Fatty acids, tallow, compds. with triethanolamine

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:

28 April 2016 to 15 July 2016

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

Remarks:

LC-MS/MS TOF analysis

Details on sampling:

- Concentrations: At the start of the test (0 hours), ca. 20 mL samples of freshly prepared test media were taken from the control, 1.0, 3.2, 10, 32 and 100 % saturated solution test media preparation flasks for chemical analysis.
At 72 hours, replicate test vessels at each treatment level were pooled and sampled. Samples (ca. 20 mL) of each were taken for chemical analysis. At each sampling occasion, duplicate samples were taken. One for immediate chemical analysis and one as a ‘back up’ sample should further analysis be required.
- Sampling method: Samples were analysed by injection onto a liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) system.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Initial solubility work was carried out under a separate study.
The large variability and inconsistency of achieved concentration observed at both stages of the study (range-finder and the definitive test) were considered to be due to the poor solubility of the compound, the UVCB (Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Materials) nature of the compound, in addition, it is considered that filtration of the saturated solutions may have also impacted on the achieved concentrations.
- Method: At the start of the definitive test, the 100 % saturated solution test concentration was prepared by weighing ca. 100 mg of test substance and adding to ca. 1000 mL of EC medium. This was then stirred for ca. 24 hours and filtered through 0.45µm syringe filters, a serial dilution was then performed from the resulting test media to give the remaining test concentrations. After which it was observed to be a colourless solution.
- Controls: A control treatment was prepared by adding EC medium only to the control vessels.
- Chemical name of vehicle: Not applicable, the test material was added to EC medium.
- Evidence of undissolved material: The control media was observed to be colourless solutions throughout the duration of the test.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Strain 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Oban, UK.
- Method of cultivation: On receipt from the CCAP, the slope culture was stored in the fridge at 2 - 8 °C. A typical shelf life for each slope was 8 months, after which the slopes were discarded. Semi-axenic cultures of Pseudokirchneriella subcapitata were maintained in liquid culture. These were used to inoculate starter liquid cultures, which in turn were used to inoculate test vessels containing control and test media at the start of each test.
Prior to each test, a sub-sample (ca. 200 µL) of a current liquid slope culture was added to two starter cultures (conical flasks) containing 100 mL EC medium.
Each culture was incubated (under the same conditions as used in the test) for at least 72 hours prior to the start of each test. Following incubation, the cell density of a single starter culture was established using a particle counter (Z2 Coulter Counter®) and confirmed manually using a haemocytometer.
An inoculum volume was then calculated and added to each test vessel, to achieve a starting alga cell density of 1 × 10^4 cells/mL. All flasks for a particular test were inoculated from a single starter culture. The second (unused) starter culture (incubated as a contingency) was discarded after the test was started.
Regular tests are conducted using a reference toxicant to ensure that cultures are of the highest quality and sensitivity.

ACCLIMATION
- Culturing media and conditions: Prior to testing, duplicate starter cultures were prepared and incubated under test conditions for at least 72 hours prior to the start of each test to obtain sufficient algal cells in exponential growth and to achieve a starting algae cell density of 1 × 10^4 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 - 24 °C

pH:

7.40 - 7.63 at test initiation.

Nominal and measured concentrations:

Range-finding Test: Nominal test substance concentrations of 0.1, 1.0, 10 and 100 % saturated solutions.
Definitive test: Nominal test concentrations of 1.0, 3.2, 10, 32 and 100 % saturated solutions (0.0523, 0.151, 0.395, 0.509 and 1.93 mg/L based on geometric mean measured concentrations)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile autoclaved glass 250 mL Erlenmeyer (conical) flasks.
- Type: Closed. Sterile foam bungs were used to cover the top of the vessels. All flasks were loosely-capped.
- Material, size, headspace, fill volume: 100 mL of the appropriate control or test media was added
- No. of organisms per vessel: Starting algal cell concentration of 1 × 10^4 cells/mL.
- No. of vessels per concentration: Three test vessels prepared for each of the five test concentrations. An additional vessel was included at each test concentration which was not inoculated at the start of the test.
- No. of vessels per control: Six test vessels were prepared for the control vessels. An additional vessel was included at which was not inoculated at the start of the test.

