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Diss Factsheets

Ecotoxicological information

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:
08 October 2008 to 11 February 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was conducted in accordance with the relevant international guideline (OECD 201). The study report presented all information required to assess the validity of the data generated and the study methodology wo be wholly reproduceable.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Regulation (1999)

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
417-560-0
EC Name:
-
Cas Number:
67881-98-5
Molecular formula:
C11H22NO6P
IUPAC Name:
trimethyl[2-({2-[(2-methylprop-2-enoyl)oxy]ethyl phosphonato}oxy)ethyl]azanium
Details on test material:
- Name of test material (as cited in study report): HEMA-Pc
- Substance type: Pure Active Substance
- Physical state: Solid (White Powder)
- Analytical purity: 99 %
- Purity test date: 29 September 2008
- Lot/batch No.: SS148076
- Expiration date of the lot/batch: 27 August 2009 (Assigned by Testing Laboratory)
- Stability under test conditions: Stable
- Storage condition of test material: ca -20 degrees celsius in the dark under nitrogen.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
N/A

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
For the purpose of chemical analysis during the definitive test, duplicate aliquots of ca 10-15 mL from each prepared concentration were removed at the commencement of the test (0 h samples) and placed in vials. At 72 h, the contents of each replicate vessel for a given concentration were combined and duplicate aliquots (ca 10-15 mL) removed and placed in vials.
An additonal flask was prepared at nominal HEMA-Pc concentrations of 6.25 and 100 mg/L. and these incubated (without algal cells) alongside the test and control flasks. At 72 h duplicate aliquots (ca 20 mL) were removed from the additional flasks, to determine if any adsorpoon/absorption of HEMA-Pc to algal cells had occurred.
These samples were analysed according to the procedures established and validated under Charles River Protocol No. 425242. Analytical Method No. 2524.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Based on the previous experimental work carried out on HEMA-Pc. the test was conducted at nominal HEMA-Pc concentrations of 6.25, 12.5, 25, 50 and 100 mg HEMAPc/L. together with a control.

Due to the hygroscopic nature of HEMA-Pc all dose preparation procedures were carried out using an Atmos bag. This allowed for the preparation of a controlled reduced moisture environment using Nitrogen. All apparatus required for the preparation of the dose solutions was placed within the Atmos bag and the bag then sealed. Any existing air within the bag was removed using a vacuum pump and the bag inflated using moisture reduced Nitrogen. Relative humidity was recorded as 3 % using a VEI model DTH10 Relative Humidity meter.

Test solutions were prepared by serial dilution of a stock solution of HEMA-Pc prepared in 1 L of growth medium. The control contained algal medium only. Refer to Table 1 for details on stock solution preparation.

Three acid washed 250 ml Erlenmeyer flasks were prepared for each test item treatment grop and six for the control group. Each flask was labelled with the study number, treatment group, replicate number and date. An aliquot of 100 ml of the appropriate test solutions was added to each flask. An inoculum volume of 1.0 ml of a concentrated algal culture (9.7 x 10^5 cells/ml) was added to each flask, giving an initial cell concentration of 0.97 x 10^4 cells/ml in each vessel. The flasks were capped using foil and placed Sanyo Gallenkamp Orbital Incubator.

- Eluate: OECD Freshwater Algal Medium
- Controls: OECD Freshwater Algal Medium only control
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Algae
- Strain: 278/4
- Source (laboratory, culture collection): Culture Centre of Algae and Protozoa (CCAP)
- Age of inoculum (at test initiation): Not reported
- Method of cultivation: axenic culturing techniques


ACCLIMATION
- Acclimation period: N/A (in-house culture)
- Culturing media and conditions (same as test or not): Same as test conditions
- Any deformed or abnormal cells observed: None

