by adding 0.56820 g of the test material to 500 mL of algal nutrient media in a 500 mL glass volumetric and stirring for 2 h. The individual test solutions were prepared by adding an appropriate amount of the stock solution into a 500 mL volumetric flask making this up to volume in algal nutrient media. All test solutions were stirred for 15 - 25 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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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: 0.313, 0.625, 1.25, 2.5, 5, 10, 20 and 40 mg/L
Measured (0 hours): 0.28, 0.57, 1.13, 2.26, 4.62, 9.82, 19.3 and 42.02 mg/L
Measured (72 hours): 0.29, 0.56, 1.22, 2.61, 4.27, 3.84, 19.15 and 35.37 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: 5460-6770 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 0.313, 0.625, 1.25, 2.5, 5, 10, 20 and 40 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 four nominal concentrations of the test material including 0.1, 1, 10 and 100 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.:

21.25 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % C.L: 16.27-25.13 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.83 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: cell yield

Remarks on result:

other: 7.04-8.66 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.26 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and cell yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.62 mg/L

Nominal / measured:

meas. (initial)

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, measured and Time-Weighted Mean (TWM) concentrations of the test material determined on samples collected at the beginning and end of the test are provided in Table 1. In some instances, test concentrations were less than the MDL at test end, so measured concentrations at 0 hours were used. 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. Prior to analysis, samples collected at the end of the test were centrifuged to remove the algae; however the results of a duplicate sample without algae added indicated only a small difference in measured concentrations (i.e., ~1%). Nominal concentrations of 100 mg/L achieved a TWM concentrations of 59.6 mg/L (i.e., approximately 59.6% of the nominal concentration).
- Results of Range-Finding Test: Results of the range-finding test are summarised in Table 2. 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. The NOEC and LOEC were estimated to be between < 0.09 and 0.09 mg/L, respectively for both average specific growth rate and cell yield. In terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be between 0.92 and 59.6 mg/L, while for cell yield the same endpoints were estimated to be between < 0.09 and 9.51 mg/L.

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. As concentrations at the test end were similar to test start it was not appropriate to calculate Time-Weighted Mean concentrations for the definitive test. The spike recovery was 95.5% which was acceptable (between 80 and 100%). Prior to analysis, samples collected at the end of the test were centrifuged to remove the algae; however the results of a duplicate sample without algae added indicated measured concentrations were below the MDL of 0.09 mg/L. This is in contrast with the range-finding results where the duplicate samples with and without algae added resulted in similar concentrations of the test material. This discrepancy is likely a function of the higher test concentration in the range-finding test, either due to chemical interactions with the nutrient media or causing algal cell death before the test material could be biologically incorporated. 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. The test met all of the test validity criteria. Results of the definitive test showed that there were adverse effects on growth of algae in terms of average specific growth rate and cell yield at concentrations of the test material at and above 2.27 mg/L. The NOEC and LOEC were estimated to be 2.26 and 4.62 mg/L, respectively for both average specific growth rate and cell yield. In terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be 6.14, 9.83 and 21.25 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 2.27, 3.72 and 7.83 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 eleven reference toxicant tests.
- In conjunction with the definitive test, the 72-hour EC25 for the P. subcapitata reference toxicant test was 537 mg/L.

Reported statistics and error estimates:

All endpoints for cell yield and average specific growth rate, from the range-finding test, were generated using Time-Weighted Mean (TWM) concentrations calculated from measured concentrations of the test material at t=0h and t=72h. For the definitive test, results were generated using measured concentrations of the test material at test initiation, also 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 rangefinding test, the NOEC and LOEC endpoints for average specific growth rate and cell yield were determined using ANOVA/ Williams 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/Williams Multiple Comparison Test (α=0.05). Growth inhibition values were estimated from inhibition of average specific growth rate (ErCx) and cell yield (EyCx) compared to the control, and were calculated using Linear Interpolation since Non-Linear Regression Analysis did not yield a valid analysis. As stimulation was observed in the two lowest test concentrations (0.28 and 0.57 mg/L, measured at 0 h), the growth rate data in these concentrations were replaced with control data prior to statistical analysis.

Table 1: Summary of the Nominal and Measured (at T=0 hours) Concentrations of the Test Material in the Range-Finding Test Solutions

Nominal Concentration (mg/L)	Measured Concentration (mg/L)		Time-Weighted Mean Concentration (mg/L)
	0 Hours	72 Hours
100	92.77e	35.42fg	59.6
10	9.51	< MDL	9.51i
1	0.92	< MDL	0.92i
0.1	0.09	< MDL	0.09i
0 (Negative Control)	< MDL	< MDL	< MDL

e Average of duplicate analyses. Relative percent difference (RPD) was 2.8%

f Average of duplicate analyses. RPD was 10.5%

g Sample was duplicated (same concentration) but with no algae added and was measured at 33.78 mg/L.

