Amines, C12-14-alkyl, isooctyl phosphates

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 20 November 2014 and 17 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Amines, C12-14-tert-alkyl, isooctyl mono phosphates

IUPAC Name:

Amines, C12-14-tert-alkyl, isooctyl mono phosphates

Constituent 2

Reference substance name:

Unknown, including minor amounts of diphosphate species, water. Possibly residual unreacted starting material and unknown byproducts.

IUPAC Name:

Unknown, including minor amounts of diphosphate species, water. Possibly residual unreacted starting material and unknown byproducts.

Test material form:

liquid: viscous

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Experimental Preparation
Nominal amounts of test item (20, 32 and 20 mg) were each separately added to the surface of 20, 10 and 2.0 liters of culture medium to give the 1.0, 3.2 and 10 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0, 3.2 and 10 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. A series of dilutions was made from the 1.0 mg/L loading rate WAF to give further stock solutions of 0.32 and 0.10 mg/L loading rate WAF.

An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (2.4 mL) to give the required test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L loading rate WAF.

Test organisms

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1 °C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 1E+03 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 1E+04 – 1E+05 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

The temperature within the incubator was recorded daily. Temperature was maintained at 24 ± 1 ºC throughout the test.

pH:

The pH of the control and each test concentration was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The pH value of the control cultures was observed to increase from pH 7.9 at 0 hours to pH 8.2 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:

Range-finding test: Nominal test concentrations of 10 and 100 mg/L loading rate WAF.
Definitive test: Nominal test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L loading rate WAF.

Details on test conditions:

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Procedure
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.


Validation of Mixing Period
Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.


Range-Finding Tests
The loading rates to be used in the definitive test were determined by preliminary range-finding tests.

The tests were conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.

The initial range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 10 and 100 mg/L for a period of 72 hours.

Nominal amounts of test item (20 and 200 mg) were each separately added to the surface of 2 liters of culture medium to give the 10 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (6.1 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF.

The control group was maintained under identical conditions but not exposed to the test item.

At the start of the range-finding tests a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

The results of the initial range-finding test showed significant inhibition of growth occurred at both 10 and 100 mg/L loading rate WAF and so a second range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 0.010, 0.10 and 1.0 mg/L for a period of 72 hours. Due to the need to test at relatively low test concentrations, the 0.010 and 0.10 mg/L loading rate WAFs were prepared as serial dilutions of the 1.0 mg/L loading rate WAF.

A nominal amount of test item (20 mg) was added to the surface of 20 liters of culture medium to give the 1.0 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. A series of dilutions was made from the 1.0 mg/L loading rate WAF to give further stock solutions of 0.10 and 0.010 mg/L loading rate WAF.

An aliquot (450 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (9.2 mL) to give the required test concentrations of 0.010, 0.10 and 1.0 mg/L loading rate WAF.

The control group was maintained under identical conditions but not exposed to the test item.

Exposure conditions and cell density determinations at 0 and 72 hours in the second range-finding test were the same as those in the initial test.


A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours from both the initial and second range-finding tests in order to determine the stability of the test item over the test duration. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

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 and three flasks each containing 100 mL were used for each treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1060000 cells per mL. Inoculation of 500 mL of test medium with 2.4 mL of this algal suspension gave an initial nominal cell density of 5000 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® Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Evaluations
Test Organism Observations
Samples were taken at 0, 24, 47 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

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.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period.

Data Analysis
Comparison of Growth Rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:

µ = (1n Nn – 1n N1) / (tn – t1)

Where:

µ = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement

The average specific growth rate over the test duration was calculated for each replicate control and test item vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.

In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.

Percentage inhibition of growth rate for each replicate test item vessel was calculated using the following equation:

Ir = ((µc - µt) / µc) x 100

Where:

Ir = percentage inhibition of average specific growth rate
µc = mean average specific growth rate for the control cultures
µt = average specific growth rate for the test culture

Comparison of Yield
Yield is calculated as the increase in biomass over the exposure period using the following equation:

Y = Nn – N0

Where:

Y = yield
N0 = cell concentration at the start of the test
Nn = cell concentration at the end of the test

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:

Iy = ((Yc – Yt) / Yc) x 100

Where:

Iy = percentage inhibition of yield
Yc = mean value for yield in the control group
Yt = mean value for yield for the treatment group

Determination of ELx Values
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ELx values were then determined from the equation for the fitted line.

Where appropriate 95% confidence limits for the EL50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence limits of 0.72 - 0.89 mg/L loading rate WAF

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range-finding Tests
The results showed no effect on growth at 0.010 and 0.10 mg/L loading rate WAF. However, growth was observed to be reduced at 1.0, 10 and 100 mg/L loading rate WAF.

Based on this information loading rates of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were selected for the definitive test.

Chemical analysis of the 0.10, 1.0 and 10 mg/L loading rate WAF test preparations at 0 hours showed measured test concentrations of 0.074, 0.81 and 7.8 mg/L respectively were obtained. There was no significant change in the measured test concentrations after 72 hours indicating that the test item was stable over the test duration.