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
N/A

Test conditions

Hardness:
Not applicable
Test temperature:
Refer to Table 2.
pH:
Refer to Table 2.
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal Concentrations: 6.25, 12.5, 25, 50, 100 mg/L together with an untreated control.
For data on measured concentrations, refer to Table 3.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml capacity glass Erlenmeyer flasks
- Type (delete if not applicable): Closed (capped with foil lids)
- Material, size, headspace, fill volume: 100 mL of test solution was used for each replicate flask within each treatment
- Aeration: None
- 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: 0.97 x 10^4 cells/ml
- Control end cells density: 1.57 x 10^6
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/A


GROWTH MEDIUM
- Standard medium used: yes (OECD algal growth medium)
- Detailed composition if non-standard medium was used: N/A


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water
- 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: N/A
- Intervals of water quality measurement: pH was measured at 0 hours and 72 hours. Maximum and minimum temperatures were measured on a daily basis.


OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: None
- Photoperiod: 24 hour light - No photoperiod
- Light intensity and quality: Light intensity = 7623 lux
- Salinity (for marine algae): N/A


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Improved neubauer counting chamber
- Chlorophyll measurement: N/A
- Other:


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study
- Test concentrations: No range finding test conducted
- Results used to determine the conditions for the definitive study: N/A
Reference substance (positive control):
no
Remarks:
Not Applicable

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 102 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: N/A
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
102 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: N/A
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 102 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: N/A
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
102 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: N/A
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): None
- Unusual cell shape: N/A
- Colour differences: N/A
- Flocculation: N/A
- Adherence to test vessels: N/A
- Aggregation of algal cells: N/A
- Other: N/A
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
N/A
Reported statistics and error estimates:
No statistical analyses were conducted on the raw data generated during this study.

Any other information on results incl. tables

Table 2. Environmental Parameters During the Definitive Test

a) Minimum / Maximum Daily Temperature

Time (h)

Temperature (degrees Celsius)

Maximum

Minimum

24

23.7

23.1

48

23.4

23.1

72

23.9

23.1

b) pH Values

Nominal HEMA-Pc Concentration (mg/L)

Overall Measured HEMA-Pc Concentration (mg/L)

pH

0 h

Replicate Flask

72 h

Control

Not Detected

8.01

1

2

3

4

5

6

8.88

8.88

9.44

9.48

8.54

8.55

6.25

5.68

7.91

1

2

3

9.52

7.68

9.42

12.5

11.9

7.74

1

2

3

9.53

7.69

7.63

25

24.5

7.81

1

2

3

7.87

7.72

8.60

50

47.5

7.89

1

2

3

8.52

7.80

9.04

100

102

8.01

1

2

3

7.85

7.49

7.47

Limit of Quantification: 0.905 mg/L

Table 3. Measured Concentrations of HEMA-Pc in Test Solutions at 0 h and 72 h During the Definitive Test

Nominal Concentration HEMA-Pc (mg/L)

0 h

Mean Measured Concentration of HEMA-Pc

72 h

Mean Measured Concentration of HEMA-Pc

(mg/L)

% Target

(mg/L)

% Target

Control

Not Detected

N/A

Not Detected

N/A

6.25

6.16

98.6

5.19

83.0

12.5

12.3

98.4

11.5

92.0

25

24.9

99.6

24.0

96.0

50

50.3

101

44.6

89.2

100

102

102

102

102

Flasks Not Inoculated with Algal Cells

6.25

6.16

98.6

6.3

101

100

102

102

103

103

Table 4. Mean Cell Concentrations at 0, 24, 48 and 72 h During the Definitive Test

Nominal HEMA-Pc Concentration (mg/L)

Overall HEMA-Pc Concentration

(mg/L)

Mean Algal Cells/mL

0 h

24 h

48 h

72 h

Control

Not Detected

9.70E+03

5.67E+04

3.42E+05

1.57E+06

6.25

5.68

9.70E+03

6.75E+04

3.48E+05

1.74E+06

12.5

11.9

9.70E+03

6.00E+04

4.31E+05

2.43E+06

25

24.5

9.70E+03

7.42E+04

4.08E+05

1.90E+06

50

47.5

9.70E+03

6.33E+04

4.32E+05

1.83E+06

100

102

9.70E+03

7.33E+04

4.28E+05

2.07E+06

Table 5. Inhibition of Growth

a) Average Specific Growth Rate (Compared with Control)

