1,6-Hexanediamine, N1,N6-bis(1,2,2-trimethylpropyl)-

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:

15 to 23 October 2007 for experimental phase

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Followed existing guideline and GLP requirements

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

yes

Remarks:

TOC of algae media not measured at initiation of definitive test, but was in preliminary; Control pH at test end exceeded 1.5 times initial pH, but cell densities at 72 hours indicate cell growth not affected

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 10 mg a.i./L primary stock solution prepared prior to test initiation by placing 0.0209 g (0.0203 g active ingredient) of test article in 2.0 L volumetric flask and bringing to volume with AAP medium. Solution was mixed for 1 hour with Teflon coated stir bar and magnetic stirring plate.
- Controls: AAP medium only
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No evidence of undissolved material seen

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Pseudokirchneriella subcapitata strain 1648, Class Chlorophyceae
- Source (laboratory, culture collection): Springborn Smithers culture laboratory originally obtained from University of Texas, Austin, Texas
- Age of inoculum (at test initiation): three days since previous transfer
- Method of cultivation: Culture medium was Algal Assay Procedure (AAP medium. Stock cultures grown in 250 ml Erlenmeyer glass flasks containing 100 ml medium. Flasks covered with stainless steel caps that allowed gas exchange. If necessary, medium had pH adjusted to 7.5 +/- 0.1 with 0.1 N hydrochloric acid or 0.1 N sodium hydroxide. Stock cultures maintained at a shaking rate of 100 +/- 10 rpm, a temperature of 24 degrees C, and continuous illumination at medium surface of 4500 to 5900 lux (420 - 550 footcandles supplied by fluorescent bulbs.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

24 +/- 1 degrees C; See tables in materials and methods for monitored values

pH:

See tables in materials and methods for monitored values. pH of test and control solutions ranged from 7.0 to 7.3 at initiation, and at 96 hours, pH of test and control solutions ranged from 7.3 to 9.5. The increase in pH is common in static algal cultures and is due to photosynthesis

Nominal and measured concentrations:

Nominal concentrations of 0, 0.032, 0.1, 0.32, 1.0, 3.2, and 10.0 mg a.i./L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: capped with stainless steel caps that allowed gas exchange
- Material, size, headspace, fill volume: sterile, 250 ml glass flasks containing 100 ml test solution
- Initial cells density: 1.03 mL innoculum contaning about 97 x 10^4 cells/mL. This allowed initial cell density of about 1 x 10^4 cells/mL
- Control end cells density: See tables in results section
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP medium


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile, deionized water
- Total organic carbon: AAP media assayed for TOC monthly. Values for March to September 2007 ranged from 0.29 to 0.56 mL.
- Metals: dilution water tested regularly for toxic metals
- Pesticides: dilution water tested regularly for pesticides
- Conductivity: See tables in material and methods section for initial and final values of conductivity nd pH

OTHER TEST CONDITIONS
- Sterile test conditions: flasks and dilution water were sterile
- Adjustment of pH: initial pH of medium was adjusted if necessary to 7.5 +/- 0.1 prior to use. Stock test solution was adjusted from 8.6 to 7.1 by adding 1.6 mL of 0.10 N hydrochloric acid solution
- Photoperiod: continuous illumination
- Light intensity and quality: 4500 to 5900 lux (420 to 550 footcandles)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: hemacytometer and compound microscope

TEST CONCENTRATIONS
- Test concentrations: 0, 0.032, 0.1, 0.32, 1.0, 3.2 and 10.0 mg a.i./L nominal
- Results used to determine the conditions for the definitive study: In preliminary study, cell fragments observed at 10 mg a.i./L, and cell densities were reduced at 0.10 mg a.i./l and higher.

Reference substance (positive control):

yes

Remarks:

potassium dichromate used as positive control monthly in assessing lab's culture

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 0.085 to 0.18 95% CL

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.083 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: 0.081 to 0.085 95% CL

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Remarks on result:

other: 0.093 to 0.17 95% CL

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.083 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Remarks on result:

other: 0.080 to 0.086 95% CL

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.14 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks:

0 to 72 hour

Remarks on result:

other: 0.089 to 0.25 95% CL

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.088 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks:

0 to 96 hour

Remarks on result:

other: 0.086 to 0.091 95% CL

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

17 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks:

0 to 72 hour

Remarks on result:

other: CL not applicable

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks:

0 to 96 hour

Remarks on result:

other: CL not applicable

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.032 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: cell numbers, yield, biomass and growth rate

Remarks on result:

other: None

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.032 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: cell numbers. yield, biomass and growth rate

Remarks on result:

other: None

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: cell numbers, yield, biomass, and growth rate

Remarks on result:

other: None

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

0.1

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: cell numbers, yield, biomass, and growth rate

Remarks on result:

other: None

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): yes, cell bloating and fragments seen in some test concentrations
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no precipitation seen in any test concentration
- Effect concentrations exceeding solubility of substance in test medium: no, compared to water solubility

Results with reference substance (positive control):

- Results with reference substance valid? Yes. but not concurrent in this test. Potassium dichromate was used as the reference toxicant in assessing performance of the laboratories' culture.
- EC50: 72 hr EC50 0.64 mg Cr/L in September, 2007 test as compared to 0.44 mg Cr/L (0.38 to 0.49 mg Cr/L) in May reference test

Reported statistics and error estimates:

See materials and methods for details on statistical analysis parameters. Mean values for all endpoints and time periods and 95% confidence levels can be found in tables in results section.