GROWTH MEDIUM
- Standard medium used: EC medium
- Detailed composition if non-standard medium was used: See Table 1 Preparation of Algal Medium.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No. At the start of the test, the pH of freshly prepared test media was determined. The pH in each test vessel was also determined at the end of the test.
- Photoperiod: 72 Hours
- Light intensity and quality: The vessels were placed on an orbital shaker (ca. 100 rpm) under conditions of constant light (4440 to 8880 Lux), using LED lights, emitting light across the visible portion of the spectrum (400 - 700 nm). The light intensity within the test area was monitored at the start and end of the test.

EFFECT PARAMETERS MEASURED: At approximately 24-hour intervals after the start of the incubation period, pre-determined volumes of test media (1.0 mL at 24 hours and 0.5 mL at 48, and 72 hours) were removed from each incubated test vessel, and transferred to individually identified cell counting vials. The contents of each vial were diluted to a 10 mL final volume with an electrolyte solution. The cell density of the vial contents was then determined using a particle counter (Z2 Coulter Counter®).
The appearance, colour and behaviour of the test substance in the test media were recorded at the start and end of the test. At 0 hours the control and 1.0, 3.2, 10, 32 and 100 % saturated solution test groups were observed as being colourless solutions. At the end of the test (72 hours) the control and the 1.0, 3.2, 10, 32 % saturated solution concentration were observed as being green homogenous hazy dispersions of algal cells, and the 100 % saturated solution test groups were observed as light green homogeneous hazy dispersions of algal cells.
A media blank (EC media or appropriate test media only) was prepared at control and each test concentration to establish background counts on each sampling occasion. Background counts were subtracted from the cell counting results for each of the inoculated test vessels. The resulting cell counts were then used to calculate the area under the growth curves, yield and the corresponding specific growth rates. The media blank vessels were also analysed at 72 hours as algal appeared to be adhering to the test substance in the range-finder test.

TEST CONCENTRATIONS
- Range finding study: Yes.
- Test concentrations: Nominal test substance concentrations of 0.1, 1.0, 10 and 100 % saturated solutions. Due to the low solubility of the test substance and based on the inhibition results of the first range-finder test it was considered justifiable to run the range-finder as a range-finder limit test, including six vessels at 100 % solution. All vessels were inolculated with sufficient algal cells to acheive an initial algae cell concentration of 1 x 10^4 cells/mL.
- Results used to determine the conditions for the definitive study: Yes. Based on the results of a range-finding limit test for which only the key finding have been reported, the definitive test was conducted at nominal test concentrations of 1.0, 3.2, 10, 32 and 100 % saturated solutions (0.0523, 0.151, 0.395, 0.509 and 1.93 mg/L based on geometric mean measured concentrations).

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.93 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

Based on nominal loading rate, the 72-hour EyC50, EbC50 and ErC50 values were calculated to be >100 mg/L.
The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 100 mg/L based on the nominal loading rate.
- Any observations that might cause a difference between measured and nominal values: The freshly prepared test media at 0 hours appeared as colourless solutions. At the end of the test (72 hours) the control and the 1.0, 3.2, 10, 32 % saturated solution concentrations were observed as being green homogenous hazy dispersions of algal cells, and the 100 % saturated solution test groups were observed as light green homogeneous hazy dispersions of algal cells.

