Aluminum, benzoate C16-18 fatty acids complexes - high aluminium

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:

24 January 2013 to 25 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant, guideline study, available as an unpublished report. .

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Aluminum, benzoate C16-18-fatty acids complexes

EC Number:

303-385-6

EC Name:

Aluminum, benzoate C16-18-fatty acids complexes

Cas Number:

94166-87-7

Molecular formula:

C23H37AlO5, C25H41AlO5

IUPAC Name:

Aluminum, benzoate C16-18-fatty acids complexes

Test material form:

other: Solid

Details on test material:

- Physical state: Pale Yellow Solid
- Purity: Not applicable - UVCB
- Substance identity: Aluminum, benzoate C16-18-fatty acids complexes
- Batch number: 11074091 + Benzoic acid
- Carbon Content: 65.1%
- Analysis code: A118/99
- Date received: 07 June 2012
- Expiration date: 01 July 2013
- Storage of test material: Room temperature in the dark

Specific details on test material used for the study:

The substance is the Source of the read-across.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Total organic carbon and aluminium analysis: Samples were collected at 0 and 72 hours from the uninoculated and pooled treatment replicates and stored at -20°C until analysis. Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

- Test water: Prepared from reverse osmosis purified water, pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl and water hardness adjusted to 150 mg/L as CaCO3.
- Preparation: 250 mg aluminum, benzoate C16-18-fatty acids complexes was added to the surface of 2.5 L of culture medium, to give a loading rate of 100 mg/L, and stirred using a magnetic stirrer at a rate that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. 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 and the WAFs removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the WAF.
- Controls: Test control: test medium only. Positive control: potassium dichromate at concentrations; 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

Test organisms

Test organisms (species):

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

Details on test organisms:

- Source: Liquid cultures (strain CCAP 278/4) were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Scottish Marine Institute, Oban, Argylll, Scotland and maintained in the laboratory under constant aeration and illumination at 21°C.
- Method of cultivation: Prior to the start of the test, approximately 100 mL of culture media, with a cell density of approximately 10+03 cells/mL, were added to conical flasks which were plugged with polyurethane foam stoppers. The flasks were kept under constant agitation by orbital shaker at 100-150 rpm and constant illumination at 24 ± 1°C until algal cell density was approximately 10E+04 to 10E+05 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Test conditions

Hardness:

At the request of the sponsor, the water hardness was changed from that of the culture medium of 15 mg/L to approximately 150 mg/L CaCO3.

Test temperature:

The temperature was maintained at 24 ± 1°C throughout the test.

pH:

The pH of the control cultures was 7.6 at 0 hours and 7.8 at 72 hours. For the treatment group the pH was 7.7 at 0 hours and 7.8 at 72 hours.

Dissolved oxygen:

No data reported

Salinity:

Not applicable

Nominal and measured concentrations:

- Preliminary test: The loading rates to be used in the definitive test were determined on the basis of a range finding study at 10 and 100 mg/L nominal loading rates showing no effects.
- Nominal concentration: The test was conducted as a limit test with a single nominal loading rate of 100 mg/L (WAF).
- Measured aluminium concentrations: The aluminium concentration was 0.13 mg/L at both 0 and 72 hours.
- Measured total organic carbon: Total Organic Carbon (TOC) analysis of the test preparation showed measured concentrations of less than the limit of quantitation (LOQ) (assessed to be 1.0 mg C/L for the method employed) at 0 hours and less than the control value at 72 hours.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass conical flask.
- Type : Flask plugged with polyurethane foam bungs.
- Material, size, headspace, fill volume: 250 mL glass concical flask containing 100 mL of test preparation.
- Aeration: Not reported
- Initial cells density: 5.45 x 10E+03 cells/mL.
- Control end cells density: 4.94 x 10E+05
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:Prepared from reverse osmosis purified water, pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl and water hardness adjusted to 150 mg/L as CaCO3.
- Total organic carbon: < 1.0 mg C/L (- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of the control and 100 mg/L loading rate WAF was determined at initiation of the test and after 72 hours exposure. The temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: No reported
- Adjustment of pH: pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Photoperiod: Continuous illumination
- Light intensity and quality: Approximately 7000 lux provided by warm white lighting (380 - 730 nm)
- Other conditions: Flask constantly shaken at approximately 150 rpm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and cell density determined using a Coulter Multisizer Particle Counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test at 100 mg/L (WAF).
- Range finding study
- Test concentrations: 10 and 100 mg test item/L.
- Results used to determine the conditions for the definitive study: No effect seen during range finding study, therefore definitive test carried out as limit test at 100 mg/L (WAF).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accommodated Fraction

