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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 May - 22 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with International guidelines and in accordance with GLP. All guideline validty criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark.
- Stability under test conditions: N/A, substance is a UVCB.
- Solubility and stability of the test substance in the solvent/vehicle: Test solution prepared as a Water Accomodated Fraction (WAF) in culture medium at a nominal rate of 100 mg/L.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: No
- Final preparation of a solid: No

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Test solution prepared as a Water Accomodated Fraction (WAF) in culture medium at a nominal rate of 100 mg/L.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) : N/A

OTHER SPECIFICS: N/A
Analytical monitoring:
yes
Remarks:
One component of the UVCB substance was selected as a marker anlayte. The marker analyste was used to confirm the concentration of the the UVCB substance in the test system by extapolation.
Details on sampling:
- Concentrations: 100 mg/L "limit test"
- Sampling method: Samples were taken from the control and the 100 mg/L loading rate WAF 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.
- Sample storage conditions before analysis: Stored 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 (200 mg) was added to the surface of 2 liters of algae 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 95 hours and the mixture allowed to stand for 1-Hour. Microscopic observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 4 cm in length). 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). Further filtration through one sheet of filter paper was required to remove as much undissolved test item as possible. Microscopic observations of the WAF were performed, after filtering and showed no micro-dispersions of test item to be present.
An aliquot (1 liter) of the 100 mg/L loading rate WAF was inoculated with algal suspension (12.1 mL) to give the required test concentration of 100 mg/L loading rate WAF.
- Controls: Negative control = Algae culture medium. Positive control (potassium dichromate) conducted under a separate GLP study on 06-09 Nov 2017 with a concentration range of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
- 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.): Not after filtration.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Freshwater green algae (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. Cultures of this original source were maintained in the Test Facility.
- Age of inoculum (at test initiation): Not reported
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master 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
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
24 +/- 1 ºC
pH:
7.9 - 8.6
Dissolved oxygen:
Not reported
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal loading rate: 100 mg/L (UVCB)
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flask
- Type (delete if not applicable): Capped with polyurethane foam plug
- Material, size, headspace, fill volume: 250 mL glass conical flasks filled to 100 mL volume.
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 5.0 x 10^3 cells per mL
- Control end cells density: 1.23 x 10^6 cells per mL
- No. of organisms per vessel: N/A
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/A

GROWTH MEDIUM
- Standard medium used: Yes
- Detailed composition if non-standard medium was used: N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: NaNO3 (25.5 mg/L), MgCl2.2H2O (12.16 mg/L), CaCl2.2H2O (4.41 mg/L), MgSO4.7H2O (14.6 mg/L), K2HPO4 (1.044 mg/L), NaHCO3 (15.0 mg/L), H3BO3 (0.186 mg/L), MnCl2.4H2O (0.415 mg/L), ZnCl2 (0.00327 mg/L), FeCl3.6H2O (0.160 mg/L), CoCl2.6H2O (0.00143 mg/L), Na2MoO4.2H2O (0.00726 mg/L), CuCl2.2H2O (0.000012 mg/L), Na2EDTA.2H2O (0.30 mg/L). The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of the control and the 100 mg/L loading rate WAF was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: No
- Photoperiod: Continuous light
- Light intensity and quality: 7000 lux @ 380-730 nm
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell concentration measured at 22, 48 and 72 h.
- Determination of cell concentrations: Coulter Multisizer Particle Counter
- Chlorophyll measurement: N/A
- Other: N/A

TEST CONCENTRATIONS
- Spacing factor for test concentrations: range
N/A
- Justification for using less concentrations than requested by guideline: Limit test with a UVCB substance conducted, based on the results of the preliminary test.
- Range finding study
- Test concentrations: Loading rates of 10 and 100 mg/L prepared.
- Results used to determine the conditions for the definitive study: Yes, no effects observed at 100 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate. The positive control test was conducted between 06 November 2017 and 09 November 2017
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
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: No
- Any stimulation of growth found in any treatment: No
- 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
Results with reference substance (positive control):
- Results with reference substance valid
- EC50: 1.6 mg/L (for growth rate)
- Other: NOEC = 0.25 mg/L (for growth rate)
Reported statistics and error estimates:
A Student’s t-test 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 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).

