Hexanamide, N-(2-hydroxyethyl)-3,5,5-trimethyl-

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:

11 December 2012 - 01 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Mean measured concentrations: 0, 1.03, 3.22, 10.1, 30.9, 96.5 mg/L
- Sampling method: duplicate aliquots (ca. 18 mL) were removed from each prepared concentration at the commencement of the test as 0 h samples. At termination of the definitive test, an aliquot (ca 15 mL) was removed from each replicate flask at each concentration and combined. Duplicate aliquots (ca 20 mL) were removed from the combined solutions as 72 h samples.

An additional flask was prepared at nominal concentrations of 1 and 100 mg of the substance coating additives/L in the definitive test and these incubated (without algal cells) alongside the test and control flasks. At 72 h, duplicate aliquots (ca 20 mL) were removed from these flasks and taken for analysis, to determine any differences in measured concentrations between cell-free medium and medium containing cells.

- Sample storage conditions before analysis: samples analysed on receipt.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test solutions were prepared by dilution of a 100 mg/L nominal stock solution of the substance coating additives, with algal medium. The 100 mg/L stock solution was prepared by adding the substance coating additives (100.19 mg) to algal medium (1 L) followed by ultrasonication for ca 10 min to aid dissolution. The control contained algal medium only. Three replicate flasks at each test concentration and six replicate control flasks were included in the test. Each flask contained 100 mL of test solution and was inoculated with ca 0.89 x 10E+04 cells/mL P. subcapitata
- Controls: ISO algal medium

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Centre of Algae and Protozoa (CCAP), Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, Argyll, UK.
- Age of inoculum (at test initiation): ca 72 h
- Method of cultivation: subculturing using fresh Iso Algal Medium (Sanyo Gallenkamp Orbital Incubator)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Algal cell concentrations were measured in the inoculum and in each flask at 24, 48 and 72 h in the test. During the test, at 24 and 48 h, aliquots were removed from test and control flasks by pipette, and cell concentrations determined using a compound light microscope and Improved Neubauer counting chambers. During the test, at the 72 h time point, aliquots (5 mL) were sampled from each flask and Lugol Solution (100 µl) was added to each to preserve the sample for storage. These aliquots were stored at ca 4C until cell concentrations were determined within 3 days of sampling. Cell concentration was determined using a compound light microscope and Improved Neubauer ruling counting chambers. The pH of the prepared algal medium and test solution was measured at test initiation (0 h) and in all control and test flasks at 72 h. The maximum-minimum temperature of test solutions in the incubator was recorded at 24, 48 and 72 h.

Test temperature:

22.7-23.7°C

pH:

7.70-9.26

Nominal and measured concentrations:

Nominal concentration : 0, 1, 3.2, 10, 32 and 100 mg/L
Mean measured concentration: 0, 1.03, 3.22, 10.1, 30.9, 96.5 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass Erlenmeyer flasks (250 mL capacity) capped with foil lids, were used as the test flasks
- Type: open
- Material, size, headspace, fill volume: 100ml
- No. of organisms per vessel: ca 0.89 x 10E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: ISO Algal Medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Charles River
- Intervals of water quality measurement: pH measured at test initiation and at 72 h. Temperature measured at 24 h intervals.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: 7663 lux (cool white fluorescent tubes)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: compound light microscope and Improved Neubauer counting chambers


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0, 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study:
Growth (as indicated by cell numbers) was inhibited by 16% at 10 mg/L and by 61% at 100 mg/L, when compared to the control. There was no inhibition at 0.1 and 1 mg/L.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

66.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

40.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.22 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.22 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Reported statistics and error estimates:

The results are reported as daily cell concentrations (x 10E+05 cells/mL), µave/day and yield. In addition, the EC10, EC20 and EC50 values i.e. the estimated concentrations that result in a 10%, 20% and 50% reduction in growth and yield were calculated.

Measured Concentrations of the substance Coating Additives the Test

Nominal Concentration of the substance Coating Additives (mg/L)	Time (h)	Mean Measured Concentration of the substance Coating Additives (mg/L)	Overall Mean Measured Concentration of  the substance Coating Additives (mg/L)	Deviation of 72 h Measured Concentration from 0 h (±%)
Control	0	ND	ND	NA
	72	ND
1	0	1.01	1.03	+3
	72	1.04
	72*	1.03	1.03	NA
3.2	0	3.20	3.22	+1
	72	3.23
10	0	10.1	10.1	-1
	72	10.0
32	0	30.4	30.9	+3
	72	31.4
100	0	95.0	96.5	+3
	72	98.0
	72*	100	100	NA