EC50, NOEC and LOEC values for cell density, yield, biomass and growth rate based on nominal concentrations

Endpoint	EC50 (mg a.i./L)	95% Confidence Limits (mg a.i./L)	NOEC (mg a.i./L) a	LOEC (mg a.i./L) b
72 hr Cell Density	0.10	0.085 – 0.18	0.032	0.10
96-hr Cell Density	0.083	0.081 – 0.085	0.032	0.10
72-hr Yield	0.10	0.093 – 0.17	0.032	0.10
96-hr Yield	0.083	0.080 – 0.086	0.032	0.10
0 to 72 hr Biomass	0.14	0.089 – 0.25	0.032	0.10
0 to 96 hr Biomass	0.088	0.086 – 0.091	0.032	0.10
0 to 72 hr Growth Rate	17 c	NA d	0.032	0.10
0 to 96 hr Growth Rate	14 c	NA	0.032	0.10

a NOEC = No-Observed-Effect Concentration

b LOEC = Lowest-Observed-Effect Concentration

c Since the inhibition of growth rate was slightly less than 50%, the 72- and 96- hour ErC50 values were

extrapolated using linear regression

d NA = Not applicable

Mean cell densities after 24, 48, 72 and 96 hours exposure

Nominal Conc. (mg a.i./L)	Mean Cell Density (x 10^4cells/mL) (Standard Deviation)
	Replicates in Mean	24 hours	48 hours	72 hours	72 hour % inhibition	96 hours	96 hour % inhibition
Control	6	3.58 (1.15)	35.42 (6.15)	145.58 (31.76)	NA c	438.17 (38.16)	NA
0.032	3	4.08 (1.01)	35.75 (11.69)	175.44 (41.65)	-21	576.00 (30.12)	-31
0.10	3	3.50 (1.75)	21 (4.13)	70.42 (14.67)d	52	161.44 (17.46)d	63
0.32	3	3.67 (1.28)	17.75 (2.84)	58.00 (6.08)d	60	85.33 (20.86)d	81
1.0	3	3.42 (1.13)	13.17 (4.54)	42.42 (16.96)d	71	88.25 (27.36)def	80
3.2	3	3.67(0.95)	17.67 (2.10)e	63.83 (5.27)de	56	93.75 (8.95)def	79
10	3	2.08 (1.15)	12.00 (3.38)e	16.42 (9.71)def	89	28.75 (1.00)def	93

a Percent inhibition compared to control

b Mean, standard deviation (SD) and percent inhibition are calculated from original raw data, not from

rounded figures presented in table

c NA = Not applicable

d Significantly reduced compared to control, based on Bonferroni’s Test

e Cells observed to be slightly bloated

f Cell fragments observed

EC and NOEC values for endpoint cell density

24 hour results	EC10	EC50	EC90	NOEC	LOEC
EC (mg a.i./L) 95% CL	2.1 0.058 – 5.9	> 10 NA a	> 10 NA a
48 hour results
EC (mg a.i./L) 95% CL	0.046 0.033 – 0.055	0.51 0.094 – 3.4	> 10 NA a
72 hour results
EC (mg a.i./L) 95% CL	0.044 0.039 – 0.047	0.10 0.085 – 0.18	> 10 NA a	0.032	0.10
96 hour results
EC (mg a.i./L) 95% CL	0.042 0.042 – 0.043	0.083 0.083 – 0.085	7.7 7.1 – 8.1	0.032	0.10

a NA = Not Applicable EC value was empirically estimated, therefore, corresponding confidence limits could not be calculated

