N,N'-hexane-1,6-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenylpropionamide]

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sampling schedule: All concentration levels and the control were analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (72 hours) with and without algae. Additionally, adsorption to glass was analytically verified from all concentration levels at the end of the exposure.

Sampling and pre-treatment: At the start, samples were taken from one additional replicate of each test item concentrat ion and the control and at the end samples were taken from pooled replicates. Two samples per concentration level and control were taken, stabilized with acetonitrile with 0.2% formic acid (factor 2) and one sample was analyzed.

Vehicle:

no

Details on test solutions:

Three saturated solutions were prepared separately with nominal loading rates of 1.00 - 3.16 - 10.0 μg test item/L.
For each loading rate an appropriate amount of stock solution containing the test item in methanol was placed on a curved glass slide. The methanol was evaporated. The glass slide with the test item was inserted in a glass flask with an appropriate amount of dilution water. A slow stirring procedure was applied for 24 ± 1 hour at room temperature. The magnetic stirrer bar was placed with a fish-clip® system a few centimeters above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 30 minutes, the
aqueous phase of the saturated solution was removed by siphoning (from the approximate middle of the glass flask). The first 25 mL were discarded.
The saturated solutions were checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative.

Control
The control solution was prepared without test item following the same method as specified for the saturated solutions. Six replicates were exposed under the same conditions as the test concentrat ions.

Test organisms (species):

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

Details on test organisms:

Origin
Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK

Cultivation at test facility
Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lx for 24 hours per day.

Culture medium
Nutrient medium Z according to LÜTTGE et al. (1994)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

34 mg CaCo3/L total hardness

Test temperature:

Nominal range: 21 - 24 °C, controlled at ± 2 °C

pH:

8.1 +/- 0.2

Conductivity:

138 μS/cm

Nominal and measured concentrations:

Nominal: 1, 3.16 and 10 μg/L
Measured (geometric mean): 0.594, 0.791 and 1.3 μg/L respectively

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs
- Test volume: 100 mL
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density:
Nominal: approximately 5 x 10^3 - 10^4 cells/mL
Actual: 6864 cells/mL
- Control end cells density: 992993 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (Nutrient medium Z according to LÜTTGE et al. (1994))

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to the guidelines
- Total organic carbon: < LOQ (Limit of quantification = 2.00 mg C/L)
- Alkalinity: 0.7 mmol/L
- Acidity: 0.3 mmol/L
- MgCl2 * 6 H2O: 12 mg/L
- CaCl2 * 2 H2O: 18 mg/L
- Conductivity: 138μS/cm
- Intervals of water quality measurement: The pH-value at the start of the exposure was measured in one additional replicate of each test item concentration and the control.
At the end of the exposure, it was measured in a pooled sample of the test item concentrat ions and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lx, corresponding to 60 to 120 μE*m^-2*s^-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll afluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was observed in the preliminary range finding test at the concentrat i on
of 1 mg/L.
- Microscopic evaluation: The algae cells were evaluated microscopically at the start and the end of the incubation period. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Three test concentrations of nominal 1.00, 3.16, 10.0 μg test item/L test item solutions (factor: √10) were tested. The dilution levels are based on the results of a preliminary range finding test (non-GLP).
- Range finding study: A non-GLP preliminary range finding test under static conditions over a period of 72 hours with headspace was conducted at the test facility with a saturated solution with a nominal loading rate of 1.00 mg/L due to the low solubility in the test medium. Two saturated solutions were prepared, one as described in section 4.2 and the other with filtration (membrane filter 0.45 μm, RC, MACHEREY-NAGEL). The Tyndall effect was for both negative.
In the range finding test, two replicates per concentration and four for the control were tested. All concentration levels were visually clear and colorless throughout the exposure period.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 10 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Geometric mean EC10 > 1.3 µg/L = highest tested concentration up to the maximum solubility limit

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Geometric mean EC50 > 1.3 µg/L = highest tested concentration up to the maximum solubility limit

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: Geometric mean EC50 > 1.3 µg/L = highest tested concentration up to the maximum solubility limit

Duration:

72 h

Dose descriptor:

EC10

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

not determinable

Results with reference substance (positive control):

See below "Any other information on results incl. tables"

Table 1: Cell Densities

Nominal test item concentration [µg/L]	Replicate   No.	Cell density [cells/mL]
		0 hours	24 hours	48 hours	72 hours
	1	6864	20049	117694	763598
10.0	2 3	6864 6864	23565 16903	128831 96338	770233 625420
	Mean	6864	20172	114288	719750
	1	6864	22395	183824	900495
3.16	2 3	6864 6864	20139 11494	122957 97390	835499 787100
	Mean	6864	18009	134724	841031
	1	6864	22312	124171	897639
1.00	2 3	6864 6864	19712 21057	119763 104947	733484 790856
	Mean	6864	21027	116294	807326
	1	6864	18552	114357	851107
	2	6864	17144	116318	775046
	3	6864	23520	138268	1025247
Control	4	6864	21487	138448	1050458
	5	6864	24050	160290	1258401
	6	6864	19627	137090	997697
	Mean	6864	20730	134129	992993

