Isooctadecanoic acid, reaction products with tetraethylenepentamine

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:

supporting study

Study period:

May 1, 1997 - October 17, 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with agreed test protocols following OECD Method 201 (1984). Analytical measurements on the test material were not conducted.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The WAFs were formulated by combining the test substance and dilution water in glass mixing vessels equipped with a magnetic stirrer, mixing the solution for approximately 24 hours, settling the solution for approximately four hours, siphoning the water phase containing the water accommodated fraction (WAF), and adding the appropriate amount of micrometals.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: freshwater alga
- Strain: Selenastrum capricornutum
- Source (laboratory, culture collection):Algae used for the test was from a culture originally procured from the Culture Collection of Algae at the University of Texas at Austin.
The culture was transferred to sterile enriched media idential to media used for this test and maintained at test conditions for at least 14 days before the definitive test. The incolum used to initiate the test was 7 days old.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

The range of incubator temperature was 23.2 to 23.5 degrees Celsius

pH:

pH ranged from 7.4 for initial pH to 10.5 for final pH

Nominal and measured concentrations:

The definitive test used the WAF of five concentrations: 13, 23, 36, 60 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass Erlenmeyer flasks
- Initial cells density:10,000 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water was sterile enriched medium
- Total organic carbon: The media had 1.3 to 1.5 mg/L total organ carbon at the beginning of the test and 1.9 to 2.2 mg/L TOC at the end of the test
- Metals: not detected at or above the limit of quantitation
- Pesticides: not detected at or above the limit of quantitation
- Chloride: not detected at or above the limit of quantitation
- Intervals of water quality measurement: The measured concentrations of total organic carbon were determined in samples of test media collected at the beginning and the end of the toxicity test. Temperature of the incubator was measured and recorded daily and pH was determined in each test vessel at the beginning and end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: pH was adjusted to 7.5
- Photoperiod: 24 hour light and 9 hour dark period
- Light intensity and quality: 400 to 410 footcandles


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Determination of cell concentrations: The number of algal cells/ml in each test vessel and the occurrence of relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a haemocytometer. Cell counts were made and recorded daily during the 96 hour test.


TEST CONCENTRATIONS

- Range finding study
- Test concentrations:
1st range finding study:0.5, 1, 10 and 100 mg/L (WAF's)
2nd range finding study: 1, 10, 100 and 1000 mg/L (WAF's)

- Results used to determine the conditions for the definitive study:

At the conclusion of the 96 hour test for the second range finding test, the number of cells/ml in the test vessels equaled the following percents of the number of cells/ml in the alternate control vessels: 1.0 mg/L = 98%, 10 mg/L = 105%, 100 mg/L = 4% and 1000 mg/L =<1%

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

44 mg/L

Nominal / measured:

nominal

Basis for effect:

cell number

Remarks on result:

other: 95% confidence interval <13 to >100 mg/L

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

94 mg/L

Nominal / measured:

nominal

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence ( 61 to >100 mg/L)

Details on results:

The algal population grew well during the test, resulting in an average of 1,747,000 cells/ml in the control and 1,653,000 cells/ml in the alternate control after 96 hours. Insoluble test material was not observed in any vessels during the test.

No effects (size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells) were noted during the test.

A determination of whether toxic effects were algistatic or algicidal was performed at the conclusion of the toxicity test. After 96 hours of testing, 0.5 ml of solution from each 100 mg/L test vessel were combined into one vessel containing 100 ml of fresh media. The resulting solution was incubated under test conditions for 72 hours. During this time, algae increased from a calculated concentration of 2,200 cells/ml to 304,000 cells/ml, indicating that the effect of the test material at the highest tested concentration was algistatic rather than algicidal.

Reported statistics and error estimates:

The probit method was used for calculating the 24, 48, 72 and 96 hour effective concentrations. The 96 hour NOEC was determined using TOXSTAT 3.3 which calculated a one way analysis of variance of the number of cells/ml and of the average specific growth rate in each test vessel at the end of the test. The data was tested for normal distribution using a Shapiro Wilks test and for homogeneity of variance using a Bartletts test.

Validity criteria fulfilled:

yes

Conclusions:

The 96 hour EC50 values are 44 mg/L when calculcated using the number of cells per ml and 94 mg/l when calculated using the average specific growth rate. No effects were noted during the test. The 96 hour NOEC is 23 mg/L test material when calculated using then number cells/ml or the average specific growth rate.

Executive summary:

The acute toxicity of the water accomodated fraction (WAF) of five mixtures of the test material and water to the freshwater alga, Selenastrum capricornutum, was investigated during a study conducted at T.R. Wilbury Laboratories, Inc. The test, which was designed to determine the toxicity of the WAF of the test substance , was performed from August 11 to 15, 1997.

