Inositol phosphates

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From April 3, 2009 to June 5, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to OECD Guideline 201 referenced as Method C.3 of Commission Regulation (EC) No440/2008. GLP compliance has been claimed through conduct of the study according to UK GLP Standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). Analytical verification of nominal test material concentrations was not possible as no quantitative method was available. Dose verification was therefore conducted by Total Organic Carbon (TOC) analysis.

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

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:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI2004/0994)

Test material

Test material information

Constituent 1

Reference substance name:

Inositol phosphates derived from rice bran

IUPAC Name:

Inositol phosphates derived from rice bran

Details on test material:

- Name of test material (as cited in study report): Inositol phosphates derived from rice bran
- Physical state: Amber coloured liquid
- Lot/batch No.: 98761634JQ-002
- Storage condition of test material: Room temperature in the dark

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
None

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 1.0, 3.2, 10, 32 and 100 mg/L
- Sampling method: Samples for analytical monitoring were taken at 0 and 72 h

Test solutions

Vehicle:

no

Details on test solutions:

None

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- Method of cultivation: Master culture was added to approximately 100 mL culture media contained in conical flasks to give an initial cell density of approximately 1000 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 10,000-100,000 cells/mL.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

No

Post exposure observation period:

None

Test conditions

Hardness:

No data

Test temperature:

24 ± 1°C

pH:

Controls: 7.2 - 7.4; 1.0 mg/L: 7.2 - 7.3; 3.2 mg/L: 7.2; 10 mg/L: 6.9 - 7.1; 32 mg/L: 6.4; 100 mg/L: 3.8 - 3.9

Dissolved oxygen:

No data

Salinity:

Freshwater

Nominal and measured concentrations:

Nominal concentrations: 0, 1.0, 3.2, 10, 32 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: Closed (plugged with polyurethane foam bungs)
- Material, size, headspace, fill volume: 250 mL glass conical flask containing 100 mL test medium
- Initial cells density: 4 x 10000 cells/mL
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six


GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis purified deionised water
- Culture medium different from test medium: No


OTHER TEST CONDITIONS
- Light intensity and quality: Continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell density was determined at 0, 24, 48 and 72 h using a Coulter® Multisizer Particle Counter


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, test concentrations for the definitive study were selected on the basis of results obtained in range finding study

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

55 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 44-69 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 6.4-11 mg/L

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities:
- Colour differences: At the initiation of test - Clear colourless solutions; After 72 hr - Control: Pale green dispersions, 1.0 mg/L: pale green dispersions, 3.2 and 10 mg/L: very pale green dispersions, 32 mg/L: extremely pale green dispersions, 100 mg/L: clear colourless solutions
- Other: Cell debris - At 32 and 100 mg/L concentrations

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50 (72 hr): 0.79 mg/L
- EbC50 (72 hr): 0.30 mg/L (95% confidence limits: 0.27-0.34)
- Other:
NOEC - Growth rate: 0.25 mg/L
Yield rate: 0.25 mg/L
LOEC - Growth rate: 0.50 mg/L
Yield rate: 0.50 mg/L

Reported statistics and error estimates:

ECx values were determined from the equation for the fitted line using the Xlfit software package (lOBS). 95% confidence limits for the EC50 values were calculated using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).
One way ANOVA Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure (Dunnett 1995) were used on the determination of growth rate, yield and biomass integral data.
The experimental data was analysed using SAS computer software package (SAS 1999-2001).

Any other information on results incl. tables

Table 1: Effect of test material on the growth of Desmodesmus subspicatus over a 72-h period

Response Variable	EC50 (mg/L)	95% Confidence Limits  (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	55	44 -69	1.0	3.2
Yield	8.4	6.4 -11	1.0	3.2

Table 2: Total organic carbon (TOC) analysis results

Sample	Nominal concentration (mg/L)	Concentration of TOC (mg C/L)	Concentration of TOC corrected for control (mg C/L)
O hours	Control	<LOQ	-
	1.0	<LOQ	<LOQ
	3.2	<LOQ	<LOQ
	10	<LOQ	<LOQ
	32	1.52	1.52
	100	3.79	3.79
72 hours	Controls	<LOQ	-
	1.0	<LOQ	<LOQQ
	3.2	<LOQ	<LOQ
	10	<LOQ	<LOQ
	32	1.33	1.33
	100	3.96	3.96

LOQ: limit of quantification

The carbon content of the test substance was determined in a separate study (2787/0012) to be equivalent to 3.29%, i.e. 3.29 mg C/100 mg substance. Based on this value, the data in Table 2 shows that nominal concentrations were reached and maintained, at least at the two highest concentrations (32 and 100 mg/L). TOC values at the lower concentrations were below the limit of quantification of the method.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Control cultures: Cell concentration was increase by a factor of 97 after 72 h, mean coefficients of variation for section by section specific growth rate was 29%., coefficient of variation for average specific growth rate for 72 h was 3%

Conclusions:

The 72 h ErC50 and EbC50 for Desmodesmus subspicatus were found to be 55 mg/L and 8.4 mg/L, respectively. The test material could not be analysed quantitatively in the test medium but total organic carbon analysis indicated that nominal test concentrations were reached and maintained throughout the study, at least at the two highest concentrations. Total organic carbon concentrations at the lower doses were below the limit of quantification of the method.

Executive summary:

A study was performed to assess the effect of inositol phosphates derived from rice bran on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Algae and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 440/2008.

Desmodesmus subspicatus was exposed to the test substance at nominal concentrations of 0, 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 h, under constant illumination and shaking at a temperature of 24 ± 1^oC.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

The 72 h ErC50 and EbC50 were found to be 55 (95% Confidence Limits,44-69) mg/L and 8.4 (95% Confidence Limits, 6.4-11) mg/L, respectively. The NOEC and LOEC for both growth rate and yield were 1.0 and 3.2 mg/L, respectively.

The test material could not be analysed quantitatively in the test medium but total organic carbon analysis indicated that nominal test concentrations were reached and maintained throughout the study, at least at the two highest concentrations. Total organic carbon concentrations at the lower doses were below the limit of quantification of the method.