Octadecanoic acid, reaction products with diethylenetriamine and urea, acetates

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:

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT #: 93580/A
Batch: 0022357000
Purity: 94.3 %
Expiry date: 15 December 2015
Description at Room Temperature: White-yellowish waxy solid
Storage Conditions: Room temperature, in the dark.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Water-accommodated fractions (WAFs) with the following loading rates of the test item were tested: 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control was tested in parallel (test water without test item). Loading rates of the test item exceeding 100 mg/L were not tested, in accordance with the test guidelines.

- Sampling method: For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae), after 24 and 48 hours and at the end of the test (containing algae). At the start and at the end of the test, duplicate samples were also taken from the control.
For the 24, 48 and 72-hour stability samples, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions. This was necessary as the volume of test solution of the treatment replicates (3 x 15 mL) was too small to perform the analyses. A volume of 500 mL per sample was necessary for analytical purposes.

- Sample storage conditions before analysis: Immediately after sampling, dichloromethane (200 mL per litre sample volume) was added to each sample in order to stabilize the latter during the storage period. Thereafter, all samples were stored deep-frozen (at about -20 °C). In pre-experiments for investigation of the storage stability of the samples, the test item proved to be stable under these storage conditions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water accommodated fractions (WAF)
- Eluate: test water
- Differential loading: For preparation of the WAFs, individual dispersions of the test item were prepared by weighing in different amounts of test item into test water, applying ultrasonic treatment for 15 minutes and stirring for 96 hours at room temperature in the dark to dissolve a maximum amount of the different components of the test item in the dispersion. After the stirring period, the dispersions were filtered through membrane filters (Schleicher & Schuell, Type NC20, pore size 0.20 μm) after preconditioning the filter material with about 200 mL of test medium to avoid test item losses in the filter. The undiluted filtrates were tested as WAFs.
Loading rate 100 mg/L: 500.0 mg test item in 5000 mL water.
Loading rate 32 mg/L: 160.38 mg test item in 5000 mL water.
Loading rate 10 mg/L: 50.0 mg test item in 5000 mL water.
Loading rate 3.2 mg/L: 19.25 mg test item in 6000 mL water.
Loading rate 1.0 mg/L: dilution of loading rate 3.2 mg/L..
The stirring period of 96 hours was chosen based on the results of a pre-experiment in which the maximum concentration of the different compounds in the test media was reached after the stirring period of 96 hours.
- Controls: test water
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum)
- Strain: 61.81 SAG
- Source: Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany)
- Age of inoculum (at test initiation): An inoculum culture was set up four days before the start of the exposure.
- Method of cultivation: The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

ACCLIMATION
- Acclimation period: four days
- Culturing media and conditions: same as in the test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Test was terminated after 72 hours.

Hardness:

0.15 mmol/L (= 15 mg/L as CaCO3)

Test temperature:

24 °C

pH:

7.3-7.5 at test start
8.6-8.7 at test end

Dissolved oxygen:

Not measured and not relevant in this test design

Salinity:

not applicable

Nominal and measured concentrations:

Loading rates 1.0, 3.2, 10, 32 and 100 mg/L
Mean measured test item concentration of the loading rate of 100 mg/L: 1.27 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks
- Type: open
- Material, size, headspace, fill volume: glass, 50 mL, 35 mL, 15 mL
- Aeration: No specific aeration but during exposure, the test solutions were continuously stirred by magnetic stirrers.
- Initial cells density: 5000 algal cells/mL (based on 0.45 x 1000 relative fluorescent units)
- Control end cells density: 1.097x 1 000 000/mL (based on 98.7 x 1000 relative fluorescent units)
- No. of organisms per vessel: 91.7-11.4 x 1000 relative fluorescent units
- 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: Analytical grade salts were dissolved in sterile purified water.
- Total organic carbon: not available
- Particulate matter: not available
- Metals:
Macro-nutrients
NaHCO3: 15.0 mg/L
K2HPO4: 1.044 mg/L
MgSO4× 7 H2O: 14.6 mg/L
MgCl2 × 6 H2O: 12.16 mg/L
CaCl2 × 2 H2O: 4.41 mg/L
NaNO3: 25.5 mg/L
Trace elements
H3BO3: 186.0 μg/L
MnCl2 × 4 H2O: 415.0 μg/L
ZnCl2: 3.27 μg/L
CoCl2 × 6 H2O: 1.43 μg/L
CuCl2 × 2 H2O: 0.012 μg/L
Na2MoO4 × 2 H2O: 7.26 μg/L
FeCl3 × 6 H2O: 160.0 μg/L
Na2EDTA × 2 H2O: 300.0 μg/L
- Pesticides: not available
- Chlorine: not available
- Alkalinity: not available
- Ca/mg ratio: not available
- Conductivity: not available
- Culture medium different from test medium: no
- Intervals of water quality measurement: at test start and test end

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: permanent light
- Light intensity and quality: 6600 Lux (range: 5770-7200 Lux, measured at nine places in the experimental area). The light intensity was within a ±15 %-deviation from the average light intensity as recommended by the guideline, illumination by fluorescent tubes (Philips TLD 36W-1/840).