Mean total yield after 24, 48, 72 and 96 hours exposure

Nominal Conc. (mg a.i./L)	Mean Yield (x 10^4cells/mL) (Standard Deviation)
	Replicates in Mean	0-24 hours	0-48 hours	0-72 hours	72 hour % inhibition	0-96 hours	96 hour % inhibition
Control	6	2.58 (1.15)	34.42 (6.15)	144.58 (31.76)	NA c	437.17 (38.16)	NA
0.032	3	3.08 (1.01)	34.75 (11.69)	174.44 (41.65)	-21	575.00 (30.12)	-32
0.10	3	2.50 (1.75)	20 (4.13)	69.42 (14.67)d	52	160.44 (17.46)d	63
0.32	3	2.67 (1.28)	16.75 (2.84)	57.00 (6.08)d	61	84.33 (20.86)d	81
1.0	3	2.42 (1.13)	12.17 (4.54)	41.42 (16.96)d	71	87.25 (27.36)d	80
3.2	3	2.67(0.95)	16.67 (2.10)	62.83 (5.27)d	57	92.75 (8.95)d	79
10	3	1.08 (1.15)	11.00 (3.38)	15.42 (9.71)d	89	27.75 (1.00)d	94

a Percent inhibition compared to control

b Mean, standard deviation (SD) and percent inhibition are calculated from original raw data, not from

rounded figures presented in table

c NA = Not applicable

d Significantly reduced compared to control, based on Bonferroni’s Test

EC and NOEC values for endpoint total yield 

72 hour results	EC10	EC50	EC90	NOEC	LOEC
EC (mg a.i./L) 95% CL	0.044 0.038 – 0.048	0.10 0.093 – 0.17	9.4 8.4 - 10	0.032	0.10
96 hour results
EC (mg a.i./L) 95% CL	0.042 0.042 – 0.043	0.083 0.080 – 0.086	7.6 6.9 – 8.0	0.032	0.10

Calculated biomass (area under the curve) after 24, 48, 72 and 96 hours exposure

Nominal Conc. (mg a.i./L)	Mean Biomass (x 10^4cells-days/mL) (Standard Deviation)
	0-24 hours	24-48 hours	48-72 hours	Total biomass	72 hour % inhibition	72-96 hours	Total Biomass	96 hour % inhibition
Control (n = 6)	1.13 (0.5)	19.14 (3.25)	87.95 (15.86)	108.22 (14.38)	NA c	287.85 (14.46)	396.07 (23.79)	NA
0.032  (n = 3)	1.35 (0.44)	19.57 (6.48)	102.78 (16.18)	123.71 (13.25)	-14	370.82 (16.72)	494.53 (29.88)	-25
0.10 (n = 3)	1.10 (0.77)	11.64 (2.84)	43.93 (9.22)	56.67 (12.66)d	48	113.73 (1.83)	170.40 (11.41)d	57
0.32 (n = 3)	1.17 (0.56)	10.05 (1.67)	36.23 (3.83)d	47.45 (5.82)d	56	69.93 (13.32)	117.38 (18.74)d	70
1.0 (n = 3)	1.06 (0.5)	7.54 (2.31)	26.33d (10.51)d	34.93 (12.84)d	68	63.66 (17.53)	98.60 (27.71)d	75
3.2 (n = 3)	1.17 (0.42)	10.00 (0.6)	39.06 (2.61)d	50.23 (2.6)d	54	76.98(3.43)	127.21 (3.08)d	68
10 (n = 3)	0.48 (0.51)	6.25 (1.88)	12.98 (5.6)d	19.71 (7.63)d	82	21.36 (5.15)	41.06 (12.46)d	90

a Percent inhibition compared to control

b Mean, standard deviation (SD) and percent inhibition are calculated from original raw data, not from

rounded figures presented in table

c NA = Not applicable

d Significantly reduced compared to control, based on Bonferroni’s Test

EC and NOEC values for endpoint biomass

72 hour results	EC10	EC50	EC90	NOEC	LOEC
EC (mg a.i./L) 95% CL	0.046 0.043 – 0.049	0.14 0.089 – 0.25	> 10 NA a	0.032	0.10
96 hour results
EC (mg a.i./L) 95% CL	0.043 0.043 – 0.044	0.088 0.086 – 0.091	9.5 8.7 – 9.9	0.032	0.10

A NA = not applicable EC value was empirically estimated, therefore, corresponding 95% confidence limits could not be calculated

Mean growth rate after 24, 48, 72 and 96 hours exposure

Nominal Conc. (mg a.i./L)	Mean Growth Rate (days-1) (Standard Deviation b)
	Replicates in Mean	0-24 hours	0-48 hours	0-72 hours	72 hour % inhibitionab	0-96 hours	96 hour % inhibitionab
Control	6	1.38 (0.48)	1.86 (0.1)	1.71 (0.08)	NA c	1.56 (0.02)	NA
0.032	3	1.58 (0.3)	1.85 (0.16)	1.78 (0.08)	-4	1.64 (0.01)	-5
0.10	3	1.34 (0.53)	1.58 (0.11)	1.46 (0.08) d	15	1.31 (0.03) d	16
0.32	3	1.42 (0.45)	1.50 (0.08)	1.40 (0.04) d	18	1.14 (0.07) d	27
1.0	3	1.35 (0.4)	1.33 (0.17)	1.28 (0.13) d	25	1.14(0.09) d	27
3.2	3	1.46(0.28)	1.50 (0.06)	1.43 (0.03) d	16	1.17 (0.03) d	25
10	3	0.66 (0.85)	1.28 (0.15)	0.91 (0.27) d	47	0.86 (0.01) d	45

