Long-chain alkenyl amide

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:

17 August 2017 to 27 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Samples (approximately 150 mL) of the range-finding and definitive test solutions and controls were collected into Teflon® capped glass bottles (250 mL, no preservative).
- Samples were collected immediately prior to test commencement (0 hour) and at test termination (72 hour, from pooled replicates) for analysis.
- All samples were refrigerated until transported to ISI and kept refrigerated by ISI until analysis was conducted. All samples were analysed within 24 hours of the sample collection date.

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 nutrient media and without the use of any solubilising agent.
- Due to the low solubility of the test material and its adhesive nature, stock solutions were prepared in 1-L glass aspirator bottles as follows. The test material was weighed out onto a pre-weighed plastic weigh boat containing a monolayer of glass beads. The test material remained attached to the surface of the glass beads while both the glass beads and the test material were transferred to the glass aspirator bottle leaving none of the test material in the plastic weigh boat. The solution was then stirred for approximately 96 hours at a rate sufficient to maintain a vortex between approximately 10 - 35% of the solution depth using a stir bar and stir plate. The solution was then settled for approximately 1 hour. The first ~ 100 mL of solution removed from each glass aspirator was disposed of.
- Individual test solutions were prepared by addition of the appropriate amount of stock solution to nutrient media to achieve the desired concentrations for testing. All tests (range-finding and definitive) included a negative control consisting of dilution water with no test material added.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CPCC (UTCC) #37
- Source: The starter culture was purchased from the University of Waterloo Culture Collection (CPCC 37)
- Age: 3 - 7 days old and in exponential growth
- 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:

6.28-7.8

Nominal and measured concentrations:

Nominal concentrations: 12.5, 25, 50, 100, 200, 400, and 800 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
- Concentration of algal innoculum: 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 OECD. 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, 0.004 µg/L, 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: 5,040 – 6,250 Lux for range-finding test; 5,240 – 6,490 Lux for definitive test.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell counts were conducted using a haemocytometer and a phase-contrast microscope (at 100 – 200 times magnification).
- For all replicates, cell counts were determined at approximately 24, 48, and 72 hours. 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.

RANGE-FINDING TEST
- Nominal concentrations of 1.0, 10, 100 and 1000 mg/L were prepared by adding 0.5, 5, 50, and 500 mL of a nominal 1000 mg/L stock solution, respectively into a 500 mL volumetric flask and making this up to volume in nutrient dilution water.
- 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. The environmental conditions and testing procedures were the same for all flasks.

TEST CONCENTRATIONS
- Results of the range-finding test indicated that the 72-hour EC50 for the growth endpoints was expected to be in the range of 100 to 1000 mg/L of the test material (as nominal concentration). As such, the definitive test was conducted as a multi-concentration test.
- Nominal concentrations of 12.5, 25, 50, 100, 200, 400, and 800 mg/L were prepared by adding 6.25, 12.5, 50, 100, 200, and 400 mL of a nominal 1000 mg/L stock solution, respectively into a 500 mL volumetric flask and making this up to volume in nutrient media.

Reference substance (positive control):

yes

Remarks:

Sodium chloride

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

496 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (418 – 571 mg/L)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

370 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: (319 – 421 mg/L)

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

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

318 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (250 - 360 mg/L)

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. The test material was shown to be stable in algal nutrient media for at room temperature for 15 days. Measured concentrations were below nominal concentrations due to the low solubility of the test material. However, measured concentrations at the beginning and end of the test were similar, indicating that the test material was stable over the period of the test. Nominal concentrations of 1000 and 100 mg/L achieved TWM concentrations of 69 and 3 mg/L, respectively (i.e., approximately 6.9 and 3.0% of the nominal concentration). With respect to analytical procedures, samples collected at the beginning of the test did not contain algae, whereas those collected at test termination did. Therefore, prior to analysis, samples collected at the end of the test were centrifuged to remove the algae. To confirm this procedure did not impact results, a duplicate sample treated in the same manner (i.e., centrifuged prior to analysis), but without algae added was analysed, and indicated only a small difference in measured concentrations (i.e., ~1%).
- Results of Range-Finding Test: Results are summarised in Table 2. The test met all of the test validity criteria.