The test was performed at 24 +/- 1 degree Celsius under static conditions with a control and five nominal concentrations of test substance (13, 23, 36, 60 and 100 mg/L). In the definitive toxicity test the full compliment of micrometals was added to the WAFs after they were prepared, and before they were used in the test. This was done based on observed differences in algal growth rates in preliminary tests where micrometals were added either before WAF preperation or afterwards. It was theorized that undissolved test substance may sequester some, or all, of the micrometals from algal media during WAF preperation; i.ee, the observed algal trowth rate effects were due more to chelation of micrometals by undissolved test substance in contact with test metals than due to dissolved chemical toxicity. The WAFs were formulated by combining the test substance and dilution water in glass mixing vessels equipped with a magnetic stirrer, mixing the solution for approximately 24 hours, settling the solution for approximately four hours, siphoning the water phase containing the water accommodated fraction (WAF), and adding the appropriate amount of micrometals. The final dilution water was sterile synthetic media adjusted to a pH of 7.5. Nominal concentrations of WAF were used for all calculations.

Algae used for the test was from a culture originally procured from the Culture Collection of Algae at the University of Texas at Austin on January 30, 1997. The culture was transferred to sterile enriched media identical to media used for this test and maintained at test conditions for at least 14 days before the definitive test. Algae was incubated under continous light on a rotary shaker adjusted to 100 rpm. Cell counts were made daily with a haemocytometer. Results of the test with the WAF of test material are presented below.

Test results:

	96 hour (mg/L)
Based on Growth Rate: EC50	94
Based on Growth Rate: NOEC	23

	96 hour (mg/L)
Based on Cell Density:
EC50	44
NOEC	23

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-2022

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)

Specific details on test material used for the study:

Batch: 1000352370

Details on test solutions:

OBSERVATIONS ON TEST ITEM SOLUBILITY
At the start of the test, all control, solvent control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, solvent control and test cultures were observed to be green dispersions.

Test organisms (species):

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

Details on test organisms:

The test was carried out using Raphidocelis subcapitata strain CCAP 278/4. Liquid cultures of Raphidocelis subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
Approximately 3 to 4 days before the start of the test, inoculum cultures of algae was set up at an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 105 to 106 cells/mL.

CULTURE MEDIUM
The culture medium used for the preliminary media preparation trials, range-finding and definitive tests was the same as that used to maintain the stock culture.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 d

Test temperature:

Temperature was maintained at 24 ±1ºC throughout the test.

pH:

The pH value of the control cultures was observed to increase from pH 7.5 at 0 hours to pH 8.6 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Nominal and measured concentrations:

VERIFICATION OF TEST CONCENTRATIONS
Samples were taken from the control, solvent control and 0.0050 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Samples were also taken from the test group from additional samples ran alongside the test at 24 and 48 hours. An additional sample was taken of uninoculated media at 0 hours and stored alongside the test for analysis at 72 hours to determine if any decline observed in the inoculated samples were due to absorption to the algal cells. All samples were analyzed on the day of sampling. A set of duplicate samples were taken on each occasion and stored frozen for further analysis if necessary. (See Result section for analysis results)

Details on test conditions:

DEFINITIVE TEST
Based on the result of the range-finding tests a "limit test" was conducted at a concentration of 0.0050 mg/L to confirm that at the limit of solubility of 0.0050 mg/L, no effect on algal growth was observed. An initial definitive test was conducted however, a measured concentration of less than the LOQ was obtained at 0 hours therefore it could not be determined if the lack of toxicity seen was as a consequence of the lack of test item in the system, therefore the test was repeated. The initial results were invalid due to a calibration failure during the analysis, results from the duplicate samples were used for evaluation purposes.

EXPERIMENTAL PREPARATION
A nominal amount of test item (25 mg) was dissolved in DMF and the volume adjusted to 50 mL to give a 0.5 mg/mL stock solution. An aliquot (10 μL) of the solvent stock solution was dispersed in 1 L of culture medium with the aid of magnetic stirring for 30 minutes. After preparation the aqueous solution was centrifuged at 10000 g for 30 minutes as a precaution to ensure any undissolved test item was removed. An aliquot (800 mL) of the stock solution was inoculated with algal suspension (3.8 mL) to give an initial nominal cell density of 5.00 x 10^3 cells/mL.
The stock solutions and each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours.

EXPOSURE CONDITIONS
As in the range-finding tests 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control, solvent control and 0.0050 mg/L treatment groups. The control and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 10 μL/L of DMF. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.06 x 10^6 cells per mL. Inoculation of 800 mL of test medium with 3.8 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

TEST ORGANISM OBSERVATIONS
Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominal inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

WATER QUALITY CRITERIA
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.
The appearance of the test media was recorded daily.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

VERIFICATION OF TEST CONCENTRATIONS
Analysis of the test preparation at 0 hours (see Annex 5) showed that a measured concentration of 0.0047 mg/L was obtained and at 24, 48 and 72 hours showed measured test concentrations to be less than the Limit of Quantification (LOQ)/Limit of Detection (LOD). Analysis of uninoculated media at 72 hours showed that a measured concentration of less than the LOD was obtained suggesting that the decline was not due to absorption to algal cells.
Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.0018 mg/L) or LOD was used to enable calculation of the geometric mean measured concentration. The geometric mean measured test concentration was determined to be 0.00075 mg/L.