Reported statistics and error estimates:

Statistical analysis was performed using the CETIS program v 1.8.6.
To distinguish between ExC50 values, estimated using areas under the growth curve, final yield and growth rates, the symbols EbC50, EyC50 and ErC50 were used, respectively.
The EC10, EC20 and EC50 are defined as the concentrations that result in a 10 %, 20 % and 50 % mean reduction, respectively, relative to the control.
The area under the growth curve (A), the average specific growth rate (µ) and the final yield over specified time intervals were analysed using a Bonferroni adj t Test (Parametric-Multiple Comparison test) to determine the no observed effect concentration (NOEC).
Linear interpolation (ICPIN) analysis was performed in order to estimate EC10, EC20 and EC50 values. Where possible, 95% confidence limits were calculated for the EC10, EC20 and EC50 values.

Chemical Analysis

The limit of quantification (LOQ) was 0.1 mg/L.

Analysis of the freshly prepared test solutions at 0 hours showed measured concentrations of 0.162, 0.590, 2.02, 5.74 and 15.2 mg/L.

Analysis of the inoculated 72 hour test solutions showed measured concentrations of 0.0169, 0.0388, 0.0774, 0.0451 and 0.245 mg/L.

Analysis of the 72 hour test solutions which were not inoculated showed measured concentrations of 0.0493, 0.305, 1.36, 2.88 and 10.9 mg/L.

The results showed a decline in concentration over the 72-hour test period, it was therefore considered justifiable to base the results on geometric mean measured concentrations, which were calculated to be 0.0523, 0.151, 0.395, 0.509 and 1.93 mg/L.

During the definitive test some results were outside of the calibration range and samples were not re-diluted and analysed again in error. However, as there was no inhibition during the definitive test and the top concentration results were within the calibration range this was not considered to impact on study integrity.

The observed variation of concentrations at 72 hours between the inoculated and uninoculated vessels is considered to be due to the test substance adhering to the algae cells.

Alagal Growth Test

The 72-hour yield (EyCx), area under the growth curve (EbCx) and growth rate (ErCx) toxicity values, with corresponding no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) values, are presented in the table below.

Parameter		Toxicity Values (mg/L)		Statistical Test
		72-hours	Confidence limits (LCL-UCL)*
Yield	EyC10	>1.93 mg/L	NC	Linear Interpolation (ICPIN)
	EyC20	>1.93 mg/L	NC
	EyC50	>1.93 mg/L	NC
	NOEC	1.93 mg/L	NC	Bonferoni Adj t Test (Parametric-Multiple Comparison)
Biomass	EbC10	>1.93 mg/L	NC	Linear Interpolation (ICPIN)
	EbC20	>1.93 mg/L	NC
	EbC50	>1.93 mg/L	NC
	NOEC	1.93 mg/L	NC	Bonferoni Adj t Test (Parametric-Multiple Comparison)
Growth Rate	ErC10	>1.93 mg/L	NC	Linear Interpolation (ICPIN)
	ErC20	>1.93 mg/L	NC
	ErC50	>1.93 mg/L	NC
	NOEC	1.93 mg/L	NC	Bonferoni Adj t Test (Parametric-Multiple Comparison)
*	LCL = lower confidence limit, UCL = upper confidence limit
NC	Not calculated

Validity Criteria

All validity criteria were met therefore the test was considered valid.

Validity criteria fulfilled:

yes

Conclusions:

Based on nominal loading rate, the 72-hour EyC50, EbC50 and ErC50 values were calculated to be >100 mg/L. The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 100 mg/L based on the nominal loading rate.

Executive summary:

The acute toxicity of the test material to the freshwater alga was investigated in a study performed under GLP conditions in accordance to the standardised guideline OECD 201.

Based on the results of a range-finding limit test, the definitive test was conducted at nominal test concentrations of 1.0, 3.2, 10, 32 and 100 % saturated solutions (0.0523, 0.151, 0.395, 0.509 and 1.93 mg/L based on geometric mean measured concentrations). As evidence suggested the test substance may have been adhering to the algal cells it was considered justifiable to run additional un-inoculated vessels at each concentration as analytical vessels.

Test vessels (250 mL conical flasks) were prepared containing 100 mL of the appropriate test or control medium. Each test vessel was inoculated with 1 × 10^4 algae cells/mL and incubated at 21 - 24 °C (under 4440 - 8880 Lux light intensity) for 72 hours with cell counts at 24-hour intervals.

