Salts of 2-ethylhexyl phosphate esters with (Z)- octadec-9-enylamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Sep -19 Oct 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance OECD 201 guideline without deviation. The substance was a UVCB of varying solubilities and therefore a WAF approach has been used in accordance with OECD Series on testing and assessment No. 23. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

Principles of method if other than guideline:

- Principle of test: Water accomodated fraction (WAF) was used in accordance with OECD guidance on aquatic toxicity testing of difficult substances and mixtures (Document no. 23, 2002).
- Short description of test conditions: Due to the need to test at relatively low levels, a single WAF of a nominal loading of 1.0 mg/L was prepared from which dilutions were made to give the remaining test concentrations.. The material is left until fully dissolved (or as dissolved as possible i.e. a saturated solution of the varying componnets of the UVCB), then particulate matter is left to settle out of the water column. 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 of test item to be present. A series of dilutions was made from the 1.0 mg/L loading rate to give further stock solutions of 0.32, 0.10, 0.032 and 0.010 mg/L loading rate WAF. The concentration-response relationship is based on the initial loading rate of the vessel.
- Parameters analysed / observed: Pariculate was monitored in the WAFs via microscopic examination.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Provided by Sponsor,Confidential
- Expiration date of the lot/batch: Confidential

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: No significant change in measured test item analyte concentrations over 72 h (within 80-120 % of nomina)l. However, stability of the entire UVCB is unknown.
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

Analytical monitoring:

yes

Remarks:

analytical monitoring used during both range finding and definitive tests, but a validated method was used during definitive test only.

Details on sampling:

- Concentrations: nominal concentration 0.01, 0.032, 0.10, 0.32 and 1.0 mg/L
- Sampling method: Duplicate samples taken at 0 h and 72 h from the controls and test group. Each duplicate was pooled for analysis.
- Sample storage conditions before analysis: frozen

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 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. This was stirred for 23 hours and allowed to settle for 1 hour. The aqueous phase was removed by mid-depth siphoning (~5 cm) with the first 75 - 100 mL discarded. This gave rise to a 1 mg/L stock. This was subsequently used to make successive dilutions to achieve 0.32, 0.10, 0.032 and 0.010 mg/L.
- Eluate: N/A
- Differential loading: N/A
- Controls: AAP medium only (OECD 201, 2011)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Microscopic inspection of the WAF showed no micro-dispersions of test item to be present.

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): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): unknown
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. Cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.

ACCLIMATION
- Acclimation period: N/A
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

OECD 201 guideline recommended

Post exposure observation period:

none

Hardness:

Not reported

Test temperature:

24 ±1 ºC

pH:

7.8-8.8

Dissolved oxygen:

N/D

Salinity:

N/D

Conductivity:

N/D

Nominal and measured concentrations:

Nominal concentrations = 0.010, 0.032, 0.10, 0.32 and 1 mg/L.
0 h measured concentrations: 72 h measured concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks plugged with polyurethane foam bungs to reduce evaporation
- Material, size, headspace, fill volume: 100 mL medium-algae, or medium-algae-test item.
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): none
- Renewal rate of test solution (frequency/flow rate): none
- Initial cells density: 5 x 10^3
- Control end cells density: 1.21 x 10^6 to 1.82 x 10^6
- No. of organisms per vessel: N/A
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): no vehicle was used and no vehicle controls were required.

GROWTH MEDIUM
- Standard medium used: yes (AAP medium, OECD 201, 2011)
- Detailed composition if non-standard medium was used: N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory prepared.
- Total organic carbon: not relevant/required
- Particulate matter: not relevant/required
- Metals: not relevant
- Pesticides: not relevant
- Chlorine: not relevant/required
- Alkalinity: not relevant/required
- Ca/mg ratio: not relevant/required
- Conductivity: not relevant/required
- Culture medium different from test medium: no
- Intervals of water quality measurement: Measurements of pH-value were performed at t = 0 h and t = 72 h. Temperature was also monitored.

