Silicon carbide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-03-24 to 2016-07-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Test design was changed during testing; change not comprehensible

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

Only three concentrations tested instead of 5; test design changed without comprehensive argumentation

Principles of method if other than guideline:

ln a first experiment the validity criteria were not met. Therefore, the study was repeated. The results of the first experiment are not stated in this report, but will be kept together with the other raw data in the GLP archive of the test facility. ln the repetition of the first experiment, inhibition in all treatments was observed. Therefore, an additional experiment was performed. ln the second experiment, the stock solutions with the minerals were added after removal of the test item to check whether the test item has an influence on the composition of the test medium during preparation of the water-accommodated fractions (WAFs).

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Silicon carbide (SiC)
Batch no.: SiC Lab sample
Appearance: grey granules/grains
Composition: 98 % SiC, 1 % sand, 1 % carbon
CAS No.: 409-21-2
ElNECS-No.: 206-991-8
Molecular formula: SiC
Molecular weight: 40.0965 g/mol
Purity: 98 %
Homogeneity: homogeneous
Solubility H20: <0.1g/L
Expiry date 31. Dezember 2017
Storage Room Temperature (20 ± 5°C); keep in dry condition

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Preparations
First experiment
The water-accommodated fractions (WAFs) were prepared for the test. This was done mixing the nominal loads (1.05 / 10.10 / 100.00 mg/L real loads) with the corresponding amount of nutrient medium and shaking vigorously for 24 hours. The resulting solution was filtrated through 0.45 µm nylon filters.
Second experiment
The water-accommodated fractions (WAFs) were prepared for the test. This was done mixing the nominal loads (1.1 / 10.2 / 100.0 mg/L real loads) with the corresponding amount of demineralised water and shaking vigorously for 23.5 hours. The resulting solution was filtrated through 0.45 µm nylon filters. After this, 10 mL/L of stock solution l and 1 mL/L each of stock solutions ll; lll and IV were added.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Unicellular freshwater green alga.
Genus: Desmodesmus
Species: subspicatus
SAG Strain Number: 86.81
Taxonomic position: Chlorophyta - Chlorophyceae

Origin and Culture
The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (lnstitut fiir Pflanzenphysiologie of Universitat Gottingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture
was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.6 – 24.0 °C

pH:

7.8 to 8.1

Nominal and measured concentrations:

1, 10 and 100 mg/L loading rate (no analytical confirmation)

Details on test conditions:

First Experiment
Algae
Four days before the start of the test, an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 72 hours. The resulting culture grew exponentially. Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined.
Performance of the Study
For each treatment, 200 mL of the respective test item solution was mixed with the necessary amount of algal pre-culture (0.32 mL) to achieve a cell concentration of 2.4 – 3.2 *103 cells/mL. In this mixture, the pH-value was measured. For the blank control, 350 mL nutrient medium was used instead of test item solution and mixed with the necessary amount of algal pre-culture (0.56 mL). In this mixture, the pH-value was measured. The test vessels were filled with 45 mL of the respective test solution and incubated open (covered with perforated plastic foil) for 72 hours, shaken on an orbital shaker. Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter. After the test, the pH value in treatments and control was measured again. At the end of the test, the treatments were examined microscopically to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test item).
Experimental Conditions
Date: 13. – 16. April 2016
Treatments tested: 1 / 10 / 100 mg/L nominal concentration
Number of replicates: 6 replicates for the control, 3 replicates for each treatment
Vessels: glass flasks total volume 65 mL
Duration: 72 hours
Temperature: 22.1 – 23.4 °C
Lighting: 5300 Lux
Blank control: nutrient medium and algae
Treatments: test solution and algae

Second Experiment
Algae
Three days before the start of the test, an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours. The resulting culture grew exponentially. Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined.
Performance of the Study
For each treatment, 200 mL of the respective test item solution was mixed with the necessary amount of algal pre-culture (0.98 mL) to achieve a cell concentration of 3.3 - 3.8 *10^3 cells/mL. In this mixture, the pH-value was measured. For the blank control, 350 mL nutrient medium was used instead of test item solution and mixed with the necessary amount of algal pre-culture (1.72 mL). In this mixture, the pH-value was measured. The test vessels were filled with 45 mL of the respective test solution and incubated open (covered with perforated plastic foil) for 72 hours, shaken on an orbital shaker. Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter. After the test, the pH value in treatments and control was measured again. At the end of the test, the treatments were examined microscopically to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test item).
Experimental Conditions
Date: 10. – 13. May 2016
Treatments tested: 1 / 10 / 100 mg/L
Number of replicates: 6 replicates for the control, 3 replicates for each treatment
Vessels: glass flasks total volume 65 mL
Duration: 72 hours
Temperature: 22.6 – 24.0 °C
Lighting: 5300 Lux
Blank control: deionised water with nutrient medium and algae
Treatments: test solution and algae

Reference substance (positive control):

yes

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No analytical determination; unclear whether EC 50 of actual concentrations would be much lower

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Excel®) based on the results of the second experiment only. The estimation of the biological data, based on the third experiment, was accomplished using the software ToxRat® Professional, version 3.2.1.
Both experiments met the criteria of validity.