Analysis of the test media samples was conducted at 0 and 72 hours (both “with” and “without” algae samples were analysed at 72 hours).

Measured test concentrations were determined from samples of test solution from each treatment and control group at the beginning and end of the test. The results showed a decline in concentration over the 72-hour test period. Given this decline it was considered justifiable to base the results on geometric mean measured concentrations. These were calculated to be 0.0523, 0.151, 0.395, 0.509 and 1.93 mg/L.

Analysis of the test samples at 0 hours showed measured concentrations to range from 0.162 to 15.2 mg/L. Samples at 72 hours showed measured concentrations to range from 0.0169 to 0.245 mg/L. Analysis of the vessels which were not inoculated with algae at the start of the test (“without” algae samples) showed measured concentrations to range from 0.0493 to 10.9 mg/L. The observed variation of concentrations at 72 hours between the inoculated and un-inoculated vessels is considered to be due to the test substance adhering to the algae cells.

The variation of achieved concentrations during the range-finder and definitive phases of the study are thought to have been caused by several factors including, the insolubility of the compound, the UVCB (Substances of unknown or variable composition, complex reaction products or biological materials) nature of the compound, and the filtration of media due to potential of micro-globules, micelles passing through filters.

Based on nominal loading rate, the 72-hour EyC50, EbC50 and ErC50 values were calculated to be >100 mg/L. The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 100 mg/L based on the nominal loading rate.

Description of key information

Based on nominal loading rate, the 72-hour EyC50, EbC50 and ErC50 values were calculated to be >100 mg/L. The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 100 mg/L based on the nominal loading rate.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

Additional information

The acute toxicity of the test material to the freshwater alga was investigated in a study performed under GLP conditions in accordance to the standardised guideline OECD 201. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Based on the results of a range-finding limit test, the definitive test was conducted at nominal test concentrations of 1.0, 3.2, 10, 32 and 100 % saturated solutions (0.0523, 0.151, 0.395, 0.509 and 1.93 mg/L based on geometric mean measured concentrations). As evidence suggested the test substance may have been adhering to the algal cells it was considered justifiable to run additional un-inoculated vessels at each concentration as analytical vessels.

Test vessels (250 mL conical flasks) were prepared containing 100 mL of the appropriate test or control medium. Each test vessel was inoculated with 1 × 10^4 algae cells/mL and incubated at 21 - 24 °C (under 4440 - 8880 Lux light intensity) for 72 hours with cell counts at 24-hour intervals.

Analysis of the test media samples was conducted at 0 and 72 hours (both “with” and “without” algae samples were analysed at 72 hours).

Measured test concentrations were determined from samples of test solution from each treatment and control group at the beginning and end of the test. The results showed a decline in concentration over the 72-hour test period. Given this decline it was considered justifiable to base the results on geometric mean measured concentrations. These were calculated to be 0.0523, 0.151, 0.395, 0.509 and 1.93 mg/L.

Analysis of the test samples at 0 hours showed measured concentrations to range from 0.162 to 15.2 mg/L. Samples at 72 hours showed measured concentrations to range from 0.0169 to 0.245 mg/L. Analysis of the vessels which were not inoculated with algae at the start of the test (“without” algae samples) showed measured concentrations to range from 0.0493 to 10.9 mg/L. The observed variation of concentrations at 72 hours between the inoculated and un-inoculated vessels is considered to be due to the test substance adhering to the algae cells.

The variation of achieved concentrations during the range-finder and definitive phases of the study are thought to have been caused by several factors including, the insolubility of the compound, the UVCB (Substances of unknown or variable composition, complex reaction products or biological materials) nature of the compound, and the filtration of media due to potential of micro-globules, micelles passing through filters.

Based on nominal loading rate, the 72-hour EyC50, EbC50 and ErC50 values were calculated to be >100 mg/L. The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 100 mg/L based on the nominal loading rate.