OTHER TEST CONDITIONS
- Sterile test conditions: not required
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: approximately 7000 lux

- Salinity (for marine algae): not relevant

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
- Chlorophyll measurement: none
- Other: none

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: 5 concentrations used in accordance with OECD guideline 201
- Range finding study: Yes, conducted at 0, 0.1, 0.01 and 1 mg/L.
- Test concentrations: 0, 0.954, 3.05, 9.77, 31.3 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.032 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.084 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: -
- Any stimulation of growth found in any treatment: mild stimulation (2 %) at 0.032mg/L but not biologically significant and within normal variation.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

The following validity criteria were met as per the OECD guideline:

- The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
- The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (Days 0 to 1, 1 to 2 and 2 to 3, for 72-Hour tests) must not exceed 35%.
- The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72 hour EC50: 1.6 mg/L (growth rate) 0.77 mg/L (yield) - The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
- Other: N/A

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 all test 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).

Table 1 Cell count, Growth Rate and Yield in the Definitive Test

Loading rate	Cell numbers [10^4/mL]				Yield [10^4/mL]	Growth rate (Cells/mL/h)
[mg/L]	0 h	24 h	48 h	72 h	0 h – 72 h	0 – 72 h
Control	5.00	3.34	21.3	181	181	0.082
	5.00	3.51	21.0	155	154	0.080
	5.00	3.10	18.3	121	120	0.076
	5.00	3.17	21.7	182	182	0.082
	5.00	2.96	20.5	157	156	0.080
	5.00	3.10	19.5	129	128	0.077
Mean	5.00	3.19	20.4	154	154	0.080
0.010	5.00	3.51	25.1	168	167	0.081
	5.00	3.11	17.8	85.9	85.4	0.071
	5.00	3.03	18.8	121	121	0.076
Mean	5.00	3.22	20.6	125	124	0.076
0.032	5.00	3.35	23.2	175	174	0.081
	5.00	3.39	22.3	166	165	0.081
	5.00	2.90	20.1	129	129	0.077
Mean	5.00	3.21	21.8	157	156	0.080
0.10	5.00	2.14	17.4	120	120	0.076
	5.00	1.58	11.2	83.8	83.3	0.071
	5.00	2.63	15.5	107	107	0.075
Mean	5.00	2.11	14.7	104	103	0.074
0.32	5.00	0.188	0.549	2.46	1.96	0.022
	5.00	0.0645	0.223	0.616	0.116	0.002
	5.00	0.0645	0.123	0.331	-0.169	-0.006
Mean	5.00	0.107	0.298	1.14	0.636	0.006
1.00	5.00	0.0645	0.0968	0.202	-0.298	-0.013
	5.00	0.0821	0.132	0.217	-0.283	-0.012
	5.00	0.0499	0.150	0.155	0.345	-0.016
Mean	5.00	0.0655	0.126	0.192	-0.308	-0.014

Table 2. Inhibition of Growth rate and yield

Loading rate	Yield Inhibition (%)	Growth rate Inhibition (%)
[mg/L]	0 h – 72 h	0 – 72 h
Control	-	-
Mean
0.010	19	[1]
		11
		5
Mean		5
0.032	[2]	[1]
		[1]
		4
Mean		1
0.10	33	5
		11
		6
Mean		7
0.32	100	73
		96
		108
Mean		92
1.00	100	116
		115
		120
Mean		117

Table 3 pH in test system

Loading rate	pH
[mg/L]	0 h	72 h
Control	7.8	8.8
0.010	7.9	8.7
0.032	7.9	8.8
0.10	7.9	8.7
0.32	7.8	8.1
1.00	7.9	8.0

Table 4 Measured concentration of analyte

Loading rate	Measured analyte concentrations (mg/L)
[mg/L]	0 h	72 h
Control	< LOQ	< LOQ
0.010	< LOQ	< LOQ
0.032	< LOQ	< LOQ
0.10	0.0158	0.0142
0.32	0.0346	0.0308
1.00	0.0964	0.0932

Validity criteria fulfilled:

yes

Remarks:

Validity criteria of the guideline were all fulfilled.