GROWTH DATA
The growth rate (r) and yield (y) of Raphidocelis subcapitata (CCAP 278/4) were not affected by the presence of the test item at a geometric mean test concentration of 0.00075 mg/L over the 72-Hour exposure period.
It was considered unnecessary and unrealistic to test at concentrations in excess of the limit of solubility of 0.0050 mg/L.

INHIBITION OF GROWTH RATE
Visual observation was conducted on the growth rate data at 72 hours. There was no increase in inhibition in the 0.00075 mg/L test group when compared to the solvent control group and therefore the No Observed Effect Concentration (NOEC) based on growth rate was 0.00075 mg/L. The LOEC could not be determined.

INHIBITION OF YIELD
Visual observation was conducted on the yield data at 72 hours. There was no increase in inhibition in the 0.00075 mg/L test group when compared to the solvent control group and therefore the No Observed Effect Concentration (NOEC) based on yield was 0.00075 mg/L. The LOEC could not be determined.

OBSERVATIONS ON CULTURES
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control, solvent control or test cultures at 72 hours.

Results with reference substance (positive control):

A positive control (Labcorp study number 8481244) used potassium dichromate as the reference item at concentrations of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L.
The positive control was conducted between 06 September 2021 and 09 September 2021.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Raphidocelis subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.4 mg/L; 95% confidence limits 1.3 to 1.5 mg/L
EyC50 (0 to 72 hour) : 0.53 mg/L; 95% confidence limits 0.21 to 1.4 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

VALIDATION CRITERIA

The following data show that the cell concentration of the control cultures increased by a factor of 123 after 72 hours and the cell concentration of the solvent control cultures increased by a factor of 100 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours : 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 6.13 x 10^5 cells per mL
Nominal cell density of solvent control at 0 hours : 5.00 x 10^3 cells per mL
Mean cell density of solvent control at 72 hours : 5.01 x 10^5 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control and solvent control cultures was 7% and 8% respectively and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control and solvent control cultures over the test period (0 to 72 hour) was 3% and 5% respectively and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

 

 

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Raphidocelis subcapitata has been investigated and based on the geometric mean measured test concentration gave EC50 values of greater than 0.00075 mg/L. The No Observed Effect Concentration (NOEC) was 0.00075 mg/L.

Executive summary:

INTRODUCTION
A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation (EC) No 761/2009.

METHODS
Following preliminary range-finding tests, Raphidocelis subcapitata was exposed to an aqueous solution of the test item at a nominal concentration of 0.0050 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solution was prepared by dissolving a nominal amount (25 mg) in Dimethylformamide (DMF) and the volume adjusted to 50 mL to give a 0.50 mg/mL stock solution. An aliquot (10 μL) of the solvent stock solution was dispersed in 1 L of culture medium with the aid of magnetic stirring for 30 minutes to ensure adequate mixing and homogeneity and centrifuged at 10000 g for 30 minutes as a precaution to ensure any undissolved test item was removed.
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

RESULTS
Analysis of the test preparation at 0 hours showed that a measured concentration of 0.0047 mg/L was obtained and at 24, 48 and 72 hours showed measured test concentrations to be less than the Limit of Detection (LOD). Analysis of a sample of uninoculated media at 72 hours showed that a measured concentration of less than the LOD was obtained suggesting that the decline observed was not due to absorption to algal cells but to the instability of the test item.
Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentration in order to give a "worst case" analysis of the data. The geometric mean measured concentration was calculated to be 0.00075 mg/L.
Exposure of Raphidocelis subcapitata to the test item gave EC50 values based on the geometric mean measured test concentrations of greater than 0.00075 mg/L. The No Observed Effect Concentration (NOEC) was 0.00075 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of the limit of solubility of 0.0050 mg/L.

Description of key information

Two valid studies have been conducted in algae and according to OECD 201 test method. The study by Ward et al, 1994 was performed on a water accomadated fraction (WAF) of the test substance with a control and five nominal concentrations of 13, 23, 36, 60 and 100 mg/L. WAF is considered a preferred method for preparation of difficult to test substances like EC 701-204-9 that is a UVCB and highly insoluble in water. Algistatic effects were observed at the highest concentration and the 96 hour EC50 values were determined to be 44 mg/L when calculated using the number of cells per mL and 94 mg/L when calculated using the average specific growth rate.  The 96 hour NOEC is 23 mg/L when calculated using the number cells/mL or the average specific growth rate.

The second study used test material prepared by solvent extraction in an attempt to analyze for the test material throughout the study. No adverse effects were observed and based on the geometric mean measured test concentration gave EC50 values of greater than 0.00075 mg/L. The No Observed Effect Concentration (NOEC) was considered 0.00075 mg/L. 

Key value for chemical safety assessment

EC50 for freshwater algae:

44 mg/L

EC10 or NOEC for freshwater algae:

23 mg/L