DEFINITIVE TEST
- Confirmation of the Exposure Concentrations: Nominal, measured and TWM concentrations of the test material determined on samples collected at the beginning and end of the test are provided in Table 3. The TWM concentration of 37.1 mg/L was achieved from preparation of a nominal 800 mg/L solution of the test material used for the highest concentration of the definitive test. The spike recovery was 87.7% which was acceptable (between 80 and 100%). The measured concentrations at the test start were higher than at test end (approximately 40%); which was different than what was observed during the range-finding test. Sample preparation procedures (e.g., stirring/settling time and mixing velocity) were similar for the range-finding and definitive tests; therefore, the decrease was considered to be primarily related to the low solubility of the test material.
- Results of the Definitive Test: Results 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 at and above 74 mg/L. A summary of the measured growth endpoints is provided in Table 5. The NOEC and LOEC were estimated to be 200 and 400 mg/L, respectively for average specific growth rate and the same endpoints were estimated at 100 and 200 mg/L, respectively for cell yield. In terms of average specific growth rate, the 72-hour EC10, EC20 and EC50 were estimated to be >100 mg/L, respectively. The 72-hour EC10 for cell yield was estimated to be 74 mg/L and the EC20 and EC50 were >100 mg/L, respectively.

VALIDITY CRITERIA
All of the validity criteria for the range-finding and definitive tests were met as listed below:
- 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%.

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 12 reference toxicant tests. In conjunction with the definitive test, the 72-hour EC25 for the P. subcapitata reference toxicant test conducted on was 633 mg/L.

Reported statistics and error estimates:

- Although analytical concentrations were verified, due to the nature of the test material (UVCB), measured concentrations were not necessarily representative of the whole substance. Therefore, statistical analysis and end points are expressed in terms of nominal concentrations.
- 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.
- All data satisfied the assumptions for normality and homogeneity of variance. From the rangefinding test, the NOEC and LOEC endpoints for average specific growth rate were determined using ANOVA/ Dunnett T3 Multiple Comparison Test (CETIS, 2013) (α=0.05). Growth rate inhibition (ErCx) endpoints were estimated using Linear Interpolation (CETIS, 2013). NOEC and LOEC endpoints for cell yield were determined using ANOVA/ Williams Multiple Comparison Test (CETIS, 2013) (α=0.05). Cell yield inhibition (EyCx) endpoints were estimated using Linear Interpolation.
- From the definitive test, the NOEC and LOEC endpoints for average specific growth rate were determined using ANOVA/Dunnett Multiple Comparison Test (CETIS, 2013) (α=0.05) (MSD = 8.41%). Growth rate inhibition (ErCx) endpoints were estimated using Non-Linear Regression analysis (CETIS, 2013). NOEC and LOEC endpoints for cell yield were determined using ANOVA/ Dunnett Multiple Comparison Test (CETIS, 2013) (α=0.05), after square root transformation (MSD = 23.0%). Cell yield inhibition (EyCx) endpoints were estimated using Non- Linear Regression analysis (CETIS, 2013).

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

Nominal Concentration (mg/L)	Measured Concentration (mg/L)		Time-Weighted Mean Concentration (mg/L)
	0 Hours	72 Hours
1000	69.31d	67.86ef	69
100	5.54	1.8	3
10	0.31	<MDLg	0.3h
1	<MDL	<MDL	<MDL
0 (Negative Control)	<MDL	<MDL	<MDL

d Average of duplicate analyses. The Relative Percent Difference (RPD) was 2.7 %.

e Average of duplicate analyses. RPD was 2.3 %.

f Sample was duplicated (same concentration) but with no algae added and was measured at 66.96 mg/L.

g MDL (Method Detection Limit) = 0.1 mg/L.

h Based on a single measurement (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 Test Material

Test Endpoint	Nominal Concentration (mg/L)
	1	10	100	1000
% Inhibition in Average Specific Growth Rate (0 to 72 hours)	-0.24a	0.89	1.79	100
% Inhibition in Cell Yield (72 hour)	-1.59	3.92	9.19	100.46

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

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)		Time-Weighted Mean Concentration (mg/L)
	0 h	72 h
800	48.46d	27.62ef	37.1
400	18.16	6.41	11.3
200	7.06	3.99	5.4
100	3.27	2.08	2.6
50	1.35	0.15	0.5
25	0.52	<MDLg	0.52h
12.5	0.16	<MDL	0.16h
0 (Negative Control)	<MDL	<MDL	<MDL

d Average of duplicate analyses, RPD = 8.5%.

e Average of duplicate analyses, RPD = 6.8%.

f Sample was duplicated (same concentration) but with no algae added and was measured at 18.97 mg/L.

g MDL = 0.1 mg/L.

h Based on a single measurement (not a TWM).