Conclusions:

A test was conducted to determine the effects of the test item, AD-633 on the growth of the single cell green alga Pseudokirchneriella subcapitata. The study was conducted in accordance with the OECD guideline 201 the “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”, as the test item is a UVCB of varying solubilities a WAF approach was used in accordance with OECD series on testing and assessment no. 23. As the concentrations were low, and producing separate WAFs was impractical and would be outwith the analytical technique, serial dilutions were applied.

The 72 hour LOELR for growth rate was determined to be 0.32 mg/L (nominal), the corresponding NOELR was set at 0.10 mg/L (nominal). The EL10 was estimated to be 0.11 mg/L.
The 72 hour LOELR for yield was determined to be 0.10 mg/L (nominal), the corresponding NOELR was set at 0.032 mg/L (nominal). The EL10 was estimated to be 0.084 mg/L.
The 72 hour EL50-value for growth rate (ErL50) was determined to be 0.18 mg/L (nominal). The 72 hour EL50-value for yield (EyL50) was 0.11 mg/L (nominal).

Executive summary:

The objective of this study was to determine the effects of the test item on the growth of the single cell green alga Pseudokirchneriella subcapitata. Where possible the no observed effect loading rate (NOELR), the lowest observed effect loading rate (LOELR) and the effective loading rate (EL50), were estimated.

The study was conducted in accordance with the OECD guideline 201 the “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”, as the test item is a multi-constituent of varying solubilities a WAF approach was used in accordance with OECD series on testing and assessment no. 23.  

A range finding study was used to determine definitive test concentrations and optimal stirring and settling time for the creation of the WAF.

The definitive test concentrations were, 0.010, 0.032, 0.10, 0.32, 1.00 mg/L. A stock WAF was prepared at 1 mg/L by stirring for 23 hours at room temperature and then leaving any particulate to settle for 1 hour. The successive concentrations were prepared by dilution as it was not possible to weigh the required small amounts to allow for direct addition. No particulate or micro dispersions were noted via microscopic examination.  All water quality parameters and study validity criteria were met as per OECD guideline 201 (2011). A positive control (potassium dichromate) is routinely run at the laboratory proving sensitivity of the test organism. The reference was deemed acceptable.

The results of the test were valid and were:

The 72 hour LOELR for growth rate was determined to be 0.32 mg/L (nominal), the corresponding NOELR was set at 0.10 mg/L (nominal). The EL10 was estimated to be 0.11 mg/L.

The 72 hour LOELR for yield was determined to be 0.10 mg/L (nominal), the corresponding NOELR was set at 0.032 mg/L (nominal). The EL10 was estimated to be 0.084 mg/L.

The 72 hour EL50-value for growth rate (ErL50) was determined to be 0.18 mg/L (nominal). The 72 hour EL50-value for yield (EyL50) was 0.11 mg/L (nominal).

Description of key information

72h EL50 (growth rate) 0.18 mg/L, 72h EL10 (growth rate) 0.11 mg/L, 72h NOELR (growth rate) 0.10 mg/L (Pseudokirchnerella subcapitata); OECD 201, 2011; Vryenhoef H., 2018.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.18 mg/L

EC10 or NOEC for freshwater algae:

0.11 mg/L

Additional information

A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriellasubcapitata. 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) No 761/2009. As the substance was a UVCB a WAF approach was used in line with the OECD Guidance Document on Aquatic Toxicity Testing Of Difficult Substances and Mixture (2000). Further, as it was not possible to weigh the small quantities for the lower doses, and it was seen impractical,. in line with the guidance a diltuion of the WAF was made.

The results were:

The 72 hour LOELR for growth rate was determined to be 0.32 mg/L (nominal), the corresponding NOELR was set at 0.10 mg/L (nominal). The EL10 was estimated to be 0.11 mg/L.

The 72 hour LOELR for yield was determined to be 0.10 mg/L (nominal), the corresponding NOELR was set at 0.032 mg/L (nominal). The EL10 was estimated to be 0.084 mg/L.

The 72 hour EL50-value for growth rate (ErL50) was determined to be 0.18 mg/L (nominal). The 72 hour EL50-value for yield (EyL50) was 0.11 mg/L (nominal).