Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine

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:

6th February 2014 - 6th March 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Test item supplied in solvent to keep fluidity based on reccomendations for ecotoxicological testing and is not thought to impact the results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

Solvent control ran alongside test and oversight meaningn solvent control was prepared at 20mg/L, see principles of methods other than guideline for details.

Principles of method if other than guideline:

Solvent control (propylene glycol) was to be run alongside both the range-finding and definitive tests containing an equal amount of propylene glycol as the highest test concentration to be employed (20 mg propylene glycol/100 mg test item). Due to a technical oversight in the second range-finding test and definitive test, the solvent control was prepared at a concentration of 20 mg propylene glycol/liter, irrespective of the maximum test item concentration prepared. This oversight was considered to have had no adverse effect on the outcome of the test as the presence of the solvent had no significant effect on the growth of the algae when compared to the control cultures.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine

EC Number:

939-981-3

IUPAC Name:

Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine

Constituent 2

Reference substance name:

Propane-1,2-diol

EC Number:

200-338-0

EC Name:

Propane-1,2-diol

Cas Number:

57-55-6

IUPAC Name:

propylene glycol

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: Not supplied
- Date Prepared: 14 May 2013

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under storage conditions: Stable
- Stability under test conditions: Stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prepared as Water Accomodated Fraction

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.10, 0.32, 1.0, 3/2 and 10 mg/L
- Sampling method: Samples were taken at 0, 20, 48 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter
- Sample storage conditions before analysis: Frozen. The 0-Hour test samples were thawed with the aid of sonication, whilst the 72-Hour test samples
were analyzed on the day of receipt

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: WAF
- Controls:
- solvent control: prepared at a concentration of 20 mg propylene glycol/liter
-A positive control (Harlan Study Number 41303826) 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.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Microscopic 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 W AFs by filtering through a glass wool plug (2-4 cm in length).

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Ohan, Argyll, Scotland.

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 103 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 104 - 105 cells/mL.

Culture Medium: The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 with O.lN NaOH or HCl. See composition below.
NaN03 25.5 mg/L
MgCh.6H20 12.16 mg/L
CaCh.2H20 4.41 mg/L
MgS04.7H20 14.6 mg/L
K1HP04 1.044 mg/L
NaHC03 15.0 mg/L
H3B03 0.186 mg/L
MnCh.4H20 0.415 mg/L
Zn Ch 0.00327 mg/L
FeCh.6H20 0.160 mg/L
C0Ch.6H20 0.00143 mg/L
Na2Mo04.2H20 0.00726 mg/L
CuCh.2H20 0.000012 mg/L
Na2EDTA.2H20 0.30 mg/L

Study design

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 1 °C

pH:

7.4-7.8

Nominal and measured concentrations:

The preliminary range finding tests had nominal loading rates of 10 and 100mg/L
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.
Given that toxicity cannot be attributed to a single component or mixture of components, but to the test item as a whole, and that the analytical method employed was specific to only the 108 m/z species present in the test item and not the test item as a whole, the results were based on nominal loading rates alone.

Details on test conditions:

TEST SYSTEM
- Aeration: Master cultures had constant aeration.
- Initial cells density:
Control - 4.59E+03 cells per mL
Solvent Control - 4.69E+03 cells per mL
0.1mg/L - 5.18E+03 cells per mL
0.32mg/L - 4.54E+03 cells per mL
1.0mg/L - 4.41E+03 cells per mL
3.2mg/L - 4.58E+03 cells per mL
10mg/L - 3.32E+03 cells per mL

- Control end cells density: 5.00E+05 cells per mL (Solvent: 5.34E+05 cells per mL)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per solvent control (replicates): 6

GROWTH MEDIUM
- Composition:
NaN03 25.5 mg/L
MgCh.6H20 12.16 mg/L
CaCh.2H20 4.41 mg/L
MgS04.7H20 14.6 mg/L
K1HP04 1.044 mg/L
NaHC03 15.0 mg/L
H3B03 0.186 mg/L
MnCh.4H20 0.415 mg/L
Zn Ch 0.00327 mg/L
FeCh.6H20 0.160 mg/L
C0Ch.6H20 0.00143 mg/L
Na2Mo04.2H20 0.00726 mg/L
CuCh.2H20 0.000012 mg/L
Na2EDTA.2H20 0.30 mg/L

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH:
- Photoperiod:
- Light intensity and quality:
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

3.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

5.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

cell number

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

1.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

cell number

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

cell number

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

cell number

Effect concentrations 9

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 10

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

cell number

Details on results:

- Observation of abnormalities (for algal test): All test, control and solvent control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control, solvent control or test cultures at
0.10, 0.32 and 1.0 mg/L loading rate W AF. Swollen cells were observed to be present in the 3.2 mg/L loading rate WAF test cultures whilst no intact cells were observed to be present in the test cultures at 10 mg/L loading rate WAF.
- Any stimulation of growth found in any treatment: No

Results with reference substance (positive control):

A positive control 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:
EC50 (0 - 72 h) 1.1 mg/L; 95% confidence limits 0.91 - 1.2 mg/L
EC50 (0 - 72 h) 0.51 mg/L; 95% confidence limits 0.45 - 0.59 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:

Statistical analysis of the growth rate data was carried out for the solvent control and all loading rates 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 solvent control, 0.10, 0.32 and 1.0 mg/L loading rate WAFs (P≥0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 1.0 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 3.2 mg/L loading rate WAF.

Statistical analysis of the yield data was carried out. There were no statistically significant decreases in yield between the solvent control, 0.10, 0.32 and 1.0 mg/L loading rate WAFs (P≥0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 1.0 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 3.2 mg/L loading rate WAF.

See 95% confidence intervals in the table.

Any other information on results incl. tables

Response Variable	EL50(mg/L Loading Rate WAF)	95% Confidence Limits (mg/L Loading Rate WAF)
Growth Rate	5.1	4.4 -5.9
Yeild	2.9	2.3 -3.6

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Growth Rate, EL50=5.1 mg/L Loading Rate WAF
Yeild, EL50=2.9mg/L Loading Rate WAF

Executive summary:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period using a water accomodated fraction

Growth Rate, EL50=5.1 mg/L Loading Rate WAF

Yeild, EL50=2.9mg/L Loading Rate WAF