MDL(Method Detection Limit) = 0.09 mg/L.

i Based on a single measurement at 0h (not a TWM).

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	10	100
	Time-Weighted Mean Concentration (mg/L)
	0.09	0.92	9.51	59.6
% Inhibition in Average Specific Growth Rate (0 to 72 hours)	3.39	4.57	35.72	110.18
% Inhibition in Cell Yield (72 hour)	16.90	22.53	86.58	100.17

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
40	42.02e	35.37f
20	19.35	19.15
10	9.82	3.84
5	4.62	4.27
2.5	2.26	2.61
1.25	1.13	1.22
0.625	0.57	0.56
0.313	0.28	0.29
0 (Negative Control)	< MDL	< MDL

e Average of duplicate analyses, RPD = 0.4%

f Sample was duplicated (same concentration) but no algae added and was measured at < MDL

MDL (Method Detection Limit) = 0.09 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)	Measured Concentration at 0 Hours (mg/L)	% Inhibition on Average Specific Growth Rate (0 to 72 hours)	% Inhibition on Cell Yield (72 hour)
40	42.02	72.31	98.00
20	19.35	46.97	91.15
10	9.82	19.77	63.86
5	4.62	4.21	19.73
2.5	2.26	1.83	9.01
1.25	1.13	0.89	4.59
0.625	0.57	-1.17	-6.30
0.313	0.28	-1.45	-7.39
0 (Negative Control)	< MDL	0.00	0.00

A negative value for % inhibition indicates stimulation as compared to the control.

MDL (Method Detection Limit) = 0.09 mg/L.

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 be 6.14, 9.83 and 21.25 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 2.27, 3.72 and 7.83 mg/L, respectively. The NOEC and LOEC were estimated to be 2.26 and 4.62 mg/L, respectively, for both average specific growth rate and cell yield.

Executive summary:

The acute 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.

Results of the range-finding test were generated using Time-Weighted Mean (TWM) concentrations calculated from measured concentrations of the test material at test initiation and test end, corrected for purity (88% test material; 12% water). In some instances, test concentrations for the range-finding test were less than the Method Detection Limit (MDL) at test end, so endpoints were generated using measured concentrations at 0 hours, also corrected for purity.

From the range-finding test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be in the range of 0.92 to 59.6 mg/L. For cell yield, the EC10, EC20 and EC50 were estimated to be between < 0.09 and 9.51 mg/L. The NOEC and LOEC from the range-finding test were estimated to be between < 0.09 and 0.09 mg/L, respectively for both average specific growth rate and cell yield.

Results of the definitive test were generated using measured concentrations of the test material at test initiation, corrected for purity.

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 be 6.14, 9.83 and 21.25 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 2.27, 3.72 and 7.83 mg/L, respectively. The NOEC and LOEC were estimated to be 2.26 and 4.62 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 be 6.14, 9.83 and 21.25 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 2.27, 3.72 and 7.83 mg/L, respectively. The NOEC and LOEC were estimated to be 2.26 and 4.62 mg/L, respectively, for both average specific growth rate and cell yield.

Key value for chemical safety assessment

EC50 for freshwater algae:

21.25 mg/L

EC10 or NOEC for freshwater algae:

2.26 mg/L

Additional information

The acute toxicity of the test material to algae was determined in accordance with the standardised guideline OECD 201, under GLP conditions. 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.

Results of the range-finding test were generated using Time-Weighted Mean (TWM) concentrations calculated from measured concentrations of the test material at test initiation and test end, corrected for purity (88% test material; 12% water). In some instances, test concentrations for the range-finding test were less than the Method Detection Limit (MDL) at test end, so endpoints were generated using measured concentrations at 0 hours, also corrected for purity.

From the range-finding test, in terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be in the range of 0.92 to 59.6 mg/L. For cell yield, the EC10, EC20 and EC50 were estimated to be between < 0.09 and 9.51 mg/L. The NOEC and LOEC from the range-finding test were estimated to be between < 0.09 and 0.09 mg/L, respectively for both average specific growth rate and cell yield.

Results of the definitive test were generated using measured concentrations of the test material at test initiation, corrected for purity.

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 be 6.14, 9.83 and 21.25 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 2.27, 3.72 and 7.83 mg/L, respectively. The NOEC and LOEC were estimated to be 2.26 and 4.62 mg/L, respectively, for both average specific growth rate and cell yield.