EFFECT PARAMETERS MEASURED: A small volume of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced.
- Determination of cell concentrations: The algal biomass in the samples was determined by fluorescence measurement (BIO-TEK Multi-Detection Microplate Reader, Model FLx800, wavelength: excitation 440 nm, emission 680 nm). The measurements were performed at least in duplicate.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: spacing factor between the loading rates 3.2
- Range finding study: yes
- Test concentrations: undiluted filtrate after 96 hours of stirring, dilutions 1:10, 1:100 and 1:1000
- Results used to determine the conditions for the definitive study: effects in the higher test concentrations

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1.27 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.27 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The results were based on the loading rate of 100 mg/L and on the mean measured concentration of 1.27 μg/L (calculated as arithmetic mean of the geometric means of the test item conc. measured at the start and the end of each test medium renewal period).

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid : yes
For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in April 2013 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 1.5 mg/L (Harlan Study Number D74222), range of the 72-hour EC50 for the growth rate from 2000 to 2013: 0.71-1.7 mg/L).

Reported statistics and error estimates:

Growth rate and yield were calculated for each test flask. The mean values for growth rate and yield were calculated for each treatment. The tabulated values represent rounded results obtained by calculation using the exact raw data.
The 72-hour EL10/EC10, EL20/EC20 and EL50/EC50 values of the test item could not be determined because of the absence of a significant inhibitory effect of the test item on the algal growth at the tested concentrations.
For the determination of the LOEL/LOEC and NOEL/NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Dunnett t-test [Dunnett, 1955; Dunnett, 1964].

Table 1 Analytical results of the test samples

Time point	Loading Rate of Test Item	Measured Concentration of Test Item			Sample Preparation Factor (F)	Determined Average Concentration of Test Item (c)
		Sample 1	Sample 2	Average
[day]	[mg/L]	[µg/L]				[µg/L]
0	Control	*	-	<LOQ	0.06	<LOQ
	100	12.1	18.7	15.4	0.21	3.23
1	100	5.25	9.12	7.19	0.21	1.51
2	100	14.4	-	n.a.	0.06	1.27
	100	-	8.01	n.a.	0.21
3	Control	*	-	<LOQ	0.06	<LOQ
	100	10.1	3.78	6.95	0.06	0.417

*: the measured concentration was below the lowest calibration concentration

LOQ: 0.217 µg test item/L

n.a.: not applicable

Table 2 Biomass of algae

Loading rate	Rep. no.	Biomass of algae*
[mg/L]		24 hours	48 hours	72 hours
Control	1	4.9	24.9	103.5
	2	4.2	22.8	111.4
	3	3.4	22.8	91.7
	4	4.4	23.4	100.9
	5	4	18.9	91.7
	6	3.3	21	93.2
	Mean	4	22.3	98.7
	SD	0.6	2.1	8
1	1	3.8	23.2	95
	2	4.6	22.5	109.5
	3	3.1	19.3	90.3
	Mean	3.8	21.7	98.3
	SD	0.8	2.1	10
3.2	1	3.9	23.7	100.5
	2	4.3	21.6	89.7
	3	2.9	15.5	75.2
	Mean	3.7	20.3	88.5
	SD	0.7	4.3	12.7
10	1	3.9	20.7	96.8
	2	4	21.9	96.1
	3	3.3	19	86.7
	Mean	3.7	20.5	93.2
	SD	0.4	1.4	5.6
32	1	3.9	18.7	92.4
	2	3.7	22.2	92.7
	3	3.6	15.1	82.1
	Mean	3.8	18.7	89.1
	SD	0.1	3.6	6.1
100	1	3.3	15.5	86
	2	3.4	20.1	89.6
	3	3.6	20.3	86.2
	Mean	3.4	18.6	87.3
	SD	0.2	2.7	2

SD: Standard deviation

*: The biomass was determined by fluorescence measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units (x 103). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 0.45 x 1000 relative fluorescence units.

