Ashes (residues), nonhazardous municipal solid waste

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:

11.1.-24.1.2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

The study was perfomed without the analytical determination of the test substance concentrations. For all evaluations and results nominal concentrations of the test substance were used.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ashes (residues)

EC Number:

268-627-4

EC Name:

Ashes (residues)

Cas Number:

68131-74-8

Test material form:

solid: bulk

Details on test material:

- Name of test material (as cited in study report): Ashes (residues)
- Substance type: technical product
- Physical state: solid
- Main components: SiO2 (42.12%), Al2O3 (32.16%), Fe2O3 (10.88%), TiO2, MnO, MgO, CaO, Na2O, K2O, SO3 - constituents with content less than 10%
- Expiration date of the lot/batch: unlimited
- Appearance: greyish black powder

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION OF SOLUTIONS OF TEST SUBSTANCE:
Since the test substance is poorly soluble in test medium at concentrations needed for the test, the saturated solution of the test substance in test medium was used as stock solution. 100 mg of the test substance was weighted into 1000 mL of test medium for preliminary and limit test. This mixture was stirred on a shaft stirrer throughout three days, ultrasonicated for 10 minutes and filtered through 0.45 micron filter. The concentrations of solutions used in the preliminary and limit test were obtained by dilution of the stock saturated solution with test medium.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM:
- Strain: Brinkmann 1953/SAG 86.81
- Source (laboratory, culture collection): The collection of autotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: The strain culture was preinoculated from the stock solution and cultivated in flasks with the test medium on indirect daylight at laboratory temperature. Algae inoculum for the test was sampled from exponentially growing inoculum culture. The strain culture was always set to pre-culturing of cells for 3 days before the start of the test. Inoculum culture was kept 3 days under conditions at which the test was performed. After this period the culture was in the state of the exponential growth and had the cell density suitable for the performance of the test. The cell density of the pre-culture was measured just before the start of the test and the needed inoculum volume was calculated.


ACCLIMATION
- Acclimation period: no
- Any deformed or abnormal cells observed: none

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

23+/-2oC

pH:

Preliminary test: 7.6
Limit test: 7.7

Nominal and measured concentrations:

Nominal concentration (preliminary test): 100, 50, 10, 5, 1 mg.L-1
Nominal concentration (limit test): 100, 91, 83, 75, 68, 62 mg.L-1

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks with stoppers venting air
- Type (delete if not applicable): open
- Material: glass
- Aeration: without aeration
- Initial cells density: 10000 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
The water with conductivity smaller than 5 μS.cm-1 was used for the preparation of solutions. All chemicals used were of analytical-grade.

Basic stock solutions
According to EU Method C.3 were prepared four basic stock solutions. The solutions were kept in the dark at temperature of 4+/-1 °C in fridge. The temperature was checked every day.

Preparation of test medium
The test medium was prepared by admixing of basic stock solutions in distilled water.

OTHER TEST CONDITIONS
- Lighting: continuous, min 6000 lux, max. 10000 lux
- Volume of tested mixture: 50 mL
- Agitation of algae culture by shaking.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- At the beginning and the end of the test, the pH of the test mixtures was measured. The lighting intensity and temperature were measured every 24 hours. The density of algae culture was evaluated microscopically at 24, 48 and 72 hours.
- Determination of cell concentrations: Burker´s counting chamber

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: Since the test substance is poorly soluble in test medium it was not possible to achieve the concentration of 100 mg.L-1. For this reason a saturated solution of the test substance in test medium was prepared for preliminary and limit test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: EbC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

other: NOECb

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 4

Duration:

72 h

Dose descriptor:

other: NOECr

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

The sensitivity of the test species and correctness of test performance is periodically verified on a three-month period by testing the reference substance, potassium dichromate.
The results of the verification test with K2Cr2O7 are the following:

72 hour - EbC50 = 0.33 mg.L-1 (95% confidence limit: 0.27 – 0.40 mg.L-1)
72 hour - ErC50 = 0.67 mg.L-1 (95% confidence limit: 0.53 – 0.81 mg.L-1)

The inhibition of test organisms in the reference substance solution meets criteria determined by the interlaboratory tests.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The highest inhibition of growth rate in limit test was 0.8 %. Therefore exact values of EbC50 and ErC50 could not be calculated and the values of EC are given in the form of a range.
The determination of NOEC values was done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of statistical software QC.Expert 2.5 © 1998-2000 (TriloByte, Czech Republic).
The nominal concentrations of the test substance were used for all evaluations and results.

Executive summary:

The test substance, Ashes (residues), was tested for growth inhibition on algae.

The test was performed according to EU Method C.3, Algal inhibition test, Directive 92/69/EEC, Published in OJ L 383A, 1992.

Since the test substance is poorly soluble in test medium it was not possible to achieve the concentration of 100 mg.L-1. For this reason a saturated solution of the test substance in test medium was prepared for preliminary and limit test.

The preliminary test was performed in range of test substance nominal concentrations 1 – 100 mg.L-1. Based on no toxicity of the test substance found in the preliminary test, the limit test was performed in appropriate concentration range. The limit test was performed in range of test substance nominal concentrations 62 – 100 mg.L-1.

The study was performed without the analytical determination of the test substance concentrations. The nominal concentrations of the test substance were used for all evaluations and results.

Test results:

72 hour - EbC50 > 100 mg.L-1 (nominal concentration)

72 hour - ErC50 > 100 mg.L-1 (nominal concentration)

72 hour - NOECb = 100 mg.L-1 (nominal concentration)

72 hour - NOECr = 100 mg.L-1 (nominal concentration)

Classification of the test substance on the basis of hazard to aquatic environment was performed according to the Directive 93/21/EEC, Annex IV (article 5.2.1.).

According to the classification criteria given in quoted Directive the test substance, Ashes (residues), is not assigned to any of quoted toxicity categories.