 

Table 2: Evaluation after 72 hours

Nominal test item concentration   [µg/L]	Replicate     No.	Growth rate     [d-1]	Inhibition of growth rate   [%]	Yield     [cells/mL]	Inhibition of yield   [%]
	1	1.57	5	756734	23
10.0	2 3	1.57 1.50	5 9	763369 618556	23 37
	Mean	(s)             1.55	6	(s)        712886	28
	1	1.63	2	893631	9
3.16	2 3	1.60 1.58	3 4	828635 780236	16 21
	Mean	(ns)           1.60	3	(ns)       834167	15
	1	1.62	2	890775	10
1.00	2 3	1.56 1.58	6 4	726620 783992	26 21
	Mean	(ns)           1.59	4	(ns)       800462	19
	1	1.61		844243
	2	1.58		768182
	3	1.67		1018383
Control	4	1.68		1043594
	5	1.74		1251537
	6	1.66		990833
	Mean	1.65		986129

 

Table 3: Section-by-Section and Average Specific Growth Rates of the Control Group (0 - 72 hours)

	Replicate No.	Specific Growth Rate [d^-1] section-by-section			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		0 - 24 hours	24 - 48 hours	48 - 72 hours
	1	0.99	1.82	2.01	1.61	0.539	33.5
	2	0.92	1.92	1.90	1.58	0.572	36.3
Control	3 4	1.23 1.14	1.77 1.86	2.00 2.03	1.67 1.68	0.396 0.471	23.7 28.1	29.7
	5	1.25	1.90	2.06	1.74	0.426	24.5
	6	1.05	1.94	1.99	1.66	0.528	31.8
				Mean	1.65
				SD ±	0.06
				CV [%]	3.43

SD = Standard deviation CV = Coefficient of variation

 

Table 4: Measured Concentrations of the Test Item during the Definitive Test with Algae

Sampling date	Start of the Exposure, 0 hours (with algae)		End of the Exposure, 72 hours (with algae)	Geometric mean measured test item concentration^1)
Nominal test item concentration [µg/L]	Test item
	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	[µg/L]
10.0	3.37	34	˂ LOQ	1.30
3.16	1.25	40	˂ LOQ	0.791
1.00	0.705	70	˂ LOQ	0.594
Control	˂ LOQ		˂ LOQ
QC	92%		99%

 

Table 5: Measured Concentrations of the Test Item during the Definitive Test without Algae

Sampling date	Start of the Exposure, 0 hours (without algae)		End of the Exposure, 72 hours (without algae)	End of the Exposure, 72 hours (Glass adsorption)
Nominal test item concentration [µg/L]	Test item
	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	Meas. conc. [µg/L]
10.0	4.60	46	˂ LOQ	˂ LOQ
3.16	1.47	47	˂ LOQ	˂ LOQ
1.00	0.710	71	˂ LOQ	˂ LOQ
Control	˂ LOQ		˂ LOQ

Table 6: Environmental Conditions

Nominal test item concentration	pH-values
[µg/L]	Start; 0 hours	End; 72 hours
10.0	8.06	8.93
3.16	8.08	9.05
1.00	8.10	9.26
Control	8.10	9.38
Room temperature [°C]:            min.: 22.5                       max.: 23.5                             mean value: 23.0
mean value: 5627 Light intensity                n = 22               range of the measured values: 4783 - 6340                     CV [%]: 8.01 [lx]                                                                   equalling -15.0 to 12.7

CV = Coefficient of variation                     n = number of measuring points

 

Table 7: EC50 values of reference item

	Current Study	Valid Range (average ± 3 x SD)
	Growth Rate inhibition
ErC50	0.851	0.778 ± 0.550
95% confidence interval	0.802 – 0.931
	Yield inhibition
EyC50	0.449	0.422 ± 0.333
95% confidence interval	0.396 – 0.506

 

Table 8: Validity criteria

Validity Criterion	Required	This study
Increase of the cell growth in the control cultures	Exponentially, ≥ 16-fold corresponding to a specific growth rate of 0.92 day^-1	145-fold (specific growth rate 1.65 day-1)
Mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures	≤ 35%	29.7%
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures	≤ 7%	3.43%

Validity criteria fulfilled:

yes

Conclusions:

With high probability acutely not harmful to aquatic algae and cyanobacteria.