 

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 Test Material.

Nominal Concentration (mg/L)	% Inhibition on Average Specific Growth Rate (0 to 72 hours)	% Inhibition on Cell Yield (72 hour)
800	104.33	100.32
400	23.93	70.40
200	6.38	27.86
100	0.52	3.02
50	-4.47a	-26.11
25	0.66	3.89
12.5	1.14	6.03
0 (Negative Control)	0.00	0.00

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

 

Table 5: Effects of Test Material on Growth Inhibition (Average Specific Growth Rate and Cell Yield) of P. subcapitata Based on a 72-Hour Static Exposure

Endpoint	ECxa (95% Confidence Limits) (Nominal; mg/L)			NOEC (mg/L)	LOEC (mg/L)
	EC10	EC20	EC50
Average Specific Growth Rate (0 to 72 hours)	318 (250 – 360)	380 (355 – 402)	496 (418 – 571)	200	400
Cell Yield (72 hour)	74 (27 – 121)	148 (105 – 191)	370 (319 – 421)	100	200

a Effective concentration causing a specified x % inhibition.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 72 h EC50 for the specific growth rate and cell yield were 496 and 370 mg/L, respectively. The 72h EC10 for the specific growth rate and cell yield were 318 and 74 mg/L, respectively.

Executive summary:

The aquatic toxicity of the test material to algae was investigated in accordance with the standardised guideline OECD 201, under GLP conditions using a Growth Inhibition Test with the Freshwater Green Alga, Pseudokirchneriella subcapitata.

Test solution preparation included continuous stirring for approximately 96 hours, followed by a settling period of approximately 1 hour. 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 were expressed in terms of nominal concentrations only.

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

Results of the range-finding test indicated that the 72-hour EC10s, EC20s and EC50s for average specific growth rate and cell yield were estimated to be in the range of 100 – 1000 mg/L. 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 318, 380 and 496 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 74, 148 and 370 mg/L, respectively. All ECx values exceeded the OECD limit of 100 mg/L, except the EC10 for cell yield. The NOEC and LOEC were estimated to be 200 and 400 mg/L, respectively for average specific growth rate. The NOEC and LOEC were estimated to be 100 and 200 mg/L, respectively for cell yield.

Under the conditions of this study, the 72 h EC50 for the specific growth rate and cell yield were 496 and 370 mg/L, respectively. The 72h EC10 for the specific growth rate and cell yield were 318 and 74 mg/L, respectively.

Description of key information

Under the conditions of the study, the 72 h EC50 for the specific growth rate and cell yield were 496 and 370 mg/L, respectively. The 72h EC10 for the specific growth rate and cell yield were 318 and 74 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

496 mg/L

EC10 or NOEC for freshwater algae:

318 mg/L

Additional information

The aquatic toxicity of the test material to algae was investigated in accordance with the standardised guideline OECD 201, under GLP conditions using a Growth Inhibition Test with the 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).

Test solution preparation included continuous stirring for approximately 96 hours, followed by a settling period of approximately 1 hour. 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 were expressed in terms of nominal concentrations only.

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

Results of the range-finding test indicated that the 72-hour EC10s, EC20s and EC50s for average specific growth rate and cell yield were estimated to be in the range of 100 – 1000 mg/L. 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 318, 380 and 496 mg/L, respectively. The 72-hour EC10, EC20 and EC50 for cell yield were estimated to be 74, 148 and 370 mg/L, respectively. All ECx values exceeded the OECD limit of 100 mg/L, except the EC10 for cell yield. The NOEC and LOEC were estimated to be 200 and 400 mg/L, respectively for average specific growth rate. The NOEC and LOEC were estimated to be 100 and 200 mg/L, respectively for cell yield.

Under the conditions of this study, the 72 h EC50 for the specific growth rate and cell yield were 496 and 370 mg/L, respectively. The 72h EC10 for the specific growth rate and cell yield were 318 and 74 mg/L, respectively.