a Percent inhibition compared to control

b Mean, standard deviation (SD) and percent inhibition are calculated from original raw data, not from

rounded figures presented in table

c NA = Not applicable

d Significantly reduced compared to control, based on Bonferroni’s Test

EC and NOEC values for endpoint growth rate

72 hour results	EC10	EC50	EC90	NOEC	LOEC
EC (mg a.i./L) 95% CL	0.076 0.066 – 0.091	17 a NA b	> 10 NA	0.032	0.10
96 hour results
EC (mg a.i./L) 95% CL	0.071 0.067 – 0.076	14 a NA	> 10 NA	0.032	0.10

a Since the inhibition of growth rate was slightly less than 50%, the 72 and 96-hour EC50 values were

extrapolated using a linear regression

b NA = not applicable EC value was empirically estimated, therefore, corresponding 95% confidence limits

could not be calculated

Test for Algistatic/Algicidal properties: An aliquot was taken at study termination from the highest concentration tested (10 mg a.i./L, nominal) and diluted with AAP medium to reduce the test article concentration to the lowest value (0.032 mg a.i./L). The estimated cell density of that diluted sample was 0.092 x 10^4 cells/mL. The sample was maintained under test conditions for 4 days. After 4 days, cell density was 43 x 10^4 cells/mL, representing a 467 times increase in recovery. This indicated that effects seen in the study were algastatic, not algicidal.

Acceptance criteria required by algae protocol:

Acceptance criteria for Control	Study results (control)
> 16 x 10^4cells/mL at 72 hours	143.58 x 10^4cells/mL at 72 hours
Mean daily growth rate CV < 35%	32%
0 – 72 hour growth rate CV < 7%	5%

Validity criteria fulfilled:

yes

Remarks:

acceptible initial control cell numbers at 72 hours, and acceptable CV on mean daily growth rate and 0 to 72 hour growth rate

Conclusions:

In an acute static toxicity test to green algae (Pseudokirchneriella subcapitata), using nominal concentrations of test article, EC50 values at 72 hours for endpoints cell density, yield, and biomass ranged from 0.10 to 0.14 mg a.i./L; EC50 values for the same endpoints at 96 hours of exposure ranged from 0.083 to 0.088 mg a.i./L. EC50 values for growth rate were 17 mg a.i./L for the 0 to 72 hour period, and 14 mg a.i./L for the 0 to 96 hour period. Since the growth rate inhibition was slightly less than 50%, the 72 and 96 hour values were extrapolated using a linear regression. NOEC for all endpoints at 72 and 96 hours was 0.032 mg a.i./L, and LOEC for the same times and endpoints was 0.10 mg a.i./L. Recovery tests indicated that test article was algistatic, not algicidal.
This study is used as the key study for classification. A 72-h EC50 of 17 mg/L for growth reduction. As the substance was not readily biodegradable a classification as chronic Category 3 is proposed under CLP (Reg. 1907/2006 and adaptations). According to Dir. 67/5448/EC and adaptations a classification as harmful to aquatic organisms and may cause long term effects to the aquatic environment is proposed. (R52/53)

Executive summary:

In an acute static toxicity test to green algae (Pseudokirchneriella subcapitata), using nominal concentrations of test article, EC50 values at 72 hours for endpoints cell density, yield, and biomass ranged from 0.10 to 0.14 mg a.i./L; EC50 values for the same endpoints at 96 hours of exposure ranged from 0.083 to 0.088 mg a.i./L. EC50 values for growth rate were 14 mg a.i./L for the 0 to 72 hour period, and 14 mg a.i./L for the 0 to 96 hour period. Since the growth rate inhibition was slightly less than 50%, the 72 and 96 hour values were extrapolated using a linear regression. NOEC for all endpoints at 72 and 96 hours was 0.032 mg a.i./L, and LOEC for the same times and endpoints was 0.10 mg a.i./L. Recovery tests indicated that test article was algistatic, not algicidal.

Description of key information

A study was  performed with Pseudokirchneriella subcapitata  to assess the acute toxicity on green algae. The study was performed under GLP and using the OECD guideline N°201. EC50 values for growth rate was 17 mg /L (nominal concentration) for the 0 to 72 hour period.  the NOEC value for cell numbers, yield, biomass and growth rate at 72 hours was 0.032 mg/L (nominal concentration), and LOEC for the same time and endpoints was 0.10 mg/L (nominal concentration). This study is used as the key study for PNEC derivation for freshwater. 

Key value for chemical safety assessment

EC50 for freshwater algae:

17 mg/L