Definitive Test
Chemical Analysis of Test Loading Rates
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.12 to 12 mg/L whilst concentrations in the range of 0.097 to 13 mg/L were observed at 72 hours.

The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Growth Data
From the data given, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

Accordingly the following results were determined from the data:

Inhibition of growth rate
ErL10 (0 - 72 h): 0.48 mg/L loading rate WAF
ErL20 (0 - 72 h): 0.57 mg/L loading rate WAF
ErL50 (0 - 72 h): 0.80 mg/L loading rate WAF ; 95% confidence limits 0.72 – 0.89 mg/L loading rate WAF

Where ErLx is the loading rate that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control, 0.10, 0.32 and 1.0 mg/L loading rate WAF test group using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control, 0.10 and 0.32 mg/L loading rates WAF (P≥0.05), however the 1.0 mg/L loading rate was significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 0.32 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 1.0 mg/L loading rate WAF.

Inhibition of Yield
EyL10 (0 - 72 h): 0.84 mg/L loading rate WAF
EyL20 (0 - 72 h): 0.85 mg/L loading rate WAF
EyL50 (0 - 72 h): 0.88 mg/L loading rate WAF*

Where EyLx is the loading rate that reduced yield by x%.


Statistical analysis of the yield data was carried out. There were no statistically significant differences between the control, 0.10 and 0.32 mg/L loading rate WAFs (P≥0.05), however the 1.0 mg/L loading rate was significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 0.32 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 1.0 mg/L loading rate WAF.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Results with reference substance (positive control):

Positive Control
A positive control (Harlan Study Number 41403074) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h): 1.2 mg/L; 95% confidence limits 1.1 – 1.4 mg/L
EyC50 (0 – 72 h): 0.63 mg/L; 95% confidence limits 0.57 – 0.70 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration (LOEC) 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.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and the 0.10, 0.32 and 1.0 mg/L loading rates to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001). The 3.2 and 10 mg/L loading rates were not included in the analysis as visual inspection of the data showed a significant effect on growth.

Any other information on results incl. tables

Validation of Mixing Period

Preliminary investigational work indicated that there was no significant increase in the amount of dissolved test item present when the preparation period was extended for longer than 24 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 89 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.

 

Mean cell density of control at 0 hours: 5.46 x 10³cells per mL

Mean cell density of control at 72 hours: 4.86 x 10⁵cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 25% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

Observations on Cultures

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.10 mg/L loading rate WAF. At 0.32 mg/L loading rate WAF broken cells were observed, at 1.0 mg/L loading rate WAF misshapen and broken cells were observed, at 3.2 mg/L loading rate WAF enlarged and turgid cells were observed and at 10 mg/L loading rate WAF broken and enlarged/turgid cells were observed.

Water Quality Criteria

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

The pH value of the control cultures was observed to increase from pH 7.9 at 0 hours to pH 8.2 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.

Vortex Depth Measurements

The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

Observations on Test Item Solubility

Observations on the test media were carried out during the mixing and testing of the WAFs.

 

At both the start and end of the mixing period, and following a 1-Hour standing period, all loading rate WAFs were observed to have formed clear colorless media columns with oily globules of test item floating at the media surface. Microscopic examination of the WAFs showed there to be no micro-dispersions or globules of test item present.

 

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.10 and 0.32 mg/L loading rate WAF test cultures were observed to be bright green dispersions whilst the 1.0, 3.2 and 10 mg/L loading rate WAFs were observed to be clear colorless solutions.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results:
The growth rate EL50 was 0.80 mg/L (loading rate WAF) with 95 % confidence limits of 0.72 - 0.89 mg/L. The NOEL was 0.32 mg/L and the LOEL was 1.0 mg/L.
The yield EL50 was 0.88 mg/L (loading rate WAF). It was not possible to calculate 95% confidence limits for the EyL50 value as the data generated did not fit the models available for the calculation of confidence limits. The NOEL was 0.32 mg/L and the LOEL was 1.0 mg/L.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga 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" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

 

Methods

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

 

Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.10, 0.32, 1.0, 3.2 and 10 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. Due to the need to test at relatively low test concentrations, the 0.10 and 0.32 mg/L loading rate WAFs were prepared as serial dilutions of the 1.0 mg/L loading rate WAF.

 

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 preparations at 0 hours showed measured test concentrations to range from 0.12 to 12 mg/L whilst concentrations in the range of 0.097 to 13 mg/L were observed at 72 hours.

 

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

 

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Response Variable	EL50 (mg/L Loading Rate WAF)	95% Confidence Liomits (mg/L Loading Rate WAF)	No Observed Effect LoadingRate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate	0.80	0.72 - 0.89	0.32	1.0
Yield	0.88	*	0.32	1.0

*It was not possible to calculate 95% confidence limits for the E_yL₅₀value as the data generated did not fit the models available for the calculation of confidence limits.