Poly(oxy-1,2-ethanediyl), alpha-[2-[bis(2-aminoethyl)methylammonion)ethyl]-, omega-hydroxy, N,N'-di-Limnanthes alba seed oil acyl derivatives, methyl sulfates (salts)

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:

December 2016 to March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP certifiied OECD guideline study without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Remarks:

Test item information was provided after the study plan was agreed. Test item information was included in the report and was considered to have no impact on the outcome or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Poly(oxy-1,2-ethanediyl), alpha-[2-[bis(2-aminoethyl)methylammonion)ethyl]-, omega-hydroxy, N,N'-di-Limnanthes alba seed oil acyl derivatives, methyl sulfates (salts)

EC Number:

629-704-1

Cas Number:

226995-92-2

Molecular formula:

The substance is a UVCB - no specific molecular weight or formula is available.

IUPAC Name:

Poly(oxy-1,2-ethanediyl), alpha-[2-[bis(2-aminoethyl)methylammonion)ethyl]-, omega-hydroxy, N,N'-di-Limnanthes alba seed oil acyl derivatives, methyl sulfates (salts)

Test material form:

liquid

Specific details on test material used for the study:

Identification: RM1004942
Batch: RL149/17
Purity: 100% UVCB
Physical state/Appearance: amber colored paste
Expiry Date: 08 September 2018
Storage Conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Test concentrations were prepared as water accommodated fractions (WAF) due to the low solubility of the test substance.

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a haemocytometer and light microscope. The flasks were then plugged with polyurethane foam bungs and incubated for 72 hours with cell density determined using a haemocytometer and light microscope at the end of the test.

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:

RANGE FINDING TEST
The loading rates to be used in the definitive test were determined by preliminary range-finding tests. 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. The test was 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.

Nominal amounts of test item (20 and 200 mg) were each separately weighed onto a glass slide and suspended within 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. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtration. 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. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded). Filtration through a second glass wool plug and two sheets of filter paper was required to remove as much undissolved test item as possible. No micro-dispersions of test item were visible in the 10 mg/L loading rate WAF, however, visual inspection of the 100 mg/L loading rate WAF showed the preparation was a very hazy dispersion. It was considered that further filtration at this point would not have removed any more of the dispersed test item present.

An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (1.9 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. A sample of each test and control culture was removed and the cell density determined at the start and end of the test.

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, a single WAF of a nominal loading rate of 1.0 mg/L was prepared from which serial dilutions were made to give the remaining test concentrations. 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 established stirring and standing period the samples were filtered to give the 1.0 mg/L loading rate WAF. 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 (4.9 mL) to give the required test concentrations of 0.010, 0.10 and 1.0 mg/L loading rate WAF. A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours 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.

DEFINITIVE TEST
Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L.

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 to 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 0.32, 0.10, 0.032 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 (5.6 mL) to give the required test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 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:

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 kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ±1 °C laboratory.

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 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 104 to 105 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

No data

Test temperature:

24 ±1 °C

pH:

7.4 to 8.0 in the test concentrations and 7.8 to 8.0 in the control.

Dissolved oxygen:

No data

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

Modification of the standard method for the preparation of aqueous media was performed for this poorly water soluble substance. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a WAF of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

Test concentrations were measured, but reported as nominal loading rates (WAF).

Details on test conditions:

250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and 3 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 4.04 x 105 cells per ml. Inoculation of 450 ml of test medium with 5.6 ml of this algal suspension gave an initial nominal cell density of 5,000 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 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

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.:

0.065 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: loading rate (WAF)

Remarks:

0.054 to 0.078 mg/l CI

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.021 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: loading rate (WAF)

Remarks:

0.018 to 0.025 mg/l CI

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.01 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: loading rate (WAF)

Effect concentrations 4

Duration:

72

Dose descriptor:

NOELR

Effect conc.:

0.01 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: loading rate (WAF)

Details on results:

Inhibition of Growth Rate:
ErL10 (0 to 72 hour) : 0.019 mg/L loading rate WAF
ErL20 (0 to 72 hour) : 0.030 mg/L loading rate WAF
ErL50 (0 to 72 hour) : 0.065 mg/L loading rate WAF; 95% confidence limits 0.054 to 0.078 mg/L loading rate WAF

Statistical analysis of the growth rate data was carried out for the control, 0.010, 0.032 and 0.10 mg/L loading rate WAF test groups 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 and 0.010 mg/L loading rate WAF (P≥0.05), however all other loading rates were significantly different (P<0.05) and, therefore the NOEL based on growth rate was 0.010 mg/L loading rate WAF. Correspondingly the Lowest Observed Effect Loading Rate (LOEL) based on growth rate was 0.032 mg/L loading rate WAF.

Inhibition of Yield
EyL10 (0 to 72 hour) : 0.0095 mg/L loading rate WAF
EyL20 (0 to 72 hour) : 0.013 mg/L loading rate WAF
EyL50 (0 to 72 hour) : 0.021 mg/L loading rate WAF; 95% confidence limits 0.018 to 0.025 mg/L loading rate WAF

Statistical analysis of the yield data was carried out as for growth (above). There were no statistically significant differences between the control and 0.010 mg/L loading rate WAF (P≥0.05), however all other loading rates were significantly different (P<0.05) and, therefore the NOEL based on yield was 0.010 mg/L loading rate WAF. Correspondingly the LOEL based on yield was 0.032 mg/L loading rate WAF.

Validation criteria:
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 to 72 hour) was 3% 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):

A positive control (Envigo study number RD71YQ) 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 to 72 hour) : 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
- EyC50 (0 to 72 hour) : 0.77 mg/L; 95% confidence limits 0.68 to 0.87 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:

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, 0.010, 0.032 and 0.10 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 0.32 and 1.0 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

Response Variable	EL50 (mg/L Loading Rate WAF)	95% Confidence Limits (mg/L Loading Rate WAF)	No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate	0.065	0.054 - 0.078	0.010	0.032
Yield	0.021	0.018 - 0.025	0.010	0.032

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ), determined to be 0.082 mg/l, to 0.80 mg/. A decline in measured test concentrations was observed at 72 hours in the range of less than the LOQ to 0.097 mg/l. 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.

Inhibition of growth rate ErL50 (0 to 72 hour) was 0.065 mg/L loading rate WAF; 95% confidence limits 0.054 to 0.078 mg/L loading rate WAF. The NOEL based on growth rate was 0.010 mg/L loading rate WAF.

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata by following 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.

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF). Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ), determined to be 0.082 mg/l, to 0.80 mg/. A decline in measured test concentrations was observed at 72 hours in the range of less than the LOQ to 0.097 mg/l. 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.

Inhibition of growth rate ErL50 (0 to 72 hour) was 0.065 mg/L loading rate WAF; 95% confidence limits 0.054 to 0.078 mg/L loading rate WAF. The NOEL based on growth rate was 0.010 mg/L loading rate WAF.