Basis for effect:

other: Growth rate and yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Water Accommodated Fraction

Basis for effect:

other: Growth rate and yield

Details on results:

The growth rate and yield of the algae were not affected (P ≥ 0.05) by the presence aluminum, benzoate C16-18-fatty acids complexes over the 72 hour exposure period. Microscopic inspection at 72 hours showed no abnormalities in the test and control cultures. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L. Given that the toxicity cannot be attributed to a single component or a mixture of components but to aluminum, benzoate C16-18-fatty acids complexes as a whole, the results were based on nominal loading rates only.

Results with reference substance (positive control):

- Reference: Harlan study number 41205049 used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L under similar exposure conditions and data evaluation to the definitive test.
- Results: The 72 hour ErC50 was 1.1 mg/L with 95 % confidence limits of 1.0 to 1.3 mg/L and the EyC50 was 0.70 mg/L. It was not possible to calculate 95% confidence limits for the EyC50 value as the data generated did not fit the models available for the calculation of confidence limits. The 72 hour NOEC was 0.50 mg/L based on growth rate and 0.25 mg/L based on yield. The 72 hour LOEC was 1.0 mg/L based on growth rate and 0.50 mg/L based on yield. The results were within normal ranges for this reference item.

Reported statistics and error estimates:

A student's t-test incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure were carried out on the growth rate and yield data after 72 hours for the control and treatment group to determine any statistically signficiant differences.

Any other information on results incl. tables

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures were observed to be pale green dispersions.

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)			Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h		% Inhibition
Control	R1	0.063	-	4.75E+05		-
	R2	0.064		4.88E+05
	R3	0.066		5.57E+05
	R4	0.065		5.27E+05
	R5	0.064		4.80E+05
	R6	0.061		4.10E+05
	Mean	0.064		4.89E+05
	SD	0.002		5.03E+05
100	R1	0.065	[2]	5.51E+05
	R2	0.066	[3]	5.56E+05
	R3	0.066	[3]	5.79E+05
	R4	0.064	0	4.96E+05
	R5	0.063	2	4.74E+05
	R6	0.064	0	4.94E+05
	Mean	0.065	[1]	5.25E+05		[7]
	SD	0.001		4.22E+05

* In accordance with the OECD test guideline only the mean value for yield is calculated

R1 – R6 = Replicates 1 to 6

SD = Standard Deviation

[Increase in growth as compared to controls]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All validity criteria met.

Conclusions:

The toxicity of aluminum, benzoate C16-18-fatty acids complexes to algal growth showed no effects at a water accommodated fraction nominal loading rate of 100 mg/L. Therefore, the EL50 is determined to be greater than 100 mg/L loading rate (WAF) and the NOELR is 100 mg/L loading rate (WAF).

Executive summary:

The toxicity of aluminum, benzoate C16-18-fatty acids complexes to algal growth showed no effects at a water accommodated fraction nominal loading rate of 100 mg/L. Therefore, the EL50 is determined to be greater than 100 mg/L loading rate (WAF) and the NOELR is 100 mg/L loading rate (WAF). The toxicity of aluminum, benzoate C16-18-fatty acids complexes to algae (Pseudokirchneriella subcapitata) was determined in a GLP-compliant, static growth inhibition study following OECD guideline 201 (Harlan 2013). Algae were exposed for 72 hours to a control and a single concentration of 100 mg/L loading rate WAF in media adjusted to a hardness of approximately 150 mg CaCO3/L. Observations of algal biomass were made at 24, 48 and 72 hours and the test solutions analysed for aluminium content and total organic carbon at test initiation and termination. The study is considered reliable and relevant for use for this endpoint.