First Experiment

Cell Numbers

The cell numbers were determined with an electronic particle counter. The means and standard deviations of the cell numbers of the control and the treatments are presented in the following table:

Table 1: Cell Number/mL

Nominal Concentration in mg/L	Parameter	Cell number / mL
		0 h	24 h	48 h	72
Blank control	Mean	2770	12417	4069	146347
Blank control	SD	288	1014	6035	15095
1	Mean	2620	12133	31487	113040
1	SD	183	791	482	9174
10	Mean	2753	11047	25027	60507
10	SD	321	962	3628	14959
100	Mean	2620	9273	18960	39207
100	SD	183	2085	2755	11938
SD = Standard deviation

 

Inhibition

The following mean inhibition values were calculated for the Growth Rate µ and the Yield.

Nominal concentration in mg/L	Inhibition %
	Growth Rate (0-72h)	Yield (0-72h)
Blank control	0	0
1	5.11	23.09
10	22.54	59.78
100	32.49	74.52

 

 

In the second experiment no deviaiton from the control group was observed for any treatment.

In the first experiment, inhibition in all treatments was observed. Therefore, a second experiment was performed. In the second experiment, the stock solutions with the minerals were added after removal of the test item to check whether the test item has an influence on the composition of the test medium during preparation of the water-accommodated fractions (WAFs). In the second experiment, no inhibition of algal growth was observed. That means inhibition of algal growth in the second experiment was caused by an influence of the test item on the composition of the test medium and not by toxicity of the test item. Therefore, only the second experiment was used for determination of the biological results.

Validity criteria fulfilled:

yes

Conclusions:

In the first experiment, inhibition in all treatments was observed. Therefore, a second experiment was performed. In the second experiment, the stock solutions with the minerals were added after removal of the test item to check whether the test item has an influence on the composition of the test medium during preparation of the water-accommodated fractions (WAFs). In the second experiment, no inhibition of algal growth was observed. That means inhibition of algal growth in the second experiment was caused by an influence of the test item on the composition of the test medium and not by toxicity of the test item. Therefore, only the second experiment was used for determination of the biological results.

Executive summary:

In a 72 hour acute toxicity study, the cultures of Desmodesmus subspicatus were exposed to silicon carbide at nominal concentrations of 0, 1, 10 and 100 mg/L under static conditions in accordance with the OECD guideline 201. In the first experiment, inhibition in all treatments was observed. Therefore, a second experiment was performed. In the second experiment, the stock solutions with the minerals were added after removal of the test item to check whether the test item has an influence on the composition of the test medium during preparation of the water-accommodated fractions (WAFs). In the second experiment, no inhibition of algal growth was observed. That means inhibition of algal growth in the second experiment was caused by an influence of the test item on the composition of the test medium and not by toxicity of the test item. Therefore, only the second experiment was used for determination of the biological results.

  

Results Synopsis

 

Test Organism: Desmodesmus subspicatus

Test Type (Flowthrough, Static, Static Renewal): Static

 

72 h ErC 50 > 100 mg/L (loading rate, no analytical verification)

Endpoint(s) Effected:  growth rate

Description of key information

In a 72 hour acute toxicity study, the cultures of Desmodesmus subspicatus were exposed to silicon carbide at nominal concentrations of 0, 1, 10 and 100 mg/L under static conditions in accordance with the OECD guideline 201. In the first experiment, inhibition in all treatments was observed. Therefore, a second experiment was performed. In the second experiment, the stock solutions with the minerals were added after removal of the test item to check whether the test item has an influence on the composition of the test medium during preparation of the water-accommodated fractions (WAFs). In the second experiment, no inhibition of algal growth was observed. That means inhibition of algal growth in the second experiment was caused by an influence of the test item on the composition of the test medium and not by toxicity of the test item. Therefore, only the second experiment was used for determination of the biological results.

72 h ErC 50 > 100 mg/L (loading rate, no analytical verification)