Table 3 Average growth rates

Treatment / Loading rate	Average growth rate µ (day^-1) and inhibition of µ (Ir)
	0-24 h		0-48 h		0-72 h
[mg/L]	µ	Ir[%]	µ	Ir[%]	µ	Ir[%]
Control	2.17	0	1.94	0	1.79	0
1	2.12	2.3	1.93	0.7	1.79	0.1
3.2	2.09	3.9	1.89	2.8	1.75	2.1
10	2.1	3.1	1.9	2.1	1.77	1
32	2.11	2.8	1.85	4.8	1.76	1.9
100	2.02	6.8	1.85	4.7	1.75	2.2

No statistically significant effect at all loading rates compared to control (according to Dunnett t-test, one-sided smaller, alpha = 0.05)

Table 4 Yield

Treatment / Loading rate	Yield Y (x 10^3) and inhibition of Y (Iy)
	0-24 h		0-48 h		0-72 h
[mg/L]	Y	Iy[%]	Y	Iy[%]	Y	Iy[%]
Control	3.6	0	21.9	0	98.3	0
1	3.4	5.2	21.2	2.9	97.8	0.5
3.2	3.2	8.8	19.8	9.3	88	10.5
10	3.3	8.1	20.1	8.1	92.7	5.6
32	3.3	7.6	18.2	16.7	88.6	9.8
100	3	16.4	18.2	16.8	86.8	11.6

No statistically significant effect at all loading rates compared to control (according to Dunnett t-test, one-sided smaller, alpha = 0.05)

Result summary:

Parameter (0 -72h)	Growth rate	Yield
EL10[mg/L]	>100	>100
EL20[mg/L]	>100	>100
EL50[mg/L]	>100	>100
NOEL[mg/L]	100	100
LOEL[mg/L]	>100	>100

Parameter	Growth rate	Yield
(0-72h)
EC10[µg/L]	>1.27	>1.27
EC20[µg/L]	>1.27	>1.27
EC50[µg/L]	>1.27	>1.27
NOEC[µg/L]	1.27	1.27
LOEC[µg/L]	>1.27	>1.27

Validity criteria fulfilled:

yes

Conclusions:

FAT#:93580/A had no toxic effects on the algal species up to the loading rate of 100 mg/L, respectively to its solubility limit in the test water under the present test conditions.

Executive summary:

The impact of the test item FAT#:93580/A on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006) and the Commission Regulation (EC) No 761/2009, C.3.

To assess the toxicity of FAT#:93580/A containing different components to algae, water accommodated fractions (WAFs) with the loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were tested. Additionally, a control (test water without test item) group was tested in parallel. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

For preparation of the WAFs, individual dispersions of the test item with the loading rates as mentioned above were prepared. The dispersions were ultrasonic treated for 15 minutes and continuously stirred at room temperature in the dark over 96 hours to dissolve a maximum amount of the different compounds of the test item in the dispersion. Then, the dispersions were filtered through membrane filters (0.2 µm) and the undiluted filtrates were tested as WAFs. Due to technical reasons, the WAF with the lowest loading rate of 1 mg/L was prepared as a dilution of the WAF with the loading rate of 3.2 mg/L.

The measured test item concentrations in the test media with the loading rate of 100 mg/L (based on the sum of six main components) were 3.23 µg/L (day 0), 1.51 µg/L (day 1), 1.27 µg/L (day 2) and 0.417 µg/L (day 3). The geometric mean measured test item concentrations at the loading rate of 100 mg/L over 72 hours was 1.27 µg/L. The reported biological results were based on loading rates and mean measured concentrations

(calculated as the geometric mean of the concentrations measured at all sampling points):

Endpoints based on loading rates for both growth rate and yield after 72 hours of exposure

EL10: >100 mg/L

EL20: >100 mg/L

EL50: >100 mg/L

NOEL: 100 mg/L

LOEL: >100 mg/L

Endpoints based on the geometric mean measured test concentration for both growth rate and yield after 72 hours of exposure

EC10: >1.27 µg/L

EC20: >1.27 µg/L

EC50: >1.27 µg/L

NOEC: 1.27 µg/L

LOEC: >1.27 µg/L

In conclusion, the test item FAT#:93580/A had no toxic effects on Pseudokirchneriella subcapitata up to its solubility limit in the test water under the present test conditions.

Description of key information

The study describes the impact of test item FAT#:93580/A on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 hour static test according to the OECD Guideline 201.

Endpoints based on loading rates for both growth rate and yield after 72 hours of exposure are:

EL10: >100 mg/L

EL20: >100 mg/L

EL50: >100 mg/L

NOEL: 100 mg/L

LOEL: >100 mg/L

In conclusion, the test item FAT#:93580/A had no toxic effects on Pseudokirchneriella subcapitata up to its solubility limit in the test water under the present test conditions.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L