* Flask not inoculated with Algae

ND = Not detected

NA – Not applicable

Mean Cell Concentration (x 10⁵cells/mL) at 0, 24, 48 and 72 h During the Test

Mean Measured Concentration of the substance Coating Additives (mg/L)	Replicate Flask	Mean Cell Concentrations Measured During the Definitive Test (x10^5cells/mL)
		0 h	24 h	48 h	72 h
Control	I	0.09	0.50	2.93	12.33
	II	0.09	0.70	2.95	14.68
	III	0.09	0.55	2.40	14.75
	IV	0.09	0.78	2.70	14.10
	V	0.09	0.55	2.90	13.15
	VI	0.09	0.43	3.15	14.03
	Mean	0.09	0.59	2.84	13.84
1.03	I	0.09	0.35	2.80	16.75
	II	0.09	0.45	2.45	14.70
	III	0.09	0.43	2.30	12.35
	Mean	0.09	0.41	2.52	14.60
3.22	I	0.09	0.55	2.15	13.25
	II	0.09	0.58	2.08	12.40
	III	0.09	0.43	2.43	13.65
	Mean	0.09	0.52	2.22	13.10
10.1	I	0.09	0.40	2.38	11.58
	II	0.09	0.50	2.40	12.98
	III	0.09	0.53	2.80	9.65
	Mean	0.09	0.48	2.53	11.40
30.9	I	0.09	0.28	2.35	11.18
	II	0.09	0.40	1.85	8.80
	III	0.09	0.30	2.38	10.28
	Mean	0.09	0.33	2.19	10.09
96.5	I	0.09	0.18	0.18	0.20
	II	0.09	0.15	0.15	0.35
	III	0.09	0.08	0.13	0.20
	Mean	0.09	0.14	0.15	0.25

Cell Growth Group Mean Values Determined from Daily Cell Counts During the Test

Growth Function	Time Interval (h)	Mean Measured Concentration of the substance Coating Additives (mg/L)
		Control	1.03	3.22	10.1	30.9	96.5
Mean Cell Concentration (x10^5cells/ml)	0-24	0.59	0.41	0.52	0.48	0.33	0.14
	0-48	2.84	2.52	2.22	2.53	2.19	0.15
	0-72	13.84	14.60	13.10	11.40	10.09	0.25
Average Specific Growth Rate (μave/day)	0-24	1.85	1.51	1.75	1.66	1.28	0.40
	0-48	1.72	1.66	1.60	1.67	1.59	0.26
	0-72	1.68	1.69	1.66	1.61	1.57	0.33
	24-48	1.60	1.82	1.46	1.67	1.91	0.12
	48-72	1.59	1.75	1.78	1.50	1.53	0.46
Yield	0-24	1.24	0.80	1.08	0.97	0.59	0.13
	0-48	6.87	6.07	5.33	6.09	5.26	0.16
	0-72	34.38	36.28	32.53	28.28	24.99	0.40

EC and NOEC Values During the Test

Growth Function	Time Interval (h)	ErC10/ EyC10 (mg/L)	ErC20/ EyC20 (mg/L)	ErC50/ EyC50 (mg/L)	NOEC (mg/L)
Average Specific Growth Rate (µave/day)	0–24     0–48     0-72	20.3 (5.6, 35.0)   40.9 (28.5, 53.4)   37.3 (28.1, 46.5)	29.6 (13.5, 45.7)   49.0 (37.1, 60.9)   46.2 (37.2, 55.2)	56.3 (37.0, 75.6)   66.7 (57.2, 76.2)   66.7 (59.2, 74.2)	10.1     10.1     3.22
Yield	0–24     0–48     0-72	15.5 (0.8, 30.2)   31.1 (21.1, 41.0)   24.4 (13.8, 35.0)	21.9 (7.1, 36.6)   36.4 (25.2, 47.7)   29.5 (22.0, 37.1)	39.2 (22.3, 56.1)   47.7 (27.7, 67.8)   40.9 (23.7, 58.1)	10.1     10.1     3.22

Values in brackets are 95% confidence limits

Values of pH Measured for Test Solutions at 0 h and for Replicate Flasks at 72 h During the Test

Mean Measured Concentration of the substance Coating Additives (mg/L)	Replicate Flask	pH Value
		0h	72h
Control	I	7.76	8.74
	II		9.01
	III		9.19
	IV		8.64
	V		8.87
	VI		8.38
1.03	I	7.75	8.77
	II		8.67
	III		8.56
3.22	I	7.79	8.70
	II		9.11
	III		8.88
10.1	I	7.84	9.26
	II		9.13
	III		8.77
30.9	I	7.81	8.33
	II		8.31
	III		8.30
96.5	I	7.79	7.70
	II		7.79
	III		7.86

Temperature Range of Test Solutions Throughout the Test

Time (h)	Temperature (°C)
	Maximum	Minimum
0-24	23.7	22.8
24-48	23.6	22.8
48-72	23.4	22.7

 