Nominal HEMA-Pc Concentration (mg/L)

Overall Measured HEMA-Pc Concentration (mg/L)

Inhibition (%) of Average Specific Growth Rate

0-24 h

24-48 h

48-72 h

0-72 h

6.25

5.68

0

0

0

0

12.5

11.9

0

0

0

0

25

24.5

0

0

0

0

50

47.5

0

0

0

0

100

102

0

0

0

0

b) Yield (Compared with Control)

Nominal HEMA-Pc Concentration (mg/L)

Overall Measured HEMA-Pc Concentration (mg/L)

Inhibition (%) of Yield

0-24 h

24-48 h

48-72 h

0-72 h

6.25

5.68

0

0

0

0

12.5

11.9

0

0

0

0

25

24.5

0

0

0

0

50

47.5

0

0

0

0

100

102

0

0

0

0

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 0-72 hour EC50, EC20 and EC10 (for average specific growth rate and yield) of HEMA-Pc to P. subcapitata was estimated as >102 mg HEMA-Pc/L based on overall mean measured concentrations of HEMA-Pc. The NOEC (for average specific growth rate) was estimated at 102 mg HEMA-Pc/L, under the conditions of this test.

The 0-72 hour EC50, EC20, and EC10 (yield) of HEMA-Pc to P. subcapitata was estimated as >102 mg HEMA-Pc/L based on overall mean measured concentrations of HEMA-Pc. The NOEC (yield) was estimated at 102 mg HEMA-Pc/L, under the conditions of this test.
Executive summary:

This study was designed to estimate the median effective concentration (EC50) of HEMA-Pc

to Pseudokirchneriella subcapitata, a unicellular freshwater green alga, over a 72 h period.

The study was performed to meet the requirements of OECD (2006) Guideline 201 and

Regulation EC 1907/2006.

Based on preliminary work, the definitive test was conducted with nominal concentrations of

6.25, 12.5, 25, 50 and 100 mg HEMA-Pc/L, together with an untreated control.

Test solutions were prepared by serial dilution of a stock solution of HEMA-Pc prepared by

direct addition of ca 100 mg of HEMA-Pc into 1 litre of OECD algal growth medium

followed by ultrasonic dispersion; the control was OECD algal growth medium only. Three

test vessels were prepared at each HEMA-Pc concentration and six vessels for the controls.

Each vessel contained 100 mL of test solution, inoculated with P. subcapitata to a final

concentration of 0.97e+04 cells/mL. Test vessels were incubated in an oscillating incubator

(ca100 rpm) at 23.1-23.9°C for 72 h, under continuous light (mean 7623 lux). Cell

concentrations were determined every 24 h in each vessel.

Chemical analysis of test samples conducted at 0 and 72 h indicated that measured

concentrations were maintained at between 83 and 102% of nominal throughout the test

period. The overall mean measured concentrations over the test period were 5.68, 11.9, 24.5,

47.5 and 102 mg HEMA-Pc/L for the nominal 6.25, 12.5, 25, 50 and 100 mg HEMA-Pc mg/L

concentrations, respectively. Results throughout the report are based on mean measured

concentrations.

There was no inhibition of growth in HEMA-Pc test solutions compared with the control

solutions. EC50, EC20 and EC10 concentrations could not be statistically calculated from the

data in terms of specific growth rate (ErCx) and yield (EyCx) as there was no inhibition at the

maximum concentration tested. EC values are therefore estimated at greater than 102 mg

HEMA-Pc/L, the highest tested concentration. The no observed effect concentration (NOEC)

is reported as 102 mg HEMA-Pc/L for both average specific growth rate and yield, under the

conditions of this test.