Reaction mass of 2-hexadecyl-N-(2-hydroxyethyl)-3-oxoicosanamide, 2-hexadecyl-N-(2-hydroxyethyl)-3-oxo-, N-(2-hydroxyethyl)-3-oxo-2-tetradecylicosanamide and N-(2-hydroxyethyl)-3-oxo-2-tetradecyloctadecanamide.

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 1 September 2011 and 11 October 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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:

Reaction mass of 2-hexadecyl-N-(2-hydroxyethyl)-3-oxoicosanamide, 2-hexadecyl-N-(2-hydroxyethyl)-3-oxo-, N-(2-hydroxyethyl)-3-oxo-2-tetradecylicosanamide and N-(2-hydroxyethyl)-3-oxo-2-tetradecyloctadecanamide.

EC Number:

938-695-6

Molecular formula:

C34H67NO3/C36H71NO3/C38H75NO3

IUPAC Name:

Reaction mass of 2-hexadecyl-N-(2-hydroxyethyl)-3-oxoicosanamide, 2-hexadecyl-N-(2-hydroxyethyl)-3-oxo-, N-(2-hydroxyethyl)-3-oxo-2-tetradecylicosanamide and N-(2-hydroxyethyl)-3-oxo-2-tetradecyloctadecanamide.

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Sponsor's identification: PC-9S
Description: off white powder
Purity: not applicable - complex mixture
Batch number: PS101103
Date received: 11 July 2011
Expiry date: 18 November 2012
Storage conditions room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Sample Preparation
Test samples were centrifuged at 4500 rpm for 10 minutes in glass tubes. A volume (10 ml) of test sample was diluted with a volume (10 ml) of tetrahydrofu ran and shaken to mix.

Standards
Standard solutions of test item were prepared initially in tetrahydrofuran then further diluted in tetrahydrofuran/test medium (50/50 v/v) to nominal concentrations of 0.002, 0.005, 0.0075, 0.01, 0.05 and 0.1 mg/I.

Samples from the range-finding test were also submitted for chemical analysis at 0 and 72 hours.

Samples were taken from the control and the 100 mg/l loading rate WAF test group (replicates R1 - Rs pooled) at 0 and 72 hours for quantitative analysis. All 0-Hour samples were stored at approximately -20°C prior to analysis. Duplicate samples were taken at each occasion and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

An amount of test item (200 mg) was added to the surface of 2 litres of culture medium to give the 100 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 middepth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

An aliquot (1 litre) of the 100 mg/l loading rate WAF was separately inoculated with algal suspension (6.0 ml) to give the required test concentration of 100 mg/l loading rate WAF. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation):
- Method of cultivation: not stated

ACCLIMATION
Not stated in report

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

Not stated

Test temperature:

Temperature was maintained at 24 ± 1°C throughout the test.

pH:

The pH of the control and 100 mg/l loading rate WAF test concentration was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.

The pH value of the control cultures was observed to increase from pH 7.5 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.

Dissolved oxygen:

Not stated

Salinity:

Not applicable as freshwater study was used

Nominal and measured concentrations:

Nominal - 100 mg/L

Details on test conditions:

TEST SYSTEM
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 5x10E3 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 10E4 - 10E5 cells/ml.

As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and 100 mg/l loading rate WAF treatment group.

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

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.

Samples were taken at 0, 24, 49 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

GROWTH MEDIUM
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
NaN03 - 25.5 mg/l
MgCI2.6H20 - 12.164 mg/l
CaCI2.2H20 - 4.41 mg/l
MgS04.7H20 - 14.7 mg/l
K2HP04 - 1.044 mg/l
NaHC03 - 15.0 mg/l
H3B03 - 0.1855 mg/l
MnCI2.4H20 - 0.415 mg/l
ZnCI2 - 0.00327 mg/l
FeCI3.6H20 - 0.159 mg/l
CoCI2.6H20 - 0.00143 mg/l
Na2Mo04.2H20 - 0.00726 mg/l
CuCI2.2H20 - 0.000012 mg/l
Na2EDTA.2H20 - 0.30 mg/l
Na2Se03.5H20 - 0.000010 mg/l

The above culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI.

OTHER TEST CONDITIONS
continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
- Salinity (for marine algae): freshwater study so not applicable

TEST CONCENTRATIONS
- definitive test: 100 mg/L
- Range finding study:
The loading rate to be used in the definitive test was determined by a preliminary rangefinding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal loading rates of 10 and 100 mg/l for a period of 72 hours. Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range finding test:
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1, which can be found in the attached background information section.

The results showed no effect on growth at 10 and 100 mg/l loading rate WAF.

Based on this information a single loading rate of six replicates of 100 mg/I, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.

Chemical analysis of the 100 mg/I loading rate WAF test preparation showed a measured test concentration of 0.023 mg/I was obtained at 0 hours whilst a decline in measured test concentration to 0.010mg/l was observed at 72 hours. This decline was considered to be due to either the unstable nature of the test item in the culture medium and/or adsorption of the test item to the algal cells present.

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd Project No: 41100094) 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 inthe definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC 50 (0 - 72 h) - 1.2 mg/l, 95% confidence limits 0.98 - 1.4 mg/l
EyC 50 (0 - 72 h) - 0.48 mg/l, 95% confidence limits 0.43 - 0.54 mg/l

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

A Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/l loading rate 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).

Any other information on results incl. tables

Validation criteria:

The following data show that the cell concentration of the control cultures increased by a factor of 174 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 6.04 x 10E3 cells per ml

Mean cell density of control at 72 hours 1.05 x 10E6 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 8% 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 cultures over the test period (0 - 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L. Accordingly the following results were determined from the data:

Inhibition of growth rate: EL50 = > 100 mg/L

Inhibition of yield: EL50 = > 100 mg/L

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

Chemical analysis of test loading rates:

Analysis of the 100 mg/L loading rate WAF test preparation at 0 hours showed a measured test concentration of 0.045 mg/L was obtained. A decline in measured test concentration was observed at 72 hours to 0.0047 mg/L (10% of the 0-Hour measured concentration). This decline was considered to be due to instability of the test item in the culture medium and/or adsorption of the test item to the algal cells present.

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.

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 and gave EL*so values of greater than 100 mg/l loading rate WAF. The No Observed Effect Loading Rate was 100 mg/iloading rate WAF.

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) 440/2008.

Methods. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. 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. Exposure of Pseudokirchneriella subcapitata to the test item gave EL*50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was determined to be 100 mg/l loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Analysis of the 100 mg/L loading rate WAF test preparation at 0 hours showed a measured test concentration of 0.045 mg/L was obtained. A decline in measured test concentration was observed at 72 hours to 0.0047 mg/L (10% of the 0-Hour measured concentration). This decline was considered to be due to instability of the test item in the culture medium and/or adsorption of the test item to the algal cells present.

However, 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.