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72 h ErC50, ErC10 and NOEC values of the test substance based on effects on growth rate of aquatic algae were determined to be 66.7, 37.3 and 3.22 mg/L (measured; arithmetic mean) respectively

Executive summary:

A study was conducted to evaluate the toxicity of the test substance, onthe unicellular green algae Pseudokirchneriella subcapitata (Selenastrum capricornutum)according to OECD Guideline 201, in compliance with GLP. The study was conducted over a 72 h period under static conditions. Based on the results of a range finding test, the definitive test was conducted at nominal substance coating additives concentrations of 1, 3.2, 10, 32 and 100 mg/L, with an untreated control. Three test vessels were prepared for each test concentration and 0.89 x 10E+04 cells of the unicellular green algae/mL were added to each. The samples for analytical confirmation of actual exposure concentrations were taken at the start (0 h) and at the end of the test (72 h). Chemical analysis of test samples taken at 0 and 72 h indicated that concentrations of the test substance were maintained within the range 99-103% of the initial measured values. Based on these results, the mean measured concentrations of the test substance were calculated to be 1.03, 3.22, 10.1, 30.9 and 96.5 mg/L, respectively. The no observed effect concentration (NOEC) and the concentration that results in a 10, 20 and 50% reduction in growth rate (ErC10, ErC20 and ErC50, respectively) and yield (EyC10, EyC20 and EyC50, respectively) were estimated, where possible. The results are reported as average specific growth rates and yield. At 72 h, the NOEC for growth rate and yield were both determined to be 3.22 mg/L. The ErC10, ErC20 and ErC50 for effects on growth rate were determined to be 37.3, 46.2 and 66.7 mg/L respectively, based on arithmetic mean measured concentrations. The EyC10, EyC20 and EyC50 for effects on yield were determined to be 24.4, 29.5 and 40.9 mg/L respectively, based on arithmetic mean measured concentrations. The pH of test solutions and the temperature of the environmental area were maintained within protocol specification throughout the test. The pH of the test solutions ranged from 7.70-9.26 and the temperature of the test solutions ranged from 22.7-23.7°C. The average light intensity measured at flask level during the test was 7663 lux. Under the study conditions, the 72 h ErC50, ErC10 and NOEC values of the test substance based on effects on growth rate of aquatic algae were determined to be 66.7, 37.3 and 3.22 mg/L (measured; arithmetic mean) respectively (Knight, 2013).

Description of key information

The 72 h ErC50, ErC10 and NOEC of the test substance to aquatic algae, based on growth rate, were determined to be 66.7, 37.3 and 3.22 mg/L (measured; arithmetic mean) respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

66.7 mg/L

EC10 or NOEC for freshwater algae:

37.3 mg/L

Additional information

A study was conducted to evaluate the toxicity of the test substance, onthe unicellular green algae Pseudokirchneriella subcapitata (Selenastrum capricornutum)according to OECD Guideline 201, in compliance with GLP. The study was conducted over a 72 h period under static conditions. Based on the results of a range finding test, the definitive test was conducted at nominal substance coating additives concentrations of 1, 3.2, 10, 32 and 100 mg/L, with an untreated control. Three test vessels were prepared for each test concentration and 0.89 x 10E+04 cells of the unicellular green algae/mL were added to each. The samples for analytical confirmation of actual exposure concentrations were taken at the start (0 h) and at the end of the test (72 h). Chemical analysis of test samples taken at 0 and 72 h indicated that concentrations of the test substance were maintained within the range 99-103% of the initial measured values. Based on these results, the mean measured concentrations of the test substance were calculated to be 1.03, 3.22, 10.1, 30.9 and 96.5 mg/L, respectively. The no observed effect concentration (NOEC) and the concentration that results in a 10, 20 and 50% reduction in growth rate (ErC10, ErC20 and ErC50, respectively) and yield (EyC10, EyC20 and EyC50, respectively) were estimated, where possible. The results are reported as average specific growth rates and yield. At 72 h, the NOEC for growth rate and yield were both determined to be 3.22 mg/L. The ErC10, ErC20 and ErC50 for effects on growth rate were determined to be 37.3, 46.2 and 66.7 mg/L respectively, based on arithmetic mean measured concentrations. The EyC10, EyC20 and EyC50 for effects on yield were determined to be 24.4, 29.5 and 40.9 mg/L respectively, based on arithmetic mean measured concentrations. The pH of test solutions and the temperature of the environmental area were maintained within protocol specification throughout the test. The pH of the test solutions ranged from 7.70-9.26 and the temperature of the test solutions ranged from 22.7-23.7°C. The average light intensity measured at flask level during the test was 7663 lux. Under the study conditions, the 72 h ErC50, ErC10 and NOEC values of the test substance based on effects on growth rate of aquatic algae were determined to be 66.7, 37.3 and 3.22 mg/L (measured; arithmetic mean) respectively (Knight, 2013).