Table 1       Measured concentrations of AD-464

Nominal Concentration

(mg/L)

Measured Concentration

(mg/L)

0 h

72 h

Control

<LOQ

<LOQ

100

0.0176

<LOQ

LOQ: Limit of Quantification (0.0019 mg/L)

 

Table 2       Cell densities

 

Nominal Loading Rate

(mg/L)

Rep.

Cell Density (x 104cells/mL)

22 h

48 h

72 h

Control

1

2.76

15.9

134

2

2.75

19.2

141

3

2.92

19.7

125

4

2.52

17.6

105

5

2.49

17.1

111

6

2.82

15.5

125

Mean

2.71

17.5

123

100

1

2.53

16.8

130

2

1.88

15.5

110

3

1.99

15.5

100

4

2.42

20.1

131

5

2.68

18.5

130

6

2.25

17.6

140

Mean

2.29

17.3

123

 

Table 3       Growth rate and inhibition

 

Nominal Loading Rate

(mg/L)

Rep.

Growth Rate (cells/mL/h)

0-72 h

% Inhibition

Control

1

0.078

-

2

0.078

-

3

0.077

-

4

0.074

-

5

0.075

-

6

0.077

-

Mean

0.077

-

SD

0.002

-

100

1

0.077

0

2

0.075

3

3

0.074

4

4

0.077

0

5

0.077

0

6

0.078

[1]

Mean

0.076

1

SD

0.002

-

Table 4      Yield and inhibition

 

Nominal Loading Rate

(mg/L)

Rep.

Yield (cells/mL)

0-72 h

% Inhibition*

Control

1

1.34 x 106

-

2

1.40 x 106

3

1.25 x 106

4

1.04 x 106

5

1.10 x 106

6

1.24 x 106

Mean

1.23 x 106

SD

1.36 x 105

100

1

1.30 x 106

0

2

1.09 x 106

3

9.95 x 105

4

1.30 x 106

5

1.30 x 106

6

1.39 x 106

Mean

1.23 x 106

SD

1.52 x 105

* In accordance with OECD 201, only the mean value was calculated.

 

Table 5       Test solution pH

 

Nominal Loading Rate

(mL/g)

pH

0h

72 h

Control

7.9

8.4

100

NR

NR

NR: Not reported

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The NOEL was 100 mg/L loading rate WAF.
Executive summary:

OECD 201 (2018) - In a 72 hour toxicity study, the cultures of Pseudokirchneriella subcapitatawere exposed to the test item, reaction products of triphenyl phosphite, oleyl alcohol and alcohols C10 -C16 (947 -909 -7) in a limit test under static conditions in accordance with the OECD 201 guidance document.  Treatment solutions were prepared from water accommodated fractions (WAF). The nominal loading was derived from 100 mg/L. The solutions were stirred for 95 h.  The solubilized fractions were decanted and filtered to prepare the treatment solution.  Test substance concentrations were confirmed by LC-MS analysis of a "marker analyte" using a validated method at 0 h and 72 h. Test substance concentrations were calculated/extrapolated from the known marker analyte concentration calibration curve. The initial cell biomass added to each treatment flask was 5 x 103cells/mL at 0 h.  Cell biomass was determined, using a particle counter, at 22, 48 and 72 h.

Routine microscopical examination of the algae cells concluded that there was no compound related phytotoxic effects.

The ErL50 for was estimated to be >100 mg/L.  The mean percent growth inhibition in the treated algal culture as compared to the control was 1 %.

No statistically significant reductions in growth rate were observed, according to Dunnett’s multiple comparison test.  The 72 h ErL50 and NOELR for growth rate was therefore determined to be >100 mg/L and 100 mg/L, respectively.

This toxicity study is acceptable and satisfies the guideline requirements for an aquatic algae toxicity study according to the OECD 201 guideline.

Description of key information

72h EC50 (growth rate) >0.0041 mg/L, 72h EC10 (growth rate) >0.0041mg/L (Pseudokirchnerella subcapitata); OECD 201; Vryenhoef, H. (2018).