Executive summary:

A GLP guideline study according OECD 201 has been conducted. Due to the very low solubility of the test item in the test medium three concentrations have been tested that were determined in the range finder study. After 72h of exposure the EC10 and EC50 on growth rate have been determined to be above the maximum solubility of the test item. Therefore, the test item is with high probability acutely not harmful to aquatic algae and cyanobacteria.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1992-11-09 to 1992-12-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Non-GLP-guideline study. Raw data re-evaluated to derive the performance criteria listed in the current OECD 201 TG.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

no analytical dose verification

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 0.4% (Arkopal) were mixed together, by means of a marble-mill during about 72 hours. 1 mL of this blend were mixed and made up to 1000 mL with water (stock solution 1 for all test concentrations). Calculated amounts of the stock solution to produce the desired test concentrations were given into the water and were homogeneously distributed. The algae were then transfered into the flasks.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): alkylphenolpolyglykolether 0.4% (Arkopal)
- Concentration of vehicle in test medium: 0.4% (stock solution)

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green Algae
- Source (laboratory, culture collection): CIBA-GEIGY Ltd., testing facility

ACCLIMATION
- Acclimation period: 3 days under test conditions.
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: none reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

no data applicable

Test temperature:

23 + 0.5°C

pH:

7.1 - 7.9

Dissolved oxygen:

no data applicable

Salinity:

no data applicable

Nominal and measured concentrations:

1.23, 3.7, 11, 33 and 100 mg test substance/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks, stoppered with aluminium caps, on Lab-shaker
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 mL filled with 50 ml test solution
- Initial cells density: 7000 cells/mL
- Control end cells density: 1032000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: none
- Photoperiod: continuous
- Light intensity and quality: cold white fluorescent light, 121 microE/m^ sec (approx. 8000 lux)

EFFECT PARAMETERS MEASURED : cell density
- Determination of cell concentrations: Cell densities were measured at 24, 48 and 72 hours exposure on a "TOA" cell counter.
- Other: Temperature was continuously measured and maintained at 23 + 0.5°C. pH was measured at 0h and 72h exposure

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Test concentrations: 1.23, 3.7, 11, 33 and 100 mg test substance/L

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Values are based on nominal concentrations

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 35-78 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Values are based on nominal concentrations

Basis for effect:

biomass

Remarks on result:

other: 5% level

Details on results:

- Exponential growth in the control (for algal test): yes
- Flocculation: Small parts of the test substance were swimming on the surface of the test water at test concentrations of 11, 33 and 100 mg/L.
- Other: In spite of the small effect of the vehicle it is clearly visible that the EbC 50 is between 33 and 100 mg/L.
- No toxicity observed in the range of solubility.
- Clear effects were observed at 100 mg/L test substance.

Validity criteria requested by OECD TG 201 compared to the present study (re-evaluation of the raw data)

-      The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

--> The biomass in the control cultures increased by a factor of 147 in the control and 94 in the vehicle control. The validity criterion is therefore fulfilled.

 

-      The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%.

Growth rates for each day derived by re-evaluation of the raw data.

	0-24	24-48	48-72	Mean	SD	CV [%]
Control	1.66	1.31	2.02	1.66	0.35	21.21
Vehicle control	1.52	1.24	1.78	1.66	0.27	17.96

 

--> The coefficients of variation do not exceed 35%. The validity criterion is fulfilled.

 

-      The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus.

--> The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 2.13% (SD 0.04). For the vehicle control the CV was 1.52% (SD 0.02). The validity criterion is fulfilled.

The validity criteria are fulfilled and the study is regarded as valid.

Executive summary:

In a 72 hour acute toxicity study, the cultures of Scenedesmus subspicatus were exposed to the test item at nominal concentrations of 0, 1.23, 3.77, 11, 33 and 100 mg/L under static conditions in accordance with the EEC guideline 302/87 (1987).  The NOEbC and EbC50 values based on cell number were 11 and 45 mg/L, respectively. 

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for OECD 201 toxicity study. The study meets the validity criteria.

Description of key information

With high probability acutely not harmful to aquatic algae and cyanobacteria.

Additional information

A GLP guideline key study according OECD 201 using Pseudokirchneriella subcapitata has been conducted. Due to the very low solubility of the test item in the test medium three concentrations (1.00 – 3.16 – 10.0 μg test item/L) have been tested that were determined in the range finder study. After 72h of exposure the EC10 and EC50 on growth rate have been determined to be above the maximum solubility of the test item. Therefore, the test item is with high probability acutely not harmful to aquatic algae and cyanobacteria.

The results are supported by a 72 hour acute toxicity study. The cultures of Scenedesmus subspicatus were exposed to the test item at nominal concentrations of 0, 1.23, 3.77, 11, 33 and 100 mg/L under static conditions in accordance with the EEC guideline 302/87 (1987).  The NOEbC and EbC50 values based on cell number were 11 and 45 mg/L, respectively and clearly above the limit of water solubility. The study met the validity criteria of the guideline.