Key value for chemical safety assessment

Additional information

OECD 201 (2018) - In a 72 hour toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to the test item, reaction products of triphenyl phosphite, oleyl alcohol and alcohols C10 -C16 (947-909-7), which contained the major constituent dioctadec-9 -enyl phosphonate (246-626-8) at > 80 % (i.e. the substance being registered in this dossier; non-stereospecific name of the substance), in a limit test under static conditions in accordance with the OECD 201 guidance document.  Treatment solutions were prepared from water accommodated fractions (WAF). The nominal loading was derived from 100 mg/L. The solutions were stirred for 95 h.  The solubilized fractions were decanted and filtered to prepare the treatment solution.  Test substance concentrations were confirmed by LC-MS analysis of the "marker analyte - dioctadec-9 -enylphosphate" of the test item (as the test item is an UVCB), using a validated method at 0 h and 72 h. Test substance concentrations were calculated/extrapolated from the known marker analyte concentration calibration curve. The initial cell biomass added to each treatment flask was 5 x 103cells/mL at 0 h.  Cell biomass was determined, using a particle counter, at 22, 48 and 72 h.

Routine microscopical examination of the algae cells concluded that there was no compound related phytotoxic effects.

The ErL50 for was estimated to be >100 mg/L.  The mean percent growth inhibition in the treated algal culture as compared to the control was 1 %.

No statistically significant reductions in growth rate were observed, according to Dunnett’s multiple comparison test.  The 72 h ErL50 and NOELR for growth rate was therefore determined to be >100 mg/L and 100 mg/L, respectively.

This dossier is for the monoconstituent (Z,Z)-di-9-octadecenyl phosphonate (246-608-1). Dioctadec-9 -enyl phosphate (non-stereospecific name of the substance) is the major constituent of reaction products of triphenyl phosphite, oleyl alcohol and alcohols C10 -C16 (947-909-7). In the particular batch of reaction products of triphenyl phosphite, oleyl alcohol and alcohols, C10 -C16 that was use in the presented algae study, the percentage of the major constituent dioctadec-9 -enyl phosphonate was 80.56 %. Therefore, in accordance with ECHA Guidance for identification and naming of substances under REACH and CLP (2017, v2.1) this batch could be considered as both "reaction products of triphenyl phosphite, oleyl alcohol and alcohols C10 -C16", and as "dioctadec-9 -enyl phosphate (264 -626 -8)" the monoconstituent, as the batch falls within both of the substances boundary compositions, and under the definition of a monoconstituent outlined by ECHA (i.e. "a monoconstituent substance is a substance, defined by its quantitative composition, in which one main constituent is present to at least 80 % (w/w)" ECHA, 2017, v2.1). Furthermore, this was the constituent of the UVCB that was measured during chemical analysis in the aquatic toxicity tests which was confirmed by the relatively large peak compared to the others shown and similar level of water solubility in comparison to the water solubility test (ENVIGO, 2018).

However, to use the current study, and data therein, for the monoconstituent, different assumptions must be made. As such the test design description differs slightly. Firstly, for the monoconstituent the test design is considered to be a saturated solution, not a water accommodated fraction. Secondly, the analyte is not considered the marker analyte of a complex mixture but the test substance itself. As such, the effect levels should be based on either nominal concentrations, or mean measured concentrations dependent on the percentage recovery of the nominal dose. Here, less than 80 % of the test item was measured 0 hours in fresh media. Further, degradation of the test item was seen during the test with recoveries <=50 % in old media. Therefore effect concentrations should and will be based on the geometric mean measured concentrations of the test item. The geometric mean measured concentration of the test item in this limit test using a saturated solution was 0.0041 mg/L. No effects were seen at the limit of solubility.

The EC50 and NOEC for this test was therefore estimated to be > 0.0041 mg/L (i.e. > than the limit of solubility).

Routine microscopical examination of the algae cells concluded that there was no compound related phytotoxic effects.

This toxicity study is acceptable and satisfies the guideline requirements for an aquatic algae toxicity